iPSCs: A tool for understanding ‘A Beautiful Mind’?

By Angela C. H. McDonald

A young brilliant mathematician seen by his colleagues as agitated, socially withdrawn, emotionally flat and paranoid is approached by a Department of Defense agent who requests his assistance with code breaking. Following acceptance of this job, the young professor believes he is being followed and is eventually chased through his university campus, captured and sedated. When the young professor comes to, he is in a psychiatric hospital and forced to realize that his secret work for the Department of Defense is one of his schizophrenic delusions. This is the story of John Nash portrayed in the film A Beautiful Mind. Like many individuals suffering from schizophrenia (1 in every 100 Canadians), John Nash struggles throughout his life with an array of symptoms including delusions and hallucinations. 

Can induced pluripotent stem cells (iPSCs) help us understand the underlying mechanism of this devastating and complex condition?  Maybe.

Scientists have proposed the use of iPSCs for modeling human disease however; many question the usefulness of iPSCs for modeling complex neurological diseases such as schizophrenia. Last month a team of researchers led by Fred Gage at the Salk Institute published in Nature the first example of modeling a complex neurological disease in a dish.

Skin fibroblasts from schizophrenia patients were obtained from a cell bank and reprogrammed into iPSCs. Patient-derived iPSCs were differentiated into neurons to study the cellular and molecular mechanisms of schizophrenia. The researchers found that patient-derived neurons were electrophysiologically equivalent to control neurons and the levels of a number of synaptic maturation markers were unaffected. However, schizophrenia neurons did show a decrease in the number or neuronal projections, decreased neuronal connectivity and alterations in global gene expression. Over 550 genes were aberrantly expressed in schizophrenia neurons, 25 per cent of which have been previously linked to the disease.

iPSC-derived schizophrenia neurons were treated with five antipsychotic drugs in an attempt to improve neuronal connectivity in vitro. The drugs were administered for the final three weeks of neuronal differentiation. Loxapine, an antipsychotic commonly used to treat schizophrenia was the only antipsychotic drug that significantly increased neuronal connectivity in all patient iPSC derived-neurons.

Interestingly, this is not the first study to investigate the molecular mechanisms of schizophrenia in vitro. A small number of studies have been performed on cultured fibroblasts from skin biopsies of schizophrenia patients. One study identified a cell proliferation defect in patient fibroblasts. While studying patient fibroblasts may provide some insight into disease mechanisms many scientists stress the importance of studying the appropriate cell type in vitro. For example, in the Nature paper noted above, Fred Gage’s group reported increased NRG1 (a protein thought to be involved in schizophrenia) expression in schizophrenia neurons but not SCZD fibroblasts, which they suggest, demonstrates the importance of studying the relevant cell type.  

I recently attended a Stem Cell Journal Club session at the Hospital for Sick Children where this paper was presented. Stem cell and neurobiologists in attendance raised a number of concerns about this study includingthe difficulty of modeling a complex systems disease that is thought to be a dynamic process, leading to the dysfunction of many pathways in the brain. How much insight will researchers have into this disease if they are studying only a few types of neurons in a dish? Even though many scientists are skeptical, we can’t disregard that almost all insight into the molecular mechanism of schizophrenia in human patients has come from the study of postmortem brain tissue. iPSCs are the only source of live human neurons available to researchersfor studying this devastating disease and for this reason, scientists should continue to use and optimize neurons from iPSCs.

 

Science 2.0: Time to throw open the laboratory doors?

by Michelle Ly

Almost three years ago, Scientific American asked if we were entering an age of Science 2.0.  Would science now be conducted in the open access realm –- freely publishing data, drafts and even whole papers? The economic cost of academic publishing has long been considered unsustainable. As well, the lack of freely accessible papers and results is a frequently heard criticism of academic research. Animal rights groups, for example, often use the perceived absence of transparency in science to cast doubt upon ethical practices involving animal testing. 

Open access has the support of many prominent scientists. This support has certainly increased over the years with diverse voices such as stem cell pioneer and cancer researcher James Till and microbiologist Jonathan Eisen joining the fray. But have we ended up in the Science 2.0 that Scientific American speculated?  While some blogs and open access journals have flourished, the majority of published data still ends up in subscription-based journals. 

One area of biology which has consistently embraced open access models is the field of bioinformatics.  Open access tools for genome analysis, molecular modeling and data visualization are common and frequently used. Recently, researchers from the RWTH Aachen University and Kiel University in Germany, together with The Scripps Research Institute in San Diego, California, published results detailing a new method for testing stem cells.  he procedure, called PluriTest, is freely available online and may satisfy the needs of both grant-starved researchers and animal rights activists alike. 

The current standard for proving pluripotency in a new cell line is through the generation of human cell derived teratomas in immunodeficient mice. Teratomas are solid tumors which contain a mixture of histologically distinct tissue types. Pluripotency is confirmed through tissue collection and subsequent histological analysis of the teratoma to determine if the cell line was able to form tissues of all three embryonic germ layers -– ectoderm, mesoderm and endoderm.   

