A research team at the Institut de recherches cliniques de Montreal (IRCM), funded by the Foundation Fighting Blindness – Canada and the Canadian Institutes of Health Research (CIHR), discovered a novel mechanism that regulates how neural stem cells of the retina generate the appropriate cell type at the right time during normal development.  These findings, published today in the renowned journal Neuron, could influence the development of future cell replacement therapies for genetic eye diseases that cause blindness.

In their report, the scientists show that a gene called Ikaros is expressed in the most immature retinal stem cells in the mouse, which are “competent” to generate all seven different cell types that compose the retina.  But this gene is not expressed in the “older” stem cells, which are more restricted in their differentiation potential and produce only the late-born neurons.  “By studying the retina of a mouse in which the Ikaros gene was inactivated, we found that the generation of early-born retinal cell types was impaired, whereas the generation of the late-born retinal cell types was not affected,” explained Dr. Michel Cayouette who led the study.  In contrast, forcing the expression of Ikaros in older retinal stem cells, which have normally turned off its expression, was sufficient to give back the competence to these cells to generate early-born neurons.  Overall, these results indicate that the expression of Ikaros in retinal stem cells is both necessary and sufficient to confer the competence to generate early-born retinal neurons.

The identification of adult retinal stem cells in recent years has opened up the possibility that such cells could one day be used to replace damaged or lost cells in various retinal diseases such as glaucoma, macular degeneration or retinitis pigmentosa.  For such approaches to be effective, however, it is crucial that stem cells generate only the appropriate cell type for a particular condition.  This study suggests that it may be possible to manipulate the competence of retinal stem cells so that they only generate retinal cells associated to a particular temporal stage.  “For example, added Dr. Cayouette, inactivating Ikaros could favor the production of later-born neurons such as photoreceptors, which are lost progressively in retinal degenerative diseases.”  Future studies will be required to assess the usefulness of this approach for potential cell replacement therapies.

A new study uncovers a previously unrecognized, species-specific impact of circadian rhythms on the production of mobilized stem cells.  The research, published by Cell Press in the October 9th issue of the journal Cell Stem Cell, suggests that when it comes to collecting human stem cells for clinical transplantation, picking the right time of day to harvest cells may result in a greater yield.

A variety of organisms have evolved an endogenous timing system, called a circadian clock, to regulate metabolic activities in a day/night cycle.  In mice, the cells that give rise to mature blood cells, called hematopoietic stem cells (HSC), are regulated under the influence of rhythmic circadian signals that influence expression of Cxcl12, a gene involved in white blood cell migration.  “Previous research has shown that the “sympathetic” branch of the nervous system, which is involved in stress responses, tightly regulates the amount of Cxcl12 expressed in the bone marrow by circadian oscillation of noradrenaline release.  Blood stem cell patterns are basically the mirror image of Cxcl12 expression in bone marrow,” says lead study author, Dr. Paul S. Frenette from the Mount Sinai School of Medicine.

Dr. Frenette and colleagues were interested in examining whether circadian time continues to influence mobilization of HSCs when mice are treated with granulocyte-colony-stimulating factor (G-CSF), the most common stem cell mobilizer used in the clinic.  The researchers found that after stimulations with G-CSF, synchronization of blood collection with the peak circadian time produced greater HSC recovery.  Therefore, even when pharmacological manipulation is used to stimulate HSC mobilization, circadian clock genes continue to influence yield.

The researchers also demonstrated the existence of significant oscillations in the number of human HSCs and found that the circadian rhythm in humans is inverted when compared to that of the mouse.  An examination of healthy donors who were contributing HSCs for bone marrow transplantation at Mount Sinai Medical Center between the years of 2000 and 2006 revealed that the average yield was greater for those who underwent the procedure in the afternoon compared with those who were harvested in the morning.

