Some of the most challenging obstacles limiting the reprogramming of mature human cells into stem cells may not seem quite as daunting in the near future.  Two independent research papers, published by Cell Press in the September 11th issue of the journal Cell Stem Cell, describe new tools that provide invaluable platforms for elucidating the molecular, genetic, and biochemical mechanisms associated with reprogramming.  The new findings also offer considerable hope toward making the reprogramming process more therapeutically relevant.

Although scientists have successfully reprogrammed mature human skin cells into induced pluripotent stem (iPS) cells by expressing a few key transcription factors, the conversion has been extremely inefficient.  “Little is known about the mechanisms by which reprogramming occurs, in part because of the low efficiency,” says senior study author Dr. Konrad Hochedlinger from the Harvard Stem Cell Institute.  In addition, the iPS cells created thus far have been generated with retroviruses and noninducible lentiviruses, both of which have major limitations that are not compatible with clinical applications.

The Hochedlinger group created a drug-inducible viral system to generate human iPS cells that were molecularly and functionally similar to human embryonic stem cells.  This method was unique in that it allowed the researchers to create iPS cells by using the drug doxycycline to control expression of the necessary factors that had been delivered to the cells with viruses.

The researchers then found that when doxycycline was removed and these “primary” iPS cells differentiated to mature cells, another exposure to the drug reactivated the genes required for reprogramming and induced generation of “secondary” iPS cells at a frequency that was far greater that the initial “primary” conversion.  The idea of generating these secondary cells was conceived in previous experiments with mice performed in the lab of Dr. Rudolf Jaenisch from the Massachusetts Institute of Technology.

“The secondary system will enable chemical and genetic screening efforts to identify key molecular constituents of reprogramming, as well as important obstacles in this process, and will ultimately lend itself as a powerful tool in the development and optimization methods to produce human iPS cells,” explains Dr. Hochedlinger.

In a separate paper, Dr. Jaenisch’s group reports on their success in deriving human secondary iPS cells using doxycycline-inducible transgenes.  “The drug-inducible system we describe represents a novel, predictable, and highly reproducible platform to study the kinetics of iPS cell generation,” says Dr. Jaenisch.  “Further, the genetic homogeneity of secondary cells makes chemical and genetic screening approaches to enhance reprogramming efficiency or to replace any of the original reprogramming factors feasible.”

Both research teams found that generation of secondary human iPS cells required less time than the initial reprogramming.  Interestingly, the time required to generate iPS cells varied among the types of skin cells that were used.  For instance, human fibroblasts required several weeks, while keratinocytes required only about 10 days.  “The fast kinetics of reprogramming observed for keratinocytes suggests that these cells would be useful for development and optimization of methods to reprogram cells by transient delivery of factors,” suggests Dr. Hochedlinger.

The combined results from both research groups represent a major advance toward more efficient strategies for reprogramming differentiated human cells into iPS cells.  The methods described here will not only provide critical insight into the reprogramming process, but also, because of the abbreviated time frame, may lead to the generation of cells that will be amenable for therapies, as reprogramming might be achievable without the prohibitive viruses or genetic modifications.

In the wild, mammalian coat color is essential for camouflage and plays a role in social behavior.  Coat color also strongly influences the animals we choose to breed both as livestock and as pets.  Understanding the genetic determinants of coat color in livestock species such as sheep, specifically bred for their coat color, is critical for improving efficient selection of the desired trait.

Classical genetics has associated alternative forms, or alleles, of the agouti signaling protein gene (ASIP) with coat color variation in a number of mammals including mice, rats, dogs, cats, pigs, and sheep.  However, most research has been focused on the mouse, with little understood about the genetic basis for coat color in economically important livestock species such as sheep.

The wild-type coat color of sheep is typically dark-bodied with a pale belly, however sheep raisers have strongly selected for a uniformly white coat domestic sheep.  A problem for the sheep industry is a recessive black “non-agouti” allele of the ASIP gene carried by white sheep that cannot be distinguished within the flock, resulting in black coat color at a low, but persistent frequency.  Determining the exact genetic differences at the ASIP locus could assist in efficient selection for white coat color.