Despite its widespread use, teratoma assays are not standardized, raising questions and concerns about the assay’s effectiveness as a stem cell quality control and regulatory tool. As well, the procedure spans 6-8 weeks between cell implantation and histological analysis, which may be unrealistic for use in large scale production.

In contrast, PluritTest uses a pattern recognition algorithm which is able to distinguish between pluripotent and non-pluripotent cell lines without the need for animal models. The algorithm relies on a large database of gene expression patterns from known human stem cell lines and returns results in about ten minutes. One of the authors, Dr. Franz-Josef Muller, describes the standardized verification process of PluriTest as a way for researchers to “forgo data from animal testing laboratories and simultaneously achieve more precise results”. As the use of stem cells in cell therapy and regenerative medicine increases, any method which reduces the cost of cell line production and testing ultimately benefits patients.     

Will it help further the open access cause? The original paper was, ironically, published behind a Nature paywall and lack of access may limit the number of PluriTest early adopters. However, it is encouraging to see the development of free tools in stem cell research and I am hopeful that more will follow.

 

Controversial stem cell clinic closed

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Please read it on its new home, Signals Blog: Controversial stem cell clinic closed

Using stem cells in developmental disorder research

Every two hours, someone is born with Rett Syndrome (RTT), a developmental disorder seen almost always in girls, but occasionally in boys. Those with the disease usually develop normally until they reach 12-18 months, at which point development stops and oftentimes is reversed, causing previously developed skills to deteriorate.

RTT is typically placed under the autism spectrum of disorders (although some have questioned this classification), and is the only autism-spectrum disorder with a known cause: A mutation in the MECP2 gene located on the X chromosome. With the cause known, researchers have a ‘starting point’ to look at for possible ways to combat the disorder, and that’s exactly what they’ve done.

One well-known researcher in the field is Dr. In-Hyun Park from the Yale Stem Cell Center, who presented his work at the Ottawa Hospital Research Institute’s Sprott Centre for Stem Cell Research in late March.

Tiny zebrafish shows how kidney regeneration could be achieved

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Aussies review embryonic and cloning legislation

by Ubaka Ogbogu

The Australian government is currently reviewing their stem cell and cloning research laws. The review, which began on December 22, 2010 with the appointment of an independent Legislation Review Committee (LRC) chaired by a retired federal judge, is the second since the enactment in 2002 of the Prohibition of Human Cloning for Reproduction Act and the Research Involving Human Embryos Act. The Australian Parliament amended both statutes in 2006, following an initial review mandated by each statute. The amended statutes contain provisions that require the Australian Minister of Mental Health and Aging to undertake further legislative reviews. These provisions instruct the reviewers to consider, among other things, medical and scientific development in embryonic stem cell research and “the actual or potential clinical and therapeutic applications of such research.”

Results from a government survey conducted in 2010 showed that the majority of Australians are in favour of stem cell research using both adult and embryonic stem cells. This report was one of the documents submitted to the LRC by the Australian Stem Cell Centre (ASCC) as part of their recommendations in support of the current regulatory framework. The ASCC submission includes five key recommendations:

At long last… a national cord blood bank for Canada

Yesterday the Ministers of Health for the Provinces and Territories of Canada announced an investment of CAD $48M over eight years to establish a national umbilical cord blood bank for Canada. This is a very welcome, and some might say, overdue investment for which many organisations in the stem cell field have been advocating for some time.

Blood and marrow (stem cell) transplantation is an important tool in the treatment of some leukemias, disorders of the blood making system (aplastic anemia, thalassemia, sickle cell anemia) and is now being piloted for the treatment of some immune and metabolic disorders such as Crohn’s, multiple sclerosis and diabetes. This process relies on the ability to identify a donor who has an identical or near identical immune system. While a brother/sister has a one in four chance of being a match, most Canadians do not have a suitable family donor due to small family size, and currently there are around 800 patients waiting for a transplant according to Canada’s unrelated bone marrow donor registry (OneMatch.ca). 

Umbilical cord Blood is a rich source of stem cells and can be used as an alternative to adult bone marrow or blood. However, in the absence of a public cord blood bank, Canada has had to import all of the cord blood used in the treatment of these diseases, at an average cost of CAD $37,000 per cord according to Canadian Blood Services. Last year 96 such transplants were performed in Canada. However, many more potential patients were left untreated because they were unable to find a match. The multicultural melting pot in which we all live and take pride in as Canadians also means Canada is one of the most genetically diverse countries in the world, and as a consequence immune matches can be difficult to find. For example, Canada’s indigenous Métis peoples are not represented in the cord blood banks of other nations. It is on these groups that Canada’s newly funded Cord Blood Bank will focus, in order to ensure that these potentially life saving therapies are available to all Canadians, regardless of their heritage.

 

Are stem cells branded?

