To Err Is Human, To Study Errors Is Science

The family of cholesterol lowering drugs known as ‘statins’ are among the most widely prescribed medications for patients with cardiovascular disease. Large-scale clinical studies have repeatedly shown that statins can significantly lower cholesterol levels and the risk of future heart attacks, especially in patients who have already been diagnosed with cardiovascular disease. A more contentious issue is the use of statins in individuals who have no history of heart attacks, strokes or blockages in their blood vessels. Instead of waiting for the first major manifestation of cardiovascular disease, should one start statin therapy early on to prevent cardiovascular disease?

If statins were free of charge and had no side effects whatsoever, the answer would be rather straightforward: Go ahead and use them as soon as possible. However, like all medications, statins come at a price. There is the financial cost to the patient or their insurance to pay for the medications, and there is a health cost to the patients who experience potential side effects. The Guideline Panel of the American College of Cardiology (ACC) and the American Heart Association (AHA) therefore recently recommended that the preventive use of statins in individuals without known cardiovascular disease should be based on personalized risk calculations. If the risk of developing disease within the next 10 years is greater than 7.5%, then the benefits of statin therapy outweigh its risks and the treatment should be initiated. The panel also indicated that if the 10-year risk of cardiovascular disease is greater than 5%, then physicians should consider prescribing statins, but should bear in mind that the scientific evidence for this recommendation was not as strong as that for higher-risk individuals.

 

Oops button - via Shutterstock
Oops button – via Shutterstock

Using statins in low risk patients

The recommendation that individuals with comparatively low risk of developing future cardiovascular disease (10-year risk lower than 10%) would benefit from statins was met skepticism by some medical experts. In October 2013, the British Medical Journal (BMJ) published a paper by John Abramson, a lecturer at Harvard Medical School, and his colleagues which re-evaluated the data from a prior study on statin benefits in patients with less than 10% cardiovascular disease risk over 10 years. Abramson and colleagues concluded that the statin benefits were over-stated and that statin therapy should not be expanded to include this group of individuals. To further bolster their case, Abramson and colleagues also cited a 2013 study by Huabing Zhang and colleagues in the Annals of Internal Medicine which (according to Abramson et al.) had reported that 18 % of patients discontinued statins due to side effects. Abramson even highlighted the finding from the Zhang study by including it as one of four bullet points summarizing the key take-home messages of his article.

The problem with this characterization of the Zhang study is that it ignored all the caveats that Zhang and colleagues had mentioned when discussing their findings. The Zhang study was based on the retrospective review of patient charts and did not establish a true cause-and-effect relationship between the discontinuation of the statins and actual side effects of statins. Patients may stop taking medications for many reasons, but this does not necessarily mean that it is due to side effects from the medication. According to the Zhang paper, 17.4% of patients in their observational retrospective study had reported a “statin related incident” and of those only 59% had stopped the medication. The fraction of patients discontinuing statins due to suspected side effects was at most 9-10% instead of the 18% cited by Abramson. But as Zhang pointed out, their study did not include a placebo control group. Trials with placebo groups document similar rates of “side effects” in patients taking statins and those taking placebos, suggesting that only a small minority of perceived side effects are truly caused by the chemical compounds in statin drugs.

 

Admitting errors is only the first step

Whether 18%, 9% or a far smaller proportion of patients experience significant medication side effects is no small matter because the analysis could affect millions of patients currently being treated with statins. A gross overestimation of statin side effects could prompt physicians to prematurely discontinue medications that have been shown to significantly reduce the risk of heart attacks in a wide range of patients. On the other hand, severely underestimating statin side effects could result in the discounting of important symptoms and the suffering of patients. Abramson’s misinterpretation of statin side effect data was pointed out by readers of the BMJ soon after the article published, and it prompted an inquiry by the journal. After re-evaluating the data and discussing the issue with Abramson and colleagues, the journal issued a correction in which it clarified the misrepresentation of the Zhang paper.

Fiona Godlee, the editor-in-chief of the BMJ also wrote an editorial explaining the decision to issue a correction regarding the question of side effects and that there was not sufficient cause to retract the whole paper since the other points made by Abramson and colleagues – the lack of benefit in low risk patients – might still hold true. Instead, Godlee recognized the inherent bias of a journal’s editor when it comes to deciding on whether or not to retract a paper. Every retraction of a peer reviewed scholarly paper is somewhat of an embarrassment to the authors of the paper as well as the journal because it suggests that the peer review process failed to identify one or more major flaws. In a commendable move, the journal appointed a multidisciplinary review panel which includes leading cardiovascular epidemiologists. This panel will review the Abramson paper as well as another BMJ paper which had also cited the inaccurately high frequency of statin side effects, investigate the peer review process that failed to identify the erroneous claims and provide recommendations regarding the ultimate fate of the papers.

 

Reviewing peer review

Why didn’t the peer reviewers who evaluated Abramson’s article catch the error prior to its publication? We can only speculate as to why such a major error was not identified by the peer reviewers. One has to bear in mind that “peer review” for academic research journals is just that – a review. In most cases, peer reviewers do not have access to the original data and cannot check the veracity or replicability of analyses and experiments. For most journals, peer review is conducted on a voluntary (unpaid) basis by two to four expert reviewers who routinely spend multiple hours analyzing the appropriateness of the experimental design, methods, presentation of results and conclusions of a submitted manuscript. The reviewers operate under the assumption that the authors of the manuscript are professional and honest in terms of how they present the data and describe their scientific methodology.

In the case of Abramson and colleagues, the correction issued by the BMJ refers not to Abramson’s own analysis but to the misreading of another group’s research. Biomedical research papers often cite 30 or 40 studies, and it is unrealistic to expect that peer reviewers read all the cited papers and ensure that they are being properly cited and interpreted. If this were the expectation, few peer reviewers would agree to serve as volunteer reviewers since they would have hardly any time left to conduct their own research. However, in this particular case, most peer reviewers familiar with statins and the controversies surrounding their side effects should have expressed concerns regarding the extraordinarily high figure of 18% cited by Abramson and colleagues. Hopefully, the review panel will identify the reasons for the failure of BMJ’s peer review system and point out ways to improve it.