Scientists at the CSIRO Queensland Bioscience Precinct in Australia have now taken this step and identified the molecular mechanisms underlying white and black coat color in domestic sheep.  The researchers investigated the genetic architecture of the ASIP gene in several sheep breeds by sequencing the ASIP locus and measuring gene expression.  “Surprisingly what we found was in fact that the genetic cause of domestic white and black sheep involves a novel tandem duplication affecting the ovine agouti gene and two other neighboring genes, AHCY and ITCH,” explains Dr. Belinda Norris, lead author of the study.  “We discovered a novel mechanism in which the dominant white sheep is caused by the ubiquitous expression of a duplicated agouti coding sequence located immediately downstream of a duplicated ITCH gene promoter region.”  It was found that recessive black sheep harbor only poorly expressed non-duplicated agouti alleles, likely a result of a defective single-copy ancestral agouti gene promoter.  The researchers also studied the ASIP locus in Barbary sheep, an ancient species exhibiting a tan body and pale belly.  They confirmed in this ancient sheep that expression of a single-copy agouti gene determines coat color patterning, similarly to findings previously described in mice and pigs.

Norris notes that this work will aid in the development of gene copy number detection and analysis methods in the mapping and association of heritable traits in livestock animals.  For sheep raisers, this could ultimately mean a genetic test that would identify carriers of the black non-agouti allele.  Furthermore, these findings will help to unravel the events leading to the domestication of sheep, and future work may be able to pinpoint when the dominant Agouti mutation occurred, and whether it occurred as single or multiple events.

Many of the seemingly disparate mutations recently discovered in autism may share common underlying mechanisms, say researchers supported in part by the National Institute of Mental Health (NIMH), a part of the National Institutes of Health (NIH).  The mutations may disrupt specific genes that are vital to the developing brain, and which are turned on and off by experience-triggered neuronal activity.

A research team led by Christopher Walsh, M.D., Ph.D., and Eric Morrow, M.D., Ph.D., of Harvard University, found two large sections missing on chromosomes in people with autism and traced them to likely inherited mutations in such genes regulated by neuronal activity.  They report their findings in the July 11, 2008 issue of Science.  The study was also supported in part by the NIH’s National Center for Research Resources, National Human Genome Research Institute, Eunice Kennedy Shriver National Institute of Child and Human Development, and the National Institute on Neurological Disorders and Stroke.

The study breaks new ground for complex disorders like autism, taking advantage of a shortcut to genetic discovery by sampling families in which parents are cousins.  The researchers found genes and mutations associated with autism in 88 families from the Middle East, Turkey and Pakistan in which cousins married and had children with the disorder.

“The emerging picture of the genetics of autism is quite surprising.  There appear to be many separate mutations involved, with each family having a different genetic cause,” explained NIMH Director Thomas R. Insel, M.D. “The one unifying observation from this new report is that all of the relevant mutations could disrupt the formation of vital neural connections during a critical period when experience is shaping the developing brain.”

Earlier studies had suggested that the individually rare mutations are present in at least 10 percent of sporadic cases of autism, which is the most common form.

The researchers used a technique that pinpoints from a relatively small group of families genes responsible for disorders that can be amplified by parenthood among relatives, which can increase transmission of recessive diseases.  Evidence had hinted at such transmission in autism, and the large amount of genetic information obtainable from such families reduced the need for a much larger sample including many families with multiple affected members.

The ratio of females to males with autism – normally one female to four males – was less lopsided in such families in which parents share a common recent ancestor.  This ratio equalized even more in a subset of these families with more than one affected member, suggesting a doubling of the rate of autism, due to recessive causes on non-sex-linked chromosomes.  Also, autism-linked spontaneous deletions and duplications of genetic material were relatively uncommon in these families, suggesting recessive inherited causes.

The researchers found multiple different genetic causes of autism in different individuals with little overlap between the families in which parents shared ancestry.  Yet a few large inherited autism-linked deletions, likely mutations, in a minority of families stood out.  The largest turned out to be in or near genes regulated, directly or indirectly, by neuronal activity.

“Autism symptoms emerge at an age when the developing brain is refining the connections between neurons in response to a child’s experience,” explained Walsh.  “Whether or not certain important genes turn on is thus dependent on experience-triggered neural activity.  Disruption of this refinement process may be a common mechanism of autism-associated mutations.”