“Our results suggest that the human HSC yield for clinical transplantation might be greater if patients were harvested during the evening compared to the morning,” explains Dr. Frenette.  “Although prospective clinical studies are needed to ascertain the optimal time for HSC collection, it is possible that a simple adjustment in the collection time may have a significant clinical impact.  Further, the maximum release of HSCs at the beginning of the rest period for both species (early night for humans, early morning for mice), supports the intriguing possibility that this phenomenon may contribute to regeneration.”

Recent studies indicate that infusing hearts with stem cells taken from bone marrow could improve cardiac function after myocardial infarction (tissue damage that results from a heart attack).  But in a recent systematic review, Cochrane Researchers concluded that more clinical trials are needed to assess the effectiveness of stem cell therapies for heart patients, as well as studies to establish how these treatments work.

In a heart attack, blocked arteries can cut off the blood supply to areas of heart tissue.  This leads to myocardial infarction severe tissue damage caused by lack of oxygen, which is transported in the blood.

“We need more studies that look at the long term effects of these interventions, as well as at the types of cells that are used and how they actually repair the heart tissue,” says lead researcher Dr. Enca Martin-Rendon, who works in the Stem Cell Research Department, NHS Blood and Transplant, at the John Radcliffe Hospital in Oxford, UK.

The team drew together data from 13 different trials involving 811 patients.  Although these trials show that treatment with bone marrow stem cells (BMSCs) may lead to a moderate improvement in cardiac function, the researchers say there is still not enough evidence to confirm this.  They also found that BMSC treatment did not reduce the measurable area of damaged heart tissue.

Only three trials looked to see if effects lasted for more than six months after BMSC treatment.  The researchers discovered that in these trials, there was no evidence of any benefit 12 months after treatment.

Quite how BMSCs cause this short term benefit is uncertain.  One theory is that they enable extra blood vessels to develop, while another is that they release chemicals that encourage the growth of healthy heart muscle cells while decreasing the development of scar tissue in the damaged area.

“If it turns out these treatments are beneficial in any way, they could be made available to all heart attack patients.  We think infusion with stem cells may help increase blood flow into damaged heart tissues, but without more investment in this area of research, we can’t be sure,” says Martin-Rendon.

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.

Wei Tong and colleagues, at Children’s Hospital of Philadelphia, have provided new insight into the molecular control of hematopoietic stem cells (HSCs), the cells that give rise to all types of blood cell, by explaining why mice lacking an inhibitory protein known as Lnk have more HSCs than normal mice.

In the study, it was observed that a greater proportion of HSCs in mice lacking Lnk were not undergoing cell division and were said to be quiescent (i.e., in a state of inactivity or in silent mode).  Lnk was found to regulate HSC quiescence by binding a signaling protein known as JAK2 after it was activated following binding of the soluble factor TPO to its receptor Mpl.  The authors therefore hypothesize that in the absence of the inhibitory molecule Lnk, TPO-initiated signaling from Mpl to JAK2 goes unchecked and the number of HSCs produced is increased to a level at which they do not need to undergo cell division as often to maintain their population.

By injecting purified stem cells isolated from adult skeletal muscle, researchers have shown they can restore healthy muscle and improve muscle function in mice with a form of muscular dystrophy.  Those muscle-building stem cells were derived from a larger pool of so-called satellite cells that normally associate with mature muscle fibers and play a role in muscle growth and repair.

In addition to their contributions to mature muscle, the injected cells also replenished the pool of regenerative cells normally found in muscle.  Those stem cells allowed the treated muscle to undergo subsequent rounds of injury repair, they found.

“Our work shows proof-of-concept that purified muscle stem cells can be used in therapy,” said Amy Wagers of Harvard University, noting that in some cases the stem cells replaced more than 90 percent of the muscle fibers.  Such an advance would require isolation of stem cells equivalent to those in the mouse from human muscle, something Wagers said her team is now working on.

Satellite cells were first described decades ago and have since generally been considered as a homogeneous group, Wagers said.  While anatomically they look similar under a microscope, they nonetheless show considerable variation in their physiology and function.  In a previous study, Wagers’ identified a set of five markers that characterize the only subset of satellite cells responsible for forming muscle, which they also refer to as skeletal muscle precursors or SMPs.