 

To err is human, to study errors is science

All researchers make mistakes, simply because they are human. It is impossible to eliminate all errors in any endeavor that involves humans, but we can construct safeguards that help us reduce the occurrence and magnitude of our errors. Overt fraud and misconduct are rare causes of errors in research, but their effects on any given research field can be devastating. One of the most notorious occurrences of research fraud is the case of the Dutch psychologist Diederik Stapel who published numerous papers based on blatant fabrication of data – showing ‘results’ of experiments on non-existent study subjects. The field of cell therapy in cardiovascular disease recently experienced a major setback when a university review of studies headed by the German cardiologist Bodo Strauer found evidence of scientific misconduct. The significant discrepancies and irregularities in Strauer’s studies have now lead to wide-ranging skepticism about the efficacy of using bone marrow cell infusions to treat heart disease.

 

It is difficult to obtain precise numbers to quantify the actual extent of severe research misconduct and fraud since it may go undetected. Even when such cases are brought to the attention of the academic leadership, the involved committees and administrators may decide to keep their findings confidential and not disclose them to the public. However, most researchers working in academic research environments would probably agree that these are rare occurrences. A far more likely source of errors in research is the cognitive bias of the researchers. Researchers who believe in certain hypotheses and ideas are prone to interpreting data in a manner most likely to support their preconceived notions. For example, it is likely that a researcher opposed to statin usage will interpret data on side effects of statins differently than a researcher who supports statin usage. While Abramson may have been biased in the interpretation of the data generated by Zhang and colleagues, the field of cardiovascular regeneration is currently grappling in what appears to be a case of biased interpretation of one’s own data. An institutional review by Harvard Medical School and Brigham and Women’s Hospital recently determined that the work of Piero Anversa, one of the world’s most widely cited stem cell researchers, was significantly compromised and warranted a retraction. His group had reported that the adult human heart exhibited an amazing regenerative potential, suggesting that roughly every 8 to 9 years the adult human heart replaces its entire collective of beating heart cells (a 7% – 19% yearly turnover of beating heart cells). These findings were in sharp contrast to a prior study which had found only a minimal turnover of beating heart cells (1% or less per year) in adult humans. Anversa’s finding was also at odds with the observations of clinical cardiologists who rarely observe a near-miraculous recovery of heart function in patients with severe heart disease. One possible explanation for the huge discrepancy between the prior research and Anversa’s studies was that Anversa and his colleagues had not taken into account the possibility of contaminations that could have falsely elevated the cell regeneration counts.

 

Improving the quality of research: peer review and more

Despite the fact that researchers are prone to make errors due to inherent biases does not mean we should simply throw our hands up in the air, say “Mistakes happen!” and let matters rest. High quality science is characterized by its willingness to correct itself, and this includes improving methods to detect and correct scientific errors early on so that we can limit their detrimental impact. The realization that lack of reproducibility of peer-reviewed scientific papers is becoming a major problem for many areas of research such as psychology, stem cell research and cancer biology has prompted calls for better ways to track reproducibility and errors in science.

One important new paradigm that is being discussed to improve the quality of scholar papers is the role of post-publication peer evaluation. Instead of viewing the publication of a peer-reviewed research paper as an endpoint, post publication peer evaluation invites fellow scientists to continue commenting on the quality and accuracy of the published research even after its publication and to engage the authors in this process. Traditional peer review relies on just a handful of reviewers who decide about the fate of a manuscript, but post publication peer evaluation opens up the debate to hundreds or even thousands of readers which may be able to detect errors that could not be identified by the small number of traditional peer reviewers prior to publication. It is also becoming apparent that science journalists and science writers can play an important role in the post-publication evaluation of published research papers by investigating and communicating research flaws identified in research papers. In addition to helping dismantle the Science Mystique, critical science journalism can help ensure that corrections, retractions or other major concerns about the validity of scientific findings are communicated to a broad non-specialist audience.

In addition to these ongoing efforts to reduce errors in science by improving the evaluation of scientific papers, it may also be useful to consider new pro-active initiatives which focus on how researchers perform and design experiments. As the head of a research group at an American university, I have to take mandatory courses (in some cases on an annual basis) informing me about laboratory hazards, ethics of animal experimentation or the ethics of how to conduct human studies. However, there are no mandatory courses helping us identify our own research biases or how to minimize their impact on the interpretation of our data. There is an underlying assumption that if you are no longer a trainee, you probably know how to perform and interpret scientific experiments. I would argue that it does not hurt to remind scientists regularly – no matter how junior or senior- that they can become victims of their biases. We have to learn to continuously re-evaluate how we conduct science and to be humble enough to listen to our colleagues, especially when they disagree with us.

 

Note: A shorter version of this article was first published at The Conversation with excellent editorial input provided by Jo Adetunji.

 

ResearchBlogging.org
Abramson, J., Rosenberg, H., Jewell, N., & Wright, J. (2013). Should people at low risk of cardiovascular disease take a statin? BMJ, 347 (oct22 3) DOI: 10.1136/bmj.f6123

Lab Grown Organs and Artistic Computers in Fifty Years?

The Pew Research Center released the 2014 survey of U.S. adults (1,001 participants, surveyed by land-line or cell phone interviews) regarding their views on technological advancements in the next 50 years.

Robot
Robot – via Shutterstock

Over eighty percent of the participants said that “People in need of an organ transplant will have new organs custom made for them in a lab” and roughly half of the participants felt that “Computers will be as effective as people at creating important works of art such as music, novels, movies, or paintings” within the next 50 years. The vast majority did not think that humans will be able to control the weather during the next few decades.

As someone working in the field of vascular and tissue engineering, I think that the perception of scientists being able to engineer transplantable organs within 50 years is realistic. We have made quite a bit of progress in the past decade when it comes to deriving functional tissues from stem cells, but we still need more research before we will be able to build functional organs. It may take a decade or two before we can reliably generate these organs, and even longer to teat and optimize them for therapeutic purposes, and to ensure their long-term survival in transplant recipients.

50 year predictions

The reason to be optimistic about engineering organs is that we have already seen examples of engineered tissues and small organoids being implanted into animal models. There are also ongoing early clinical trials with patches of engineered tissues and engineered blood vessels. Scaling up these successes to whole organ engineering in humans will be challenging but sounds feasible.

I am surprised by the fact that half of the U.S. adults believe computers will be “effective” at creating works of art within the next 50 years. Do we have preliminary evidence – even at a small scale – that computers can currently “create” art? Perhaps this comes down to our definitions of what constitutes “creativity”. One could envision computers generating paintings, music and novels based on existing art created by humans. But is that true creativity? Then again, when humans “create” art, they also base their new product on their experiences and prior art created by other humans. Maybe computer-created art in fifty years isn’t  far-fetched after all.

