Gene appears to modify severity of autism’s social dysfunction

With the help of two sets of brothers with autism, Johns Hopkins scientists have identified a gene associated with autism that appears to be linked very specifically to the severity of social interaction deficits.

The gene, GRIP1 (glutamate receptor interacting protein 1), is a blueprint for a traffic-directing protein at synapses.

Identified more than a decade ago by Richard L. Huganir, PhD, professor and director of the Solomon H. Snyder Department of Neuroscience at the Johns Hopkins University School of Medicine, and a Howard Hughes Medical Institute investigator, GRIP1 regulates how fast receptors travel to a cell’s surface, where they are activated by a brain-signaling chemical called glutamate, allowing neurons to communicate with one another.

The new study, which tracked two versions of GRIP1 in the genomes of 480 people with autism, was published 22 March 2011 in the Proceedings of the National Academy of Sciences, and lends support to a prevailing theory that autism spectrum disorders (ASD), molecularly speaking, reflect an imbalance between inhibitory and excitatory signaling at synapses.

“The GRIP1 variants we studied are not sufficient to cause autism by themselves, but appear to be contributing factors that can modify the severity of the disease,” says Tao Wang, MD, PhD, assistant professor, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine. “GRIP1 mutations seem to contribute to social interaction deficits in the patients we studied.”

- doi: 10.1073/pnas.1102233108

Researchers find much more efficient way to create iPSCs

Researchers at the University of Pennsylvania School of Medicine have devised a totally new and far more efficient way of generating induced pluripotent stem cells (iPSCs). The researchers used fibroblast cells, which are easily obtained from skin biopsies, and could be used to generate patient-specific iPSCs for drug screening and tissue regeneration.

iPSCs are typically generated from adult non-reproductive cells by expressing four different genes called transcription factors. The generation of iPSCs was first reported in 2006 by Shinya Yamanaka, and multiple groups have since reported the ability to generate these cells using some variations on the same four transcription factors.

The promise of this line of research is to one day efficiently generate patientspecific stem cells in order to study human disease as well as create a cellular “storehouse” to regenerate a person’s own cells, for the heart or liver, for example. Despite this promise, generation of iPSCs is hampered by low efficiency, especially when using human cells.

“It’s a game changer,” says Edward Morrisey, PhD, professor in the Departments of Medicine and Cell and Developmental Biology and scientific director at the Penn Institute for Regenerative Medicine. “This is the first time we’ve been able to make induced pluripotent stem cells without the four transcription factors and increase the efficiency by 100-fold.” Morrisey led the study published April in Cell Stem Cell.

“Generating induced pluripotent stem cells efficiently is paramount for their potential therapeutic use,” noted James Kiley, PhD, director of the National Heart, Lung, and Blood Institute’s Division of Lung Diseases. “This novel study is an important step forward in that direction and it will also advance research on stem cell biology in general.”

Before this procedure, which uses microRNAs instead of the four key transcription factor genes, for every 100,000 adult cells reprogrammed, researchers were able to get a small handful of iPSCs, usually less than 20. Using the microRNA-mediated method, they have been able to generate approximately 10,000 induced pluripotent stem cells from every 100,000 adult human cells that they start with. MicroRNAs (miRNAs) are short RNA molecules that bind to complementary sequences on messenger RNAs to silence gene expression.

The Morrisey lab discovered this new approach through studies focusing on the role of microRNAs in lung development. This lab was working on a microRNA cluster called miR302/367, which plays an important role in lung endoderm progenitor development. This same microRNA cluster was reported to be expressed at high levels in embryonic stem cells, and iPSCs and microRNAs have been shown to alter cell phenotypes.

The investigators performed a simple experiment and expressed the microRNAs in mouse fibroblasts and were surprised to observe colonies that looked just like iPSCs. “We were very surprised that this worked the very first time we did the experiment,” says Morrisey. “We were also surprised that it worked much more efficiently than the transcription factor approach pioneered by Dr Yamanaka.”

Since microRNAs act as repressors of protein expression, it seems likely that they repress the repressors of the four transcription factors and other factors important for maintaining the pluripotent-stemcell state. However, exactly how the miRNAs work differently compared to the transcription factors in creating iPSCs will require further investigation.