What makes a human different from a chimp?  Researchers from the European Molecular Biology Laboratory’s European Bioinformatics Institute [EMBL-EBI] have come one important step closer to answering such evolutionary questions correctly.  In the current issue of Science they uncover systematic errors in existing methods that compare genetic sequences of different species to learn about their evolutionary relationships.  They present a new computational tool that avoids these errors and provides accurate insights into the evolution of DNA and protein sequences.  The results challenge our understanding of how evolution happens and suggest that sequence turnover is much more common than assumed.

“Evolution is happening so slowly that we cannot study it by simply watching it.  That’s why we learn about the relationships between species and the course and mechanism of evolution by comparing genetic sequences,” says Nick Goldman, group leader at EMBL-EBI.

The four letter code that constitutes the DNA of all living things changes over time; for example individual or several letters can be copied incorrectly [substitution], lost [deletion] or gained [insertion].  Such changes can lead to functional and structural changes in genes and proteins and ultimately to the formation of new species.  Reconstructing the history of these mutation events reveals the course of evolution.

A comparison of multiple sequences starts with their alignment.  Characters in different sequences that share common ancestry are matched and gains and losses of characters are marked as gaps.  Since this procedure is computationally heavy, multiple alignments are often built progressively from several pairwise alignments.  It is impossible, however, to judge if a length difference between two sequences is a deletion in one or an insertion in the other sequence.  For correct alignment of multiple sequences, distinguishing between these two events is crucial.  Existing methods, that fail to do that, lead to a flawed understanding of the course of evolution.

“Our new method gets around these errors by taking into account what we already know about evolutionary relationships,” says Ari Löytynoja, who developed the tool in Goldman’s lab.  “Say we are comparing the DNA of human and chimp and can’t tell if a deletion or an insertion happened.  To solve this our tool automatically invokes information about the corresponding sequences in closely related species, such as gorilla or macaque.  If they show the same gap as the chimp, this suggests an insertion in humans.”

Findings achieved with the new technique suggest that insertions are much more common than assumed, while the frequency of deletions has been overestimated by existing methods.  A likely reason for these systematic errors of other techniques is that they were originally developed for structural matching of protein sequences.  The focus of molecular biology is shifting, however, and understanding functional changes in genomes requires specifically designed methods that consider sequences’ histories.  Such approaches will likely reveal further bugs in our understanding of evolution in future and might challenge the conventional picture of sequence evolution.

The potentially fatal heart disease LQT-2, which is characterized by the prolongation of a specific interval of time (known as the QT interval) in the heart’s electrical cycle, is caused by mutations in the HERG gene. What triggers the changes in the electrical activity in the heart (and therefore in the beating of the heart) has not been completely determined, although loud noises and emotional stress can be triggers. In a new study, a team of researchers from the Academic Medical Centre, The Netherlands, and the University of Wisconsin, Madison, has revealed that fever can also trigger life-threatening changes in the electrical activity in the heart of patients with LQT-2.

The team, led by Arthur Wilde and Craig January, measured the electrical activity in the heart over time (something that is recorded in an ECG) of two LQT-2 patients with the same HERG mutation (A558P), and found that fever was associated with prolonged QT intervals in these individuals. When this mutation was introduced into a cultured human cell line, the cells exhibited temperature-dependent characteristics, including altered electrical currents across their cell membranes at high temperatures. The authors therefore conclude that similar changes in electrical currents occur in heart cells at the high temperatures associated with fever and that fever is a potential trigger of the potentially lethal changes in the electrical activity in the heart of patients with LQT-2.

While many university students tend to mature out of heavy-drinking behavior by the time they become young adults, some go on to develop alcohol-use disorders (AUDs). Most genetic research on an individual’s family history of alcoholism (FHA) has looked at the parents’  “usually paternal” alcohol use. New findings indicate that looking at the density of FHA  including first-, second- and third-degree relatives is much more telling.Results will be published in the August issue of Alcoholism: Clinical & Experimental Research and are currently available at OnlineEarly.

“Using a density measure of FHA can identify a greater number of individuals who may be at risk for developing an alcohol problem,” said Christy Capone, a postdoctoral research fellow at Brown University’s Center for Alcohol and Addiction Studies and the study’s first author. “The greater number of affected relatives’ the greater the potential risk of developing an AUD. Ours is the first published study to examine this measure among college students.”