 

Attitudes towards changes

Not everyone is enthusiastic about new technologies.

 

When asked whether it would be a change for the better or a change for the worse……

 

1) “If most people wear implants or other devices that constantly show them information about the world around them”

2) “If lifelike robots become the primary caregivers for the elderly and people in poor health”

3) “If personal and commercial drones are given permission to fly through most U.S. airspace”

4) “If prospective parents can alter the DNA of their children to produce smarter, healthier, or more athletic offspring”

 

…the majority of participants felt they would be worse off with these changes.

The way the questions were phrased did not leave room for a more nuanced response. For example, would it be ok to change DNA to “produce” healthier children (i.e. correct lethal genetic defects using genome editing) without necessarily “producing” smarter and more athletic children?

Conflating health, intelligence and athleticism into one question makes it difficult to ascertain how the public feels about using genome editing to help children survive versus using it to make kids run faster.

Most participants did not think they would want to eat lab grown meat or use brain implants to improve their mental capacity but roughly half of them seemed fine with using driverless cars.

 

Lab grown meat

 

When asked about what futuristic invention they would like to own, younger participants seemed most excited about time travel and other travel gadgets (flying cars, bikes and space crafts), whereas older participants wanted to inventions to prolong life or cure diseases.

What do people want

I was a bit surprised that this final question did not elicit responses such as inventions that would help reduce or reverse global warming and pollution or inventions that could remedy world hunger and the global scarcity of resources. Maybe it has to also do with how the question was phrased. Here is the actual question:

Science fiction writers have always imagined new inventions that change the world of the future. How about you? If there was one futuristic invention that you could own, what would it be?

 

Here is the actual data (PDF) of the responses people gave:

 

Improved health and longevity/Cure for diseases                        9%

Time machine/Time travel                                                           9%

Flying car/Flying bike                                                                6%

Personal robot/Robot servants                                                 4%

Personal space craft                                                                 4%

Self-driving car                                                                         3%

Teleporter/Teleportation/Transporter                                           3%

World peace/Stop wars/Improved understanding/Better planet     2%

New energy source/efficient cars/other environment                    2%

Invention to make household tasks easier                                   1%

Ability to live forever/Immortality                                                  1%

Jetpack                                                                                    1%

Money/Scheme to get rich/Ability to read future                         1%

Brain implant/Improve memory                                                   1%

Hovercar/Hoverboard                                                                1%

Hologram/Holodeck                                                                  *

Remote communications (via device or ESP)                              *

Other                                                                                        9%

None/Nothing/Not interested in futuristic inventions                     11%

 

The science fiction reference in the question may have prompted participants to think of technologies described in sci-fi novels and movies. Perhaps the majority of respondents did not think that world peace or climate-control could be achieved with specific sci-fi style inventions. Or perhaps the participants did not realize that climate change, global scarcity of food or other resources and violent conflicts are some of the biggest threats that humankind has ever faced.

Many of the responses to this final question tend to fall into the category of “how could my life become more convenient“, such as using personal robots and flying cars. But will these conveniences even matter if we cannot curb the major threats that our planet faces?

Growing Skepticism about the Stem Cell Acid Trip

In January 2014, the two papers “Stimulus-triggered fate conversion of somatic cells into pluripotency” and “Bidirectional developmental potential in reprogrammed cells with acquired pluripotency” published in the journal Nature by Haruko Obokata and colleagues took the world of stem cell research by surprise.

Since Shinya Yamanaka’s landmark discovery that adult skin cells could be reprogrammed into embryonic-like induced pluripotent stem cells (iPSCs) by introducing selected embryonic genes into adult cells, laboratories all over the world have been using modifications of the “Yamanaka method” to create their own stem cell lines. The original Yamanaka method published in 2006 used a virus which integrated into the genome of the adult cell to introduce the necessary genes. Any introduction of genetic material into a cell carries the risk of causing genetic aberrancies that could lead to complications, especially if the newly generated stem cells are intended for therapeutic usage in patients.

billboard-63978_150

Researchers have therefore tried to modify the “Yamanaka method” and reduce the risk of genetic aberrations by either using genetic tools to remove the introduced genes once the cells are fully reprogrammed to a stem cell state, introducing genes without non-integrating viruses or by using complex cocktails of chemicals and growth factors in order to generate stem cells without the introduction of any genes into the adult cells.

The papers by Obokata and colleagues at the RIKEN center in Kobe, Japan use a far more simple method to reprogram adult cells. Instead of introducing foreign genes, they suggest that one can expose adult mouse cells to a severe stress such as an acidic solution. The cells which survive acid-dipping adventure (25 minutes in a solution with pH 5.7) activate their endogenous dormant embryonic genes by an unknown mechanism. The researchers then show that these activated cells take on properties of embryonic stem cells or iPSCs if they are maintained in a stem cell culture medium and treated with the necessary growth factors. Once the cells reach the stem cell state, they can then be converted into cells of any desired tissue, both in a culture dish as well as in a developing mouse embryo. Many of the experiments in the papers were performed by starting out with adult mouse lymphocytes, but the researchers also found that mouse skin fibroblasts and other cells could also be successfully converted into an embryonic-like state using the acid stress.

My first reaction was incredulity. How could such a simple and yet noxious stress such as exposing cells to acid be sufficient to initiate a complex “stemness” program? Research labs have spent years fine-tuning the introduction of the embryonic genes, trying to figure out the optimal combination of genes and timing of when the genes are essential during the reprogramming process. These two papers propose that the whole business of introducing stem cell genes into adult cells was unnecessary – All You Need Is Acid.

 

This sounds too good to be true. The recent history in stem cell research has taught us that we need to be skeptical. Some of the most widely cited stem cell papers cannot be replicated. This problem is not unique to stem cell research, because other biomedical research areas such as cancer biology are also struggling with issues of replicability, but the high scientific impact of burgeoning stem cell research has forced its replicability issues into the limelight. Nowadays, whenever stem cell researchers hear about a ground-breaking new stem cell discovery, they often tend to respond with some degree of skepticism until multiple independent laboratories can confirm the results.

My second reaction was that I really liked the idea. Maybe we had never tried something as straightforward as an acid stress because we were too narrow-minded, always looking for complex ways to create stem cells instead of trying simple approaches. The stress-induction of stem cell behavior may also represent a regenerative mechanism that has been conserved by evolution. When our amphibian cousins regenerate limbs following an injury, adult tissue cells are also reprogrammed to a premature state by the stress of the injury before they start building a new limb.