In the new study, the researchers analyzed the stem cell and regenerative properties of those SMPs.  When engrafted into muscle of mice lacking dystrophin, purified SMPs contributed to up to 94 percent of muscle fibers, restoring dystrophin expression and significantly improving muscle structure and contractile function, they report.  (The dystrophin gene encodes a protein important for muscle integrity.  Mice lacking dystrophin, also known as mdx mice, are a model for Duchenne Muscular Dystrophy, the most prevalent form of muscular dystrophy.)

” Importantly, high-level engraftment of transplanted SMPs in mdx animals shows therapeutic value—restoring defective dystrophin gene expression, improving muscle histology, and rescuing physiological muscle function,” the researchers said.  “Moreover, in addition to generating mature muscle fibers, transplanted SMPs also re-seed the satellite cell niche and are maintained there such that they can be recruited to participate in future rounds of muscle regeneration.

“Taken together, these data indicate that SMPs act as renewable, transplantable stem cells for adult skeletal muscle.  The level of myofiber reconstitution achieved by these myogenic stem cells exceeds that reported for most other myogenic cell populations and leads to a striking improvement of muscle contraction function in SMP-treated muscles.  These data thus provide direct evidence that prospectively isolatable, lineage-specific skeletal muscle stem cells provide a robust source of muscle replacement cells and a viable therapeutic option for the treatment of muscle degenerative disorders.”

Wagers noted however that there may be complications in the delivery of cell therapy in humans, particularly for those with conditions influencing skeletal muscle throughout the body.  Even so, the new findings present an “opportunity to understand what happens [to these regenerative cells] in disease and identify factors and pathways that may boost their activity,” she said.  “We may get a handle on drugs that could target muscle impairment” not only in those with muscular dystrophies, but also in elderly people suffering from the muscle wasting that comes with age.

The ability to drive somatic, or fully differentiated, human cells back to a pluripotent or “stem cell” state would overcome many of the significant scientific and social challenges to the use of embryo-derived stem cells and help realize the promise of regenerative medicine. Recent research with mouse and human cells has demonstrated that such a transformation (“reprogramming”) is possible, although the current process is inefficient and, when it does work, poorly understood. But now, thanks to the application of powerful new integrative genomic tools, a cross-disciplinary research team from Harvard University, Whitehead Institute, and the Broad Institute of MIT and Harvard has uncovered significant new information about the molecular changes that underlie the direct reprogramming process. Their findings are published online in the journal Nature.

“We used a genomic approach to identify key obstacles to the reprogramming process and to understand why most cells fail to reprogram,” said Alexander Meissner, assistant professor at Harvard University’s Department of Stem Cell and Regenerative Biology and associate member of the Broad Institute, who led the multi-institutional effort. “Currently, reprogramming requires infecting somatic cells with engineered viruses. This approach may be unsuitable for generating stem cells that can be used in regenerative medicine. Our work provides critical insights that might ultimately lead to a more refined approach.”

Previous work had demonstrated that four transcription factors — proteins that mediate whether their target genes are turned on or off — could drive fully differentiated cells, such as skin or blood cells, into a stem cell-like state, known as induced pluripotent stem (iPS) cells. Building off of this knowledge, the researchers examined both successfully and unsuccessfully reprogrammed cells to better understand the complex process.

“Interestingly, the response of most cells appears to be activation of normal ‘fail safe’ mechanisms”, said Tarjei Mikkelsen, a graduate student at the Broad Institute and first author of the Nature paper. ”Improving the low efficiency of the reprogramming process will require circumventing these mechanisms without disabling them permanently.”