The iPSCs generated by the microRNA method in the Morrisey lab are able to generate most, if not all, tissues in the developing mouse, including germ cells, eggs and sperm. The group is currently working with several collaborators to redifferentiate these iPSCs into cardiomyocytes, hematopoietic cells, and liver hepatocytes.

“We think this method will be very valuable in generating iPSCs from patient samples in a high-throughput manner” says Morrisey. microRNAs can also be introduced into cells using synthetically generated versions of miRNAs called mimics or precursors. These mimics can be easily introduced into cells at high levels, which should allow for a non-genetic method for efficiently generating iPSCs.

“The upshot is that we hope to be able to produce synthetic microRNAs to transform adult cells into induced pluripotent stem cells, which could eventually then be redifferentiated into other cell types, for example, liver, heart muscle or nerve cells” says Morrisey.

- Ref: Morrisey Edward, et al. Highly Efficient miRNA-Mediated Reprogramming of Mouse and Human Somatic Cells to Pluripotency. Cell Stem Cell, Volume 8, Issue 4, 376-388, 8 April 2011

Stem cells survive with only short-term immunosuppression

A short-term treatment with three immunedampening drugs allowed human embryonic stem cells to survive and thrive in mice, according to researchers at the Stanford University School of Medicine. Without such treatment, the animals’ immune systems quickly destroy the transplanted cells.

The finding is important because it may allow humans to accept transplanted stem cells intended to treat disease or injury without requiring the on-going use of powerful immunosuppressant medications.

“We are very excited about the clinical potential of this finding,” said Joseph Wu, MD, PhD, associate professor of cardiovascular medicine and of radiology at Stanford and senior author of the study, published in the March issue of Cell Stem Cell. “The immunological issue is one of the most important biological problems to solve, in my opinion. Clinicians need to make sure there is no tumour formation, and also that the cells are not rejected.”

This paper, in tandem with a previous study by Wu published in February in the Journal of Clinical Investigation, helps to recast a scientific debate over the relative benefits of embryonic stem cells as compared with induced pluripotent stem cells (iPS), which can be created from a person’s own skin or other cells.

“Most people don’t realise that, although it’s possible to generate patient-specific iPS cells, the cost of doing so would likely be prohibitive for all but the most specialised applications,” said Wu. “It also takes time, time that a patient with an acute health problem like a stroke, heart attack, or neurological trauma may not have.”

- Ref: Jospeh Wu. “Short-Term Immunosuppression Promotes Engraftment of Embryonic and Induced Pluripotent Stem Cells.” Cell Stem Cell, Volume 8, Issue 3, 309-317, 4 March 2011.

Researchers take critical step forward in stem cell repair of spinal cord injury

In a critical step toward the development of improved therapies for spinal cord injury, scientists have discovered that a specific type of human cell, generated from stem cells and transplanted into spinal cord injured rats, provide tremendous benefit, not only repairing damage to the nervous system but helping the animals regain locomotor function as well.

The study, published in the journal PLoS ONE, focuses on human astrocytes - the major support cells in the central nervous system - and indicates that transplantation of these cells represents a potential new avenue for the treatment of spinal cord injuries and other central nervous system disorders.

“We've shown in previous research that the right types of rat astrocytes are beneficial, but this study brings it up to the human level, which is a huge step,” said Chris Proschel, PhD, lead study author and assistant professor of Genetics at the University of Rochester Medical Center. “What's really striking is the robustness of the effect. Scientists have claimed repair of spinal cord injuries in rats before, but the benefits have been variable and rarely as strong as what we've seen with our transplants.”

There is one caveat to the finding – not just any old astrocyte will do. Using human foetal glial precursor cells, researchers generated two types of astrocytes by switching on or off different signals in the cells. Once implanted in the animals, they discovered that one type of human astrocyte promoted significant recovery following spinal cord injury, while another did not.

“Our study is unique in showing that different types of human astrocytes, derived from the exact same population of human precursor cells, have completely different effects when it comes to repairing the injured spinal cord,” noted Stephen Davies, PhD, first author and associate professor in the Department of Neurosurgery at the University of Colorado Denver. “Clearly, not all human astrocytes are equal when it comes to promoting repair of the injured central nervous system.”

The researchers also found that transplanting the original stem cells directly into spinal cord injured rats did not aid recovery.

“It is essential to first create the most beneficial cell type in tissue culture before transplantation. It is clear that we cannot rely on the injured tissue to induce the most useful differentiation of these precursor cells,” explained researcher Mark Noble, director of the University of Rochester Stem Cell and Regenerative Medicine Institute.