The idea of stress-induced reprogramming of adult cells to an embryonic-like state also has a powerful poetic appeal, which inspired me to write the following haiku:

 

The old warrior

plunges into an acid lake

to emerge reborn.

 

(Read more about science-related haikus here)

Just because the idea of acid-induced reprogramming is so attractive does not mean that it is scientifically accurate or replicable.

A number of concerns about potential scientific misconduct in the context of the two papers have been raised and it appears that the RIKEN center is investigating these concerns. Specifically, anonymous bloggers have pointed out irregularities in the figures of the papers and that some of the images may be duplicated. We will have to wait for the results of the investigation, but even if image errors or duplications are found, this does not necessarily mean that this was intentional misconduct or fraud. Assembling manuscripts with so many images is no easy task and unintentional errors do occur. These errors are probably far more common than we think. High profile papers undergo much more scrutiny than the average peer-reviewed paper, and this is probably why we tend to uncover them more readily in such papers. For example, image duplication errors were discovered in the 2013 Cell paper on human cloning, but many researchers agreed that the errors in the 2013 Cell paper were likely due to sloppiness during the assembly of the submitted manuscript and did not constitute intentional fraud.

Irrespective of the investigation into the irregularities of figures in the two Nature papers, the key question that stem cell researchers have to now address is whether the core findings of the Obokata papers are replicable. Can adult cells – lymphocytes, skin fibroblasts or other cells – be converted into embryonic-like stem cells by an acid stress? If yes, then this will make stem cell generation far easier and it will open up a whole new field of inquiry, leading to many new exciting questions. Do human cells also respond to acid stress in the same manner as the mouse cells? How does acid stress reprogram the adult cells? Is there an acid-stress signal that directly acts on stem cell transcription factors or does the stress merely activate global epigenetic switches? Are other stressors equally effective? Does this kind of reprogramming occur in our bodies in response to an injury such as low oxygen or inflammation because these kinds of injuries can transiently create an acidic environment in our tissues?

Researchers all around the world are currently attempting to test the effect of acid exposure on the activation of stem cell genes. Paul Knoepfler’s stem cell blog is currently soliciting input from researchers trying to replicate the work. Paul makes it very clear that this is an informal exchange of ideas so that researchers can learn from each other on a “real-time” basis. It is an opportunity to find out about how colleagues are progressing without having to wait for 6-12 months for the next big stem cell meeting or the publication of a paper confirming or denying the replication of acid-induced reprogramming. Posting one’s summary of results on a blog is not as rigorous as publishing a peer-reviewed paper with all the necessary methodological details, but it can at least provide some clues as to whether some or all of the results in the controversial Obokata papers can be replicated.

If the preliminary findings of multiple labs posted on the blog indicate that lymphocytes or skin cells begin to activate their stem cell gene signature after acid stress, then we at least know that this is a project which merits further investigation and researchers will be more willing to invest valuable time and resources to conduct additional replication experiments. On the other hand, if nearly all the researchers post negative results on the blog, then it is probably not a good investment of resources to spend the next year or so trying to replicate the results.

It does not hurt to have one’s paradigms or ideas challenged by new scientific papers as long as we realize that paradigm-challenging papers need to be replicated. The Nature papers must have undergone rigorous peer review before their publication, but scientific peer review does not involve checking replicability of the results. Peer reviewers focus on assessing the internal logic, experimental design, novelty, significance and validity of the conclusions based on the presented data. The crucial step of replicability testing occurs in the post-publication phase. The post-publication exchange of results on scientific blogs by independent research labs is an opportunity to crowd-source replicability testing and thus accelerate the scientific authentication process. Irrespective of whether or not the attempts to replicate acid-induced reprogramming succeed, the willingness of the stem cell community to engage in a dialogue using scientific blogs and evaluate replicability is an important step forward.

 

ResearchBlogging.org
Obokata H, Wakayama T, Sasai Y, Kojima K, Vacanti MP, Niwa H, Yamato M, & Vacanti CA (2014). Stimulus-triggered fate conversion of somatic cells into pluripotency. Nature, 505 (7485), 641-7 PMID: 24476887

Replicability of High-Impact Papers in Stem Cell Research

I recently used the Web of Science database to generate a list of the most highly cited papers in stem cell research. As of July 2013, the search for original research articles which use the key word “stem cells” resulted in the following list of the ten most widely cited papers to date:

 

Human ESC colony – Wikimedia

1. Pittenger M et al. (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284(5411):143-147

Citations: 8,157

 

2.  Thomson JA et al. (1998) Embryonic stem cell lines derived from human blastocysts. Science 282(5391):1145-1147

Citations: 5,565

 

3. Takahashi K and Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126(4): 663-676

Citations: 5,034

 

4. Takahashi K et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861-872

Citations: 4,061

 

5. Donehower LA et al  (1992) Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356(6366): 215-221

Citations: 3,279

 

6. Al-Hajj M et al (2003) Prospective identification of tumorigenic breast cancer cells. Proceedings of the National Academy of Sciences 100(7): 3983-3988

Citations: 3,183

 

7. Yu J et al (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318(5858): 1917-1920

Citations: 3,086

 

8. Jiang YH et al (2002) Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418(6893):41-49

Citations: 2,983

 

9. Orlic D et al (2001) Bone marrow cells regenerate infarcted myocardium. Nature 410 (6829):701-705

Citations: 2,961

 

10. Lu J et al (2005) MicroRNA expression profiles classify human cancers. Nature 435(7043): 834-838

Citations: 2,917

 

Three of the articles (Donehower et al, Al-Hajj et al and Lu et al) in this “top ten list” do not focus on stem cells but are actually cancer research papers. They were probably identified by the search because the authors may have made comparisons to stem cells or used stem cells as tools.The remaining seven articles are indeed widely known in the stem cell field.

 

The Science paper by Pittenger and colleagues in 1999 provided a very comprehensive description of mesenchymal stem cells (MSCs), a type of adult stem cell which is found in the bone marrow alongside hematopoietic stem cells (HSCs). Despite the fact that MSCs and HSCs are both adult stem cells in the bone marrow, they have very different functions. HSCs give rise to circulating blood cells, whereas MSCs primarily form bone, fat and cartilage as was nicely demonstrated by Pittenger and colleagues.