Family density appears to be a promising method to identify a higher percentage of at-risk individuals, agreed John Hustad, research associate at Brown University. For example, in this study, approximately 44 percent of the at-risk participants would have been missed if a typical family-history measure had been used instead of the family-history density approach.

The study population for this research consisted of 408 undergraduate students (293 females, 115 males) from a northeastern U.S. university who were asked to complete an anonymous survey for course credit during the 2005-2006 academic year.

“Our use of a density measure identified a large proportion of students, about 29 percent, who are at potentially greater risk for development of AUDs based on their report of alcoholism among first- and second-degree relatives,” said Capone. “Our other key finding was the relationship between FHA and other potential risk factors  behavioral undercontrol, age of onset of drinking (AOD), and cigarette use.”

All of these risks factors are inter-related, added Hustad. First, family-history density was related to AOD, behavioral undercontrol, and current cigarette use which, in turn, are related to alcohol use and/or alcohol-related problems in this sample of college students. Second, behavioral undercontrol was associated with alcohol problems but not the degree of alcohol consumption; this suggests that individuals with a family-history density of AUDs and behavioral undercontrol are more likely to behave irresponsibly when drinking.

“The importance of identifying these risk factors is the idea that they can be useful markers of at-risk status and can help us to develop appropriate intervention strategies,” said Capone. “Although, given the fact that many students come to college already having experience with alcohol, I believe that preventive interventions should begin early in the high-school years or during the transition from middle school to high school.”

Hustad agreed. “Due to the relationship between earlier AOD and more alcohol-related problems during college, it is clear that education and prevention efforts should begin well before the college years,” he said. “Until that happens, the risk factors identified in this research can be easily implemented in any screening and brief intervention for incoming college students. For example, these results suggest that effective interventions addressing tobacco use may have a positive influence on both smoking and alcohol-related consequences.”

“It is important to remember that not everyone with density of familial alcoholism will go on to develop a long-term problem with alcohol themselves,” said Capone. “Alcohol dependence is a very complex disorder and FHA is but one influence on its development. However, college students who are heavy drinkers and have a greater density of familial alcoholism are certainly at higher risk of continuing to drink in a problematic fashion after the college years.”

Researchers at the Burnham Institute for Medical Research, led by Josh Luis Millhn, Ph.D., have demonstrated in mice the first successful use of enzyme replacement therapy to prevent hypophosphatasia (HPP), a primary skeletal disease of genetic origin. This discovery lays the foundation for future clinical trials for HPP patients.

Rickets is a softening of the bones that most commonly results from a lack of vitamin D or calcium and from insufficient exposure to sunlight. Hypophosphatasia is a rare, heritable form of rickets caused by mutations in a gene called TNAP, which is essential for the process that causes minerals such as calcium and phosphorus to be deposited in developing bones and teeth. The physical presentations of this disorder can vary depending on the specific mutation, with more severe symptoms occurring at a younger age of onset. The most severe form of the disease occurs at birth, which can present with absence of bone mineralization in utero, resulting in stillbirth.

Using a mouse model, Josh Luis Millhn, Ph.D. tested the hypothesis that, when administered from birth, a bone-targeted form of the TNAP gene would ease the skeletal defects of HPP. The Millhn laboratory, in collaboration with scientists from Enobia Pharma in Montreal, Canada and from the Shriners Hospitals for Children in St. Louis, Missouri, created a soluble form of human TNAP that had been shown to display a strong attraction to bone tissue. Upon injecting the enzyme into the fat layer under the skin of the mice, the treated mice maintained a healthy rate of growth and apparent well being, as well as normal bone mineral density (BMD) of the skull, femur and spine. In fact, complete preservation of skeletal and dental structures were observed after 15 days, and bone lesions were still not seen after 52 days of treatment.

“While the biochemical mechanism that leads to skeletal and dental defects of HPP is now generally understood,” said Dr. Millhn, “there is currently no established medical treatment.”

Given the success of this therapy in preventing HPP, current efforts in Dr. Millhn’s laboratory are focused on reversing the bone defects in mice once the disease is quite advanced. Future clinical trials may reveal this as the first promising therapy for patients with this genetic disorder.