- doi: 10.1371/ journal.pone.0017328

Whole-exome sequencing of skin cancer complete

A team led by researchers at the US National Institutes of Health is the first to systematically survey the landscape of the melanoma genome, the DNA code of the deadliest form of skin cancer. The researchers have made surprising new discoveries using whole-exome sequencing, an approach that decodes the 1-2% of the genome that contains protein-coding genes. The study appears in the 15 April 2011, early online issue of Nature Genetics.

Melanoma is the most serious form of skin cancer and its incidence is increasing faster than any other cancer. A major cause is thought to be overexposure to the sun, particularly ultraviolet radiation, which can damage DNA and lead to cancer-causing genetic changes within skin cells.

“It is now clear that genomic analysis will have a major impact on our ability to diagnose and treat cancer,” said National Human Genome Research Institute Director Eric D. Green, MD, PhD. “This study represents a collaboration of basic science, clinical research, genome sequencing and data analysis at its best.”

The researchers conducted a comprehensive genome analysis and explored the melanoma genome’s functional components, especially gene alterations, or mutations. They studied advanced disease – the metastatic stage – when cells have the highest accumulation of gene mutations.

“Melanoma is one of the most challenging solid cancers to work with because it has such a high rate of mutation,” said senior author Yardena Samuels, PhD, investigator in the Cancer Genetics Branch of the NHGRI”s Division of Intramural Research. “Whole-exome sequencing will help us identify the most important changes.”

The researchers discovered mutations in one particular gene, known as TRRAP. It was remarkable for occurring at the exact position in six separate individuals with melanoma. TRRAP harbours a recurrent mutation clustered in one position along the string of DNA code in about 4% of cases.

“These data suggest that TRRAP is a driver and probably an oncogene,” said Dr. Samuels. Oncogenes are cancer-causing genes that enable the cell to survive despite stressful conditions, rather than die off normally. “This was one of the most important discoveries in the study since we never expected to identify novel hot-spot mutations,” she said.

TRRAP is found in many species, suggesting its importance in normal function and that mutations in this gene would detrimentally affect protein function. To confirm a possible cell-survival function for TRRAP, the researchers disrupted the gene in mutant cell lines. The cells had an increase in cell death over time. Cancer cells normally fail to undergo cell death, which allows them to become immortal and cause disease. The test showed that TRRAP is a cancer-causing oncogene, because the mutant cell is clearly dependent on it. Dr Samuels cautioned that while this discovery is exciting, it remains a basic science finding and does not necessarily suggest a therapy.

- doi: 10.1038/ng.810

Researchers call for screening after finding mutations in iPSCs

A new study finds that the genetic material of reprogrammed pluripotent stem cell lines may in fact be compromised with mutations, and suggests that extensive genetic screening of hiPSCs (human induced pluripotent stem cells) become standard practice before these stem cells are used clinically.

Ordinary human cells reprogrammed as induced pluripotent stem cells may ultimately revolutionise personalised medicine by creating new and diverse therapies unique to individual patients. But important and unanswered questions have persisted about the safety of these cells, in particular whether their genetic material is altered during the reprogramming process.

The study was published in the 3 March 2011 issue of the journal Nature was conducted by a team of researchers in the US, co-directed by Kun Zhang, PhD, an assistant professor of bioengineering in the UC San Diego Jacobs School of Engineering. They examined 22 different hiPSC lines obtained from seven research groups that employed different methods to reprogramme skin cells into pluripotent stem cells. In all of these cell lines, the researchers found protein-coding point mutations, an estimated six mutations per exome. The exome is the part of the genome that contains the genetic instructions for making proteins and other gene products.

“Every single stem cell line we looked at had mutations. Based on our best knowledge, we expected to see 10 times fewer mutations than we actually observed,” said Zhang.

The findings help answer the question of whether reprogramming adult mammalian cells into hiPSCs affects the overall genome at the fundamental level of single nucleotides. They do. Zhang called the mutations “permanent genome scars”.

The scientists said while some of the mutations appeared to be silent; the majority did change specific protein functions, including those in genes associated with causative effects in cancers.

Their studies open a new window into the genetic behaviour of these important types of stem cells and begin to define some new and straightforward safety standards that may help accelerate their use in clinical settings.

- doi: 10.1038/ nature09805

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