The article by Thomson and colleagues was published in 1998 in the journal Science described the derivation of human embryonic stem cells (ESCs) and revolutionized the field of stem cell research. While adult stem cells have a very limited capacity in terms of lineages they can turn into, ESCs are derived from the early blastocyst stage of embryonic development (within the first 1-2 weeks following fertilization) and thus retain the capacity to turn into a very wide range of tissues, such as neurons, heart cells, blood vessel cells or liver cells. This paper not only identified the methods for isolating human ESCs, but also how to keep them in culture and expand them as undifferentiated stem cells.

The Cell paper by Takahashi and Yamanaka in 2006 represented another major advancement in the field of stem cell biology, because it showed for the first time that a mouse adult skin cell (fibroblast) could be reprogrammed and converted into a truly pluripotent stem cell (an induced pluripotent stem cell or iPSC) which exhibited all the major characteristics of an embryonic stem cell (ESC). It was as if the adult skin cell was traveling back in time, erasing its identity of having been a skin cell and returning to primordial, embryonic-like stem cell. Only one year later, Dr. Yamanaka’s group was able to demonstrate the same phenomena for adult human skin cells in the 2007 Cell paper (Takahashi et al), and in the same year a different group independently confirmed that adult human cells could be reprogrammed to the iPSC state (Science paper by Yu et al in 2007). The generation of iPSCs described in these three papers is probably the most remarkable discovery in stem cell biology during the past decade. It is no wonder that each of these three papers have been cited several thousand times even though they were published only six or seven years ago, and that Dr. Yamanaka was awarded the 2012 Nobel prize for this pioneering work.

All five of the above-mentioned stem cell papers have one thing in common: the results have been repeated and confirmed by numerous independent laboratories all over the world. However, this does not necessarily hold true for the other two highly cited stem cell papers on this list.

The 2002 Nature paper by Jiang and colleagues from Dr. Verfaillie’s laboratory at the University of Minnesota proposed that the bone marrow contained a rather special subset of adult MSCs which had a much broader differentiation potential than had been previously recognized. While adult MSCs were thought to primarily turn into bone, cartilage or fat when given the appropriate cues, this rare new cell type – referred to as MAPCs (multipotent adult progenitor cells) – appeared to differentiate into a much broader range of tissues. The paper even showed data from an experiment in which these adult mouse bone marrow stem cells were combined with embryonic cells and gave rise to a chimeric mouse. i.e. a mouse in which the tissues were in part derived from standard embryonic cells and in part from the newly discovered adult MAPCs. Such chimerism suggested that the MAPCs were embryonic-like, contributing to the formation of all the tissues in the mice. At the time of its publication, this paper was met with great enthusiasm because it proved that the adult body contained embryonic-like cells, hidden away in the bone marrow, and that these MAPCs could be used to regenerate ailing organs and tissues without having to use ethically problematic human embryonic stem cells.

There was just one major catch. Many laboratories around the world tried to replicate the results and were unable to identify the MAPCs, and even when they found cells that were MAPCs, they were unable to confirm the embryonic-like nature of the cells. In a remarkable example of investigative journalism, the science journalists Peter Aldhous and Eugenie Reich identified multiple irregularities in the publications involving MAPCs and documented the inability of researchers to replicate the findings by publishing the results of their investigation in the New Scientist (PDF).

The second high profile stem cell paper which was also plagued by an inability to replicate the results was the 2001 Nature paper by Orlic and colleagues. In this paper from Dr. Anversa’s laboratory, the authors suggested that adult hematopoietic (blood-forming) stem cells from the bone marrow could regenerate an infarcted heart by becoming heart cells (cardiomyocytes). It was a rather bold claim, because simply injecting these blood-forming stem cells into the heart seemed to be sufficient to redirect their fate. Instead of giving rise to red and white blood cells, these bone marrow cells were generating functional heart cells. If this were the case, then every patient could be potentially treated with their own bone marrow and grow back damaged heart tissue after a heart attack. Unfortunately, it was too good to be true. Two leading stem cell laboratories partnered up to confirm the results, but even after years of experiments, they were unable to find any evidence of adult bone marrow stem cells converting into functional heart cells. They published their findings three years later, also in the journal Nature:

Murry CE et al (2004) Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature 428(6983): 664-668

Citations: 1,150

 

Interestingly, the original paper which had made the claim that bone marrow cells can become functional heart cells has been cited nearly 3,000 times, whereas the refutation by Murry and colleagues, published in the same high-profile journal has been cited only 1,150 times. The vast majority of the nearly 3,000 citations of the 2001 paper by Orlic and colleagues occurred after it had been refuted in 2004! The 2001 Orlic et al paper has even been used to justify clinical trials in which bone marrow was obtained from heart attack patients and injected into their hearts. As expected after the refutation by Murry and colleagues, the success of these clinical trials was rather limited One of the largest bone marrow infusion trials in heart attack patients was recently published, showing no success of the therapy.

These claims of the two papers (Orlic et al and Jiang et al) were quite innovative and exciting, and they were also published in a high-profile, peer-reviewed journal, just like the other five stem cell papers. The crucial difference was the fact that their findings could not be replicated by other laboratories. Despite their lack of replicability, both papers had an enormous impact on the field of stem cell research. Senior scientists, postdocs and graduate students may have devoted a substantial amount of time and resources to developing projects that built on the findings of these two papers, only to find out that they could not be replicated. If there is a lesson to be learned, it is that we need to be rather cautious in terms of our enthusiasm for new claims in stem cell biology until they have been appropriately confirmed by other researchers. Furthermore, we need to streamline the replicability testing process so that we do not have to wait years before we find out that one of the most highly prized discoveries cannot be independently confirmed.

 

Update 7/24/2013: Peter Aldhous reminded me that the superb job of investigative journalism into the question of MAPCs was performed in partnership with the science writer Eugenie Reich, the author of a book on scientific fraud. I have updated the blog post to reflect this.

“Inflamm-Aging”: Inflammatory Signals in the Brain Regulate the Lifespan of Mice

The hypothalamus is located at the base of the brain and in adult humans, it has a volume of only 4cm3, less than half a percent of the total adult human brain volume. Despite its small size, the hypothalamus is one of the most important control centers in our brain because it functions as the major interface between two regulatory systems in our body: The nervous system and the endocrine (hormonal) system. It consists of many subunits (nuclei) which continuously sense inputs and then respond to these inputs by releasing neurotransmitters or hormones that regulate a broad range of vital functions, such as our metabolism, appetite, thirst, reproduction, temperature and even our internal timing system, the circadian clock. As if this huge workload wasn’t enough, researchers have now uncovered an additional role for the hypothalamus: regulating lifespan.