The researchers used next-generation sequencing technologies to generate genome-wide maps of epigenetic modifications — which control how DNA is packaged and accessed within cells — and integrated this approach with gene expression profiling to monitor how cells change during the reprogramming process. Their key findings include:

1. Fully reprogrammed cells, or iPS cells, demonstrate gene expression and epigenetic modifications that are strikingly similar, although not necessarily identical, to embryonic stem cells.
2. Cells that escape their initial fail-safe mechanisms can still become ‘stuck’ in partially reprogrammed states.
3. By identifying characteristic differences in the epigenetic maps and expression profiles of these partially reprogrammed cells, the researchers designed treatments using chemicals or RNA interference (RNAi) that were sufficient to drive them to a fully reprogrammed state.
4. One of these treatments, involving the chemotherapeutic 5-azacytidine, could improve the overall efficiency of the reprogramming process by several hundred percent.

“A key advance facilitating this work was the isolation of partially reprogrammed cells,” said co-author Jacob Hanna, a postdoctoral fellow at the Whitehead Institute, who recently led two other independent reprogramming studies. “We expect that further characterization of partially programmed cells, along with the discovery and use of other small molecules, will make cellular reprogramming even more efficient and eventually safe for use in regenerative medicine.”

CAMBRIDGE, Mass.  (April 18, 2008) – A team of researchers have demonstrated that fully mature, differentiated B cells can be reprogrammed to an embryonic-stem-cell-like state, without the use of an egg according to a study published in the April 18 issue of Cell.

In previous research, induced pluripotent stem (IPS) cells have been created from fibroblasts, a specific type of skin cells that may differentiate into other types of skin cells.  Because there is no way to tell if the fibroblasts were fully differentiated, the cells used in earlier experiments may have been less differentiated and therefore easier to convert to the embryonic-stem-cell-like state of IPS cells.

B cells are immune cells that can bind to specific antigens, such as proteins from bacteria, viruses or microorganisms.  Unlike fibroblasts, mature B cells have a specific part of their DNA cut out as a final maturation step.  “Once that piece of DNA is cut out, it can’t come back,” says Jacob Hanna, first author on the paper and a postdoctoral fellow in Whitehead Member Rudolf Jaenisch’s lab.  “Checking the genome give us a way to make sure the resulting IPS cells were not from immature cells.”

Hanna and his colleagues began the experiment by generating IPS cells from immature B cells.  Similar to the process used to create IPS cells from fibroblast cells, Hanna successfully reprogrammed the immature B cells into IPS cells by using retroviruses to transfer four genes (Oct4, Sox2, c-Myc and Klf4) into the cells’ DNA.

However, an additional factor, CCAAT/enhancer-binding-protein-?  (C/EBP?), was needed to nudge mature B cells to be reprogrammed as IPS cells.

Like IPS cells from earlier fibroblast studies, the IPS cells from both the mature and immature B cells could be used to create mice.  The mice grown from the reprogrammed mature B cells were missing the same part of their DNA as the mature B cells, demonstrating that Hanna and his colleagues had successfully reprogrammed fully differentiated cells.

In addition to demonstrating the power of reprogramming, this work offers the promise of powerful new mouse models for autoimmune diseases such as multiple sclerosis and type 1 diabetes, in which the body attacks certain types of its own cells.  For example, mature B or T cells specific for nerve cells called glia could be reprogrammed to IPS cells and then used to create mice with an entire immune system that is primed to only attack the glia cells, thereby creating a mouse model for studying multiple sclerosis.

Eventually, researchers will be able to study diseases by following a similar process with human cells, predicts Jaenisch, who is also a professor of biology at Massachusetts Institute of Technology.  “In principle, this will allow you to transfer a complex genetic human disease into a Petri dish, and study it,” he says.  “That could be the first step to analyze the disease and to define a therapy.”

Reference:

Cell, April 18, 2008 134(2). “Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency”

Jacob Hanna (1), Styliani Markoulaki (1), Patrick Schorderet (1), Caroline Beard (1), Bryce W. Carey (1), Marius Wernig (1), Menno P. Creyghton (1), Eveline J. Steine (1), (1), John P. Cassady (1), Christopher J. Lengner (1), Jessica A. Dausman (1), Rudolf Jaenisch (1,2)

1. Whitehead Institute for Biomedical Research, Cambridge, MA 02142 USA

2. Department of Biology, MIT, Cambridge, MA 02142 USA