I've heard many different adjectives used to frame discussions of stem cells: powerful, promising, controversial, cutting edge, rejuvenating, mysterious, sexy, to name but a few. All of these descriptors are used in various ways to promote particular opinions, beliefs or imagery of stem cells and many of them, I would argue, are vastly overused and in some cases, quite misleading.

While sitting in the morning session at the Understanding Stem Cell Controversies course currently taking place in Calgary, I was introduced to a new adjective in the stem cell lexicon -- branded (and, oddly enough, this descriptor was used by two presenters, Brian Kwon and Timothy Caulfield). Can a word, typically attached to such concepts as corporate identity and customer loyalty, be used in relation to the broad field of stem cells? Of course it can. There may not be a logo to solidify that brand, but the adjectives I listed are, in essence, the brand of stem cells. 

At the heart of this morning's discussions was the use (or misuse) of the stem cell brand not just to sell stem cell products such as face creams and spa treatments, but also to instil a belief that stem cell therapies are a routinely available and the best choice to be made in treating otherwise incurable injuries or disease. As an orthopaedic surgeon, Brian Kwon noted that "every one of my patients asks about stem cell treatment".

In fact, Kwon's current research found that the number of spinal cord injury patients who would choose a stem cell treatment outnumbers those who would choose a conventional drug therapy (assuming that both treatment options exist). Think about it. A spinal cord stem cell treatment would be highly invasive, requiring a major operation, exposure of the spinal cord, injection of a needle into the spinal cord itself and possibly a prolonged course of immunosuppressants. Conventional drug therapies are typically administered either orally or intravenously. 

Kwon further noted that a 2009 sample of media reports about potential treatments for spinal cord injury all used the term "stem cell" as part of the news story, even when the treatment in question had nothing to do with stem cells whatsoever. See more of Kwon's presentation:

 

Since stem cells have thus far resisted any attempt to define ownership, the brand also defies any single entity's attempt to manage it. For the scientific community, this is troubling. Timothy Caulfield spoke about a current trend to shift the stem cell brand identity from a scientific and clinical framework to the realm of "alternative medicine". Such a shift would negate the authority of scientists in their attempts to raise awareness of the dangers of stem cell tourism by distancing them from public discussions on the issue. 

-- Lisa Willemse, SCN Director of Communications

Genomic instability in iPS cells

by Chris Kamel

They're promising, but not perfect. Induced pluripotent stem cells are perhaps one of the most studied areas of stem cell research today, as researchers work to improve their method of production, but new findings out of Canada and Finland suggests that the process of reprogramming may cause unwanted and irreversible DNA damage. As such, the rush to improve iPS cell generation may, in itself, be partially responsible for decreased integrity of the cells.

To date, several methods of reprogramming have been developed with the partial aim of improving the efficiency of iPS cell generation. The reprogramming process involves the introduction of a key set of genes which, along with turning terminally differentiated cells into stem cells, can alter other aspects of cell biology. For example, one of the genes involved in reprogramming, the oncogene c-Myc, can interact with p53 and p21 activities. This has a positive effect on reprogramming efficiency but can also lead to genomic instability.

One mark of genomic instability is copy number variation (CNV), an abnormal number of copies of DNA caused by the deletion or duplication of regions of the genome. The Canadian-Finnish paper, to be published tomorrow in Nature, demonstrated that human iPS cells have more CNVs than human ES cell lines, and that the number of CNVs decreased as the induced pluripotent cells were passaged in culture. These observations were independent of gene delivery method, original cell source, and the presence or absence of c-Myc, and suggest that generation of CNVs occurs with the reprogramming process. Focussing on copy number variation caused by genomic deletions, the authors found that in "young" iPS cell populations, many deletions were found in regions responsible for mainenance of an undifferentiated state in ES cells. As cells were passaged in culture, these deletions were not found indicating these cells were being selected against and overgrown.

The study demonstrates that the reprogramming process to generate iPS cells is associated with a high mutation rate. While selection deleterious mutations in culture brings the number of CNVs in line with those in human ES cells, it does not exclude the fact that some mutations may, in fact, offer selective advantage and lead to propagation of unwanted traits. The paper offers some new insights into the reprogramming mechanisms and potential downsides of the iPS approach. Better understanding of the reprogramming process and of the genomic integrity of iPS cell lines could lead to more efficient techniques and higher quality iPS cells for use in future research and therapeutic applications.

 

Horse-derived iPS cells

by Chris Kamel

We've talked often about induced pluripotent stem cells (iPS cells) on this blog -- the transformation of adult terminally differentiated cells into stem cells that can differentiate into various lineages -- mostly in the context of discoveries in mice and potential applications in regenerative therapy for humans. One thing mentioned less often is the use of stem cell technologies for our pets and animal companions. Anew paper published by Dr. Andras Nagy's group at Mount Sinai Hospital and the University of Toronto has established equine iPS cell lines raising the possibility of stem cell therapies for horses.