The recent paper “Hypothalamic programming of systemic ageing involving IKK-β,NF-κB and GnRH” published in the journal Nature (published online May 1, 2013) by Guo Zhang and colleagues at the Albert Einstein College of Medicine in New York used elegant genetic mouse models to either continuously activate or continuously suppress the function of the NF-κB protein in the hypothalamus. This protein is a key transcription factor which is found in most organs and tissues and turns on genes in response to an inflammatory stimulus. The researchers were thus able to artificially create an internal scenario in which the hypothalamus was receiving a continuous “inflammation on” or “inflammation off” input without having to provide any external infectious or inflammatory agents. The results were quite striking. Continuous activation of the inflammatory NF-κB pathway in the hypothalamus resulted in a reduction of overall lifespan in the mice, but it also resulted in a loss of muscle mass, bone mass, and cognitive function – the mice showed signs of accelerated aging. An even more remarkable finding was that continuous suppression of the inflammatory pathway extended the lifespan of the mice when compared to their littermates that did not undergo any genetic modifications. Not only did these mice live longer (median lifespan increased by 23%), but they also exhibited significantly less physical and cognitive decline than regular mice!

To investigate the mechanism by which the suppression of inflammatory signals could result in such a profound increase in longevity and functional capacity, the researchers studied Gonadotropin Releasing Hormone (GnRH), one of the major hormones released by the hypothalamus which in turn regulates the release of reproductive hormones. They found that aging or inflammatory activation indeed suppressed GnRH release, whereas inhibition of the inflammatory signaling was able to restore GnRH levels. More importantly, simply injecting the mice with GnRH was able to prevent the physical and cognitive decline in the aging mice. How the injections of GnRH were able to restore muscle mass and even cognitive function was not evaluated in the study, but the researchers did observe that the brain showed increased evidence of neuron growth, which could explain the anti-aging effects of GnRH.

This paper is not the first to link inflammation to aging, but it is the first to show that localized inflammation signals in the hypothalamus can have such a profound effect on the lifespan of mice and it is also the first to propose that suppression of GnRH may be the reason for this inflammation-aging link. As with all important scientific papers, this study raises more questions than it answers. Is GnRH not just a regulator of sex hormones, but does it also exert effects on neurons and muscle cells that are independent of its role as a regulator of reproductive hormones? The mice with prolonged life-spans were all studied in a laboratory setting and thus not exposed to infectious agents that mice (or humans, for that matter) living in the wild commonly encounter. Would suppression of the NF-κB pathway in the hypothalamus possibly compromise their ability to fend off infections or other natural forms of inflammation? It is also not clear whether the GnRH link would apply to all mammals such humans, since aging female primates have higher, (not lower!) GnRH levels. These are all questions that lie beyond the scope of this paper and they need to be addressed in future papers.

However, there are some major limitations of this study and the proposed new hypothalamus-inflammation-GnRH-aging model. First, there is one rather obvious experiment that is missing. The researchers showed that manipulating NF-κB in the hypothalamus can have a major effect on the lifespan and the cognitive as well as physical function, but for some reason the researchers did not show the results from a rather simple experiment: Does GnRH alone extend the lifespan? If GnRH were really the main pathway by which the hypothalamus regulates aging, than giving GnRH ought to have extended the lifespan of the mice.

A second limitation of the paper is that it does not distinguish between general functional decline versus decreased regeneration. Biological aging is characterized by a gradual functional decline over time, but this is due to a combination of at least two parallel processes. Existing cells and tissues accumulate damaged and become dysfunctional and regenerative stem cells or progenitor cells become exhausted and cannot keep up with the repair. This study does not assess whether increased NF-κB activation in the hypothalamus causes more cellular dysfunction, whether it merely inhibits the regenerative repair process or whether it affects both. The researchers did not perform assessments of cellular aging, such as measuring the expression levels of the cellular aging regulator p16 or quantify oxidative stress. Therefore, it is unclear whether NF-κB activation in the hypothalamus had any impact on the cellular aging (senescence) program in the brain, muscles or elsewhere in the body.

Another key limitation is that the hypothalamus has so many functions other than GnRH release, which could all contribute to aging and changes in the lifespan of the mice. The authors themselves have previously published that NF-κB in the hypothalamus regulates the link between obesity and high blood pressure and multiple other groups have already shown that the hypothalamus may affect aging via its role in metabolic regulation. Unfortunately, the current study glosses over the potential role of metabolism and high blood pressure, which could explain the observed longevity effects and instead just focuses on the more provocative but less substantiated idea of GnRH as the aging regulator.

Due to these limitations, we still have to await additional studies that confirm the role of GnRH as the target for NF-κB activation in the hypothalamus and this link between inflammation, aging and the hypothalamus.

We should also remember that biological aging is just one aspect of aging. As André Maurois once wrote, “Old age is far more than white hair, wrinkles, the feeling that it is too late and the game finished, that the stage belongs to the rising generations. The true evil is not the weakening of the body, but the indifference of the soul.

 

Image credit: A GIF depicting the Hypothalamus “BodyParts3D”, © The Database Center for Life ScienceCreative Commons license via Wikimedia Commons, Painting by John Haberle (1856-1933) – Time and Eternity, via Wikimedia Commons

 

ResearchBlogging.org
Zhang G, Li J, Purkayastha S, Tang Y, Zhang H, Yin Y, Li B, Liu G, & Cai D (2013). Hypothalamic programming of systemic ageing involving IKK-β, NF-κB and GnRH. Nature, 497 (7448), 211-216 PMID: 23636330

Cellular Alchemy: Converting Fibroblasts Into Heart Cells

Medieval alchemists devoted their lives to the pursuit of the infamous Philosopher’s Stone, an elusive substance that was thought to convert base metals into valuable gold. Needless to say, nobody ever discovered the Philosopher’s Stone. Well, perhaps some alchemist did get lucky but was wise enough to keep the discovery secret. Instead of publishing the discovery and receiving the Nobel Prize for Alchemy, the lucky alchemist probably just walked around in junkyards, surreptitiously collected scraps of metal and brought them to home to create a Scrooge-McDuck-style money bin.  Today, we view the Philosopher’s Stone as just a myth that occasionally resurfaces in the titles of popular fantasy novels, but cell biologists have discovered their own version of the Philosopher’s Stone: The conversion of fibroblast cells into precious heart cells (cardiomyocytes) or brain cells (neurons).

 

Fibroblasts are an abundant cell type, found in many organs such as the heart, liver and the skin. One of their main functions is to repair wounds and form scars in this process. They are fairly easy to grow or to expand, both in the body as well as in a culture dish. The easy access to large quantities of fibroblasts makes them analogous to the “base metals” of the alchemist. Adult cardiomyocytes, on the other hand, are not able to grow, which is why a heart attack which causes death of cardiomyocytes can be so devastating. There is a tiny fraction of regenerative stem-cell like cells in the heart that are activated after a heart attack and regenerate some cardiomyocytes, but most of the damaged and dying heart cells are replaced by a scar – formed by the fibroblasts in the heart. This scar keeps the heart intact so that the wall of the heart does not rupture, but it is unable to contract or beat, thus weakening the overall pump function of the heart. In a large heart attack, a substantial portion of cardiomycoytes are replaced with scar tissue, which can result in heart failure and heart failure.

A few years back, a research group at the Gladstone Institute of Cardiovascular Disease (University of California, San Francisco) headed by Deepak Srivastava pioneered a very interesting new approach to rescuing heart function after a heart attack.  In a 2010 paper published in the journal Cell, the researchers were able to show that plain-old fibroblasts from the heart or from the tail of a mouse could be converted into beating cardiomyocytes! The key to this cellular alchemy was the introduction of three genes – Gata4, Mef2C and Tbx5 also known as the GMT cocktail– into the fibroblasts. These genes encode for developmental cardiac transcription factors, i.e. proteins that regulate the expression of genes which direct the formation of heart cells. The basic idea was that by introducing these regulatory factors, they would act as switches that turn on the whole heart gene program machinery. Unlike the approach of the Nobel Prize laureate Shinya Yamanaka, who had developed a method to generate stem cells (induced pluripotent stem cells or iPSCs) from fibroblasts, Srivastava’s group bypassed the whole stem cell generation process and directly created heart cells from fibroblasts. In a follow-up paper published in the journal Nature in 2012, the Srivastava group took this research to the next level by introducing the GMT cocktail directly into the heart of mice and showing that this substantially improved heart function after a heart attack. Instead of merely forming scars, the fibroblasts in the heart were being converted into functional, beating heart cells – cellular alchemy with great promise for new cardiovascular therapies.

As exciting as these discoveries were, many researchers remained skeptical because the cardiac stem cell field has so often seen paradigm-shifting discoveries appear on the horizon, only to later on find out that they cannot be replicated by other laboratories. Fortunately, Eric Olson’s group at the University of Texas, Southwestern Medical Center also published a paper in Nature in 2012, independently confirming that cardiac fibroblasts could indeed be converted into cardiomyocytes. They added on a fourth factor to the GMT cocktail because it appeared to increase the success of conversion. Olson’s group was also able to confirm Srivastava’s finding that directly treating the mouse hearts with these genes helped convert cardiac fibroblasts into heart cells. They also noticed an interesting oddity. Their success of creating heart cells from fibroblasts in the living mouse was far better than what they would have expected from their experiments in a dish. They attributed this to the special cardiac environment and the presence of other cells in the heart that may have helped the fibroblasts convert to beating heart cells. However, another group of scientists attempted to replicate the findings of the 2010 Cell paper and found that their success rate was far lower than that of the Srivastava group. In the paper entitled “Inefficient Reprogramming of Fibroblasts into Cardiomyocytes Using Gata4, Mef2c, and Tbx5” published in the journal Circulation Research in 2012, Chen and colleagues found that very few fibroblasts could be converted into cardiomyocytes and that the electrical properties of the newly generated heart cells did not match up to those of adult heart cells. One of the key differences between this Circulation Research paper and the 2010 paper of the Srivastava group was that Chen and colleagues used fibroblasts from older mice, whereas the Srivastava group had used fibroblasts from newly born mice. Arguably, the use of older cells by Chen and colleagues might be a closer approximation to the cells one would use in patients. Most patients with heart attacks are older than 40 years and not newborns.

These studies were all performed on mouse fibroblasts being converted into heart cells, but they did not address the question whether human fibroblasts would behave the same way. A recent paper in the Proceedings of the National Academy of Sciences by Eric Olson’s laboratory (published online before print on March 4, 2013 by Nam and colleagues) has now attempted to answer this question. Their findings confirm that human fibroblasts can also be converted into beating heart cells, however the group of genes required to coax the fibroblasts into converting is slightly different and also requires the introduction of microRNAs – tiny RNA molecules that can also regulate the expression of a whole group of genes. Their paper also points out an important caveat.  The generated heart-like cells were not uniform and showed a broad range of function, with only some of the spontaneously contracting and with an electrical activity pattern that was not the same as in adult heart cells.

Where does this whole body of work leave us? One major finding seems to be fairly solid. Fibroblasts can be converted into beating heart cells. The efficiency of conversion and the quality of the generated heart cells – from mouse or human fibroblasts – still needs to be optimized. Even though the idea of cellular alchemy sounds fascinating, there are many additional obstacles that need to be overcome before such therapies could ever be tested in humans. The method to introduce these genes into the fibroblasts used viruses which permanently integrate into the DNA of the fibroblast and could cause genetic anomalies in the fibroblasts. It is unlikely that such viruses could be used in patients. The fact that the generated heart cells show heterogeneity in their electrical activity could become a major problem for patients because patches of newly generated heart cells in one portion of the heart might be beating at a different rate of rhythm than other patches. Such electrical dyssynchony can cause life threatening heart rhythm problems, which means that the electrical properties of the generated cells need to be carefully understood and standardized. We also know little about the long-term survival of these converted cells in the heart and whether the converted cells maintain their heart-cell-like activity for months or years. The idea of directly converting fibroblasts by introducing the genes into the heart instead of first obtaining the fibroblasts, then converting them in a dish and lastly implanting the converted cells back into the heart sounds very convenient. But this convenience comes at a price. It requires human gene therapy which has its own risks and it is very difficult to control the cell conversion process in an intact heart of a patient. On the other hand, if cells are converted in a dish, one can easily test and discard the suboptimal cells and only implant the most mature or functional heart cells.

This process of cellular alchemy is still in its infancy. It is one of the most exciting new areas in the field of regenerative medicine, because it shows how plastic cells are. Hopefully, as more and more labs begin to investigate the direct reprogramming of cells, we will be able to address the obstacles and challenges posed by this emerging field.

 

Image credit: Painting in 1771 by Joseph Wright of Derby – The Alchymist, In Search of the Philosopher’s Stone via Wikimedia Commons

 

ResearchBlogging.org
Nam, Y., Song, K., Luo, X., Daniel, E., Lambeth, K., West, K., Hill, J., DiMaio, J., Baker, L., Bassel-Duby, R., & Olson, E. (2013). Reprogramming of human fibroblasts toward a cardiac fate Proceedings of the National Academy of Sciences, 110 (14), 5588-5593 DOI: 10.1073/pnas.1301019110

Bone Marrow Cell Infusions Do NOT Improve Cardiac Function After Heart Attack

For over a decade, cardiologists have been conducting trials in patients using cells extracted from the bone marrow and infusing them into the blood vessels of the heart in patients who have suffered a heart attack. This type of a procedure is not without risks. It involves multiple invasive procedures in patients who are already quite ill, because they are experiencing a major heart attack:

1) Patients with a major heart attack (also referred to as ST-elevation Myocardial Infarction or STEMI) usually undergo an immediate angiogram of the heart to treat the blockage that is causing the heart attack by impeding the blood flow. This is the standard of care for heart attack patients in the developed world.

2) Patients enrolled in an experimental cell therapy trial are then brought back for a second procedure during which bone marrow is extracted with a needle under local anesthesia.

3) The research patients then undergo another angiogram of the heart using a catheter which allows for the infusion of bone marrow cells into the heart.

The hope is that the stem cells contained within the bone marrow are able to help regenerate the heart, either by turning into heart cells (cardiomyocytes), blood vessel cells (endothelial cells) or releasing factors that protect the heart and prevent the formation of a large scar. Unfortunately, there is very limited scientific evidence that bone marrow stem cells can actually turn into functional heart cells. The trials that have been conducted so far have yielded mixed results – some show that infusing the bone marrow cells indeed improves heart function, others show that patients who just receive the standard therapy with cell infusions do just as well. Most of the trials have been quite small – often studying only 10-50 patients.

The SWISS-AMI cell therapy trial, published online on April 17, 2013 in the world’s leading cardiovascular research journal Circulation, addressed this question in a randomized, controlled trial, which enrolled 200 patients who had suffered a major heart attack. The published paper is entitled “Intracoronary Injection of Bone Marrow Derived Mononuclear Cells, Early or Late after Acute Myocardial Infarction: Effects on Global Left Ventricular Function” and was conducted in Switzerland.

The researchers assigned the patients to three groups: a) Standard heart attack treatment, b) Standard heart attack treatment and infusion of bone marrow cells 5-7 days after the heart attack or c) Standard heart attack treatment and infusion of bone marrow cells 3-4 weeks after the heart attack. They assessed heart function four months later using cardiac magnetic resonance imaging, one of the best tools available to determine heart function. The results were rather disappointing: Neither of the two cell treatment groups showed any improvement in their cardiac function.

This trial had some important limitations: Even though this study enrolled 200 patients and was thus larger than most other cell therapy trials for heart attack patients, it is still a rather small study when compared to other cardiovascular studies, which routinely enroll thousands of patients. Furthermore, this study only assessed heart function after four months and it is possible that if they had waited longer, they might have seen some benefit of the cell therapy. Despite these limitations, the trial will dampen the general enthusiasm for injecting bone marrow cells into heart attack patients.

Is this study a set-back for cardiac stem cell treatments? Not really. As the authors reveal in their data analysis, most of the cells contained in the bone marrow preparation that they used for the infusion were plain old white blood cells and NOT stem cells. Actually, only 1% of the infused cells were hematopoietic stem cells (stem cells that give rise to blood cells) and there was an undisclosed percentage of other stem cell types (such as mesenchymal stem cells) contained in the infused bone marrow extract. As I point out in the accompanying editorial “Bone Marrow Tinctures for Cardiovascular Disease: Lost in Translation“, using such a mixture of poorly defined cells is ill-suited to promote cardiac regeneration or repair. Therefore, this important study is not a set-back for cardiac stem cell therapy, but a well-deserved setback for injections of undefined cells, most of which are not true stem cells!

Even if the majority of infused cells had been stem cells, there is no guarantee that merely infusing them into the heart would necessarily result in the formation of new heart tissue. Regenerating heart tissue from adult stem cells requires priming or directing stem cells towards becoming heart cells and ensuring that the cells can attach and integrate into the heart, not just infusing or injecting them into the heart.

It is commendable that the journal published this negative study, because too many treatments are being marketed as “stem cell therapies” without clarifying whether the injected cells are truly efficacious. Hopefully, the results of this trial will lead to more caution when rushing to perform “stem cell treatments” in patients without carefully defining the scientific characteristics and therapeutic potential of the cells that are being used.

 

Link to the original paper:  “Intracoronary Injection of Bone Marrow Derived Mononuclear Cells, Early or Late after Acute Myocardial Infarction: Effects on Global Left Ventricular Function

Link to the editorial: “Bone Marrow Tinctures for Cardiovascular Disease: Lost in Translation

Image credit: Surgeon extracting bone marrow from a patient (Public Domain image via Wikimedia)

ResearchBlogging.org
Surder, D., Manka, R., Lo Cicero, V., Moccetti, T., Rufibach, K., Soncin, S., Turchetto, L., Radrizzani, M., Astori, G., Schwitter, J., Erne, P., Zuber, M., Auf der Maur, C., Jamshidi, P., Gaemperli, O., Windecker, S., Moschovitis, A., Wahl, A., Buhler, I., Wyss, C., Kozerke, S., Landmesser, U., Luscher, T., & Corti, R. (2013). Intracoronary Injection of Bone Marrow Derived Mononuclear Cells, Early or Late after Acute Myocardial Infarction: Effects on Global Left Ventricular Function Four months results of the SWISS-AMI trial Circulation DOI: 10.1161/CIRCULATIONAHA.112.001035
ResearchBlogging.org
Rehman, J. (2013). Bone Marrow Tinctures for Cardiovascular Disease: Lost in Translation Circulation DOI: 10.1161/CIRCULATIONAHA.113.002775