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.

Contrary to a long-standing assumption that blood vessel cells in healthy tissues and those associated with tumors are similar, a new study unequivocally demonstrates that tumor blood vessel cells are far from normal.  The research, published by Cell Press in the September issue of the journal Cancer Cell, identifies tumor-specific blood vessel cells that are atypically stem cell-like and have the potential to differentiate into cartilageor bone-like tissues.

Although it has been known for some time that tumors can be eradicated in mice by targeting their blood supply, very little is known about the biology of the endothelial cells that line tumor blood vessels (TECs).  “A primary assumption of antiangiogenesis therapy is that TECs are normal and derived from nearby, preexisting vessels,” explains senior author Dr. Michael Klagsbrun from Children’s Hospital Boston and Harvard Medical School.  “However, we and other groups have shown that there are several key differences between normal and tumor endothelium.”

Dr. Klagsbrun and lead author Dr. Andrew Dudley isolated TECs from mice that spontaneously develop prostate tumors very similar to human prostate cancers.  The researchers found that the TECs were multipotent, meaning that they were not fully mature and had the potential to differentiate into multiple different types of cells.  The isolated TECs differentiated to form cartilageand bone-like tissues.  “These results suggest that TECs possess a stem/progenitor cell property that distinguishes them from Ecs throughout the normal vasculature and undergo atypical differentiation,” explains Dr. Klagsbrun.

The researchers went on to demonstrate blood vessel calcification in human and mouse prostate tumor specimens.  This bone-like calcification has also been described in diseased blood vessels and is likely to have clinical significance in prostate cancer.  “It is possible that calcification of tumor blood vessels could impair blood flow or enable tumor cell entry into the bloodstream, facilitating metastasis,” offers Dr. Klagsbrun.  “Further, the expression of bone-specific proteins in prostate tumor cells may enable their survival once they reach the bone microenvironment.”

Additional research is required to determine how the atypical properties of TECs are associated with the tortuous, leaky vessels characteristic of tumors and whether vascular calcification does indeed encourage tumor cell metastasis.  It is also possible that vascular calcification, which is easily discernible histologically, may be a useful diagnostic criterion.

Scientists have identified about two dozen genes that control embryonic stem cell fate.  The genes may either prod or restrain stem cells from drifting into a kind of limbo, they suspect.  The limbo lies between the embryonic stage and fully differentiated, or specialized, cells, such as bone, muscle or fat.

By knowing the genes and proteins that control a cell’s progress toward the differentiated form, researchers may be able to accelerate the process – a potential boon for the use of stem cells in therapy or the study of some degenerative diseases, the scientists say.

Their finding comes from the first large-scale search for genes crucial to embryonic stem cells.  The research was carried out by a team at the University of California, San Francisco and is reported in a paper in the July 11, 2008 issue of “Cell.”

“The genes we identified are necessary for embryonic stem cells to maintain a memory of who they are,” says Barbara Panning, PhD, associate professor of biochemistry and biophysics at UCSF, and senior author on the paper.  “Without them the cell doesn’t know whether it should remain a stem cell or differentiate into a specialized cell.”

The scientists used a powerful technique known as RNA interference, or RNAi, to screen more than 1,000 genes for their role in mouse embryonic stem cells.  The technique allows researchers to “knock down” individual genes, reducing their abundance in order to determine the gene’s normal role.

The research focused on proteins that help package DNA.  In the nucleus, DNA normally wraps around protein complexes called nucleosomes, forming a structure known as chromatin.  This is what makes up chromosomes.

They found 22 proteins, each of which is essential for embryonic stem cells to maintain their consistent shape, growth properties, and pattern of gene expression.

Most of the genes code for multi-protein complexes that physically rearrange, or “remodel” nucleosomes, changing the likelihood that the underlying genes will be expressed to make proteins.

The main player they identified is a 17-protein complex called Tip60-p400.  This complex is necessary for the cellular memory that maintains embryonic stem cell identity, Panning explains.  Without it, the embryonic stem cells turned into a different cell type, which had some features of a stem cell but many features of a differentiated cell.

The scientists believe that Tip60-p400 is necessary for embryonic stem cells to correctly read the signals that determine cell type.  These findings are not only important for understanding cellular memory in embryonic stem cells, but will also likely be relevant to other cell types, they say.

Inactivation of other genes disrupted embryonic stem cell proliferation.  These genes were already known to have only slight influence on viability of mature cells in the body.  This suggests that embryonic stem cells are “uniquely sensitive to certain perturbations of chromatin structure,” the scientists report.

If other types of stem cells are also found to be sensitive to these chromatin perturbations, this could lead to novel cancer therapies in the future, Panning says.

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.

Researchers at the Joslin Diabetes Center have demonstrated for the first time that transplanted muscle stem cells can both improve muscle function in animals with a form of muscular dystrophy and replenish the stem cell population for use in the repair of future muscle injuries.

“I’m very excited about this,” said lead author Amy J. Wagers, Ph.D., Principal Investigator in the Joslin Section on Developmental and Stem Cell Biology, principal faculty member at the Harvard Stem Cell Institute and Assistant Professor of Stem Cell and Regenerative Biology at Harvard University.  “This study indicates the presence of renewing muscle stem cells in adult skeletal muscle and demonstrates the potential benefit of stem cell therapy for the treatment of muscle degenerative diseases such as muscular dystrophy.”

The study was designed to test the concept that skeletal muscle precursor cells could function as adult stem cells and that transplantation of these cells could both repair muscle tissue and regenerate the stem cell pool in a model of Duchenne muscular dystrophy, she said.  The research is published in the July 11 issue of Cell.

Duchenne muscular dystrophy is the most common form of the disease and is characterized by rapidly progressing muscle degeneration.  The disease is caused by a genetic mutation and there is currently no cure.

The data from this new study demonstrate that regenerative muscle stem cells can be distinguished from other cells in the muscle by unique protein markers present on their surfaces.  The authors used these markers to select stem cells from normal adult muscle and transferred the cells to diseased muscle of mice carrying a mutation in the same gene affected in human Duchenne muscular dystrophy.

“Once the healthy stem cells were transplanted into the muscles of the mice with muscular dystrophy, they generated cells that incorporated into the diseased muscle and substantially improved the ability of the treated muscles to contract,” said Wagers.  “At the same time, the transplantation of the healthy stem cells replenished the formerly diseased stem cell pool, providing a reservoir of healthy stem cells that could be re-activated to repair the muscle again during a second injury.”

According to the paper, these cells provide an effective source of immediately available muscle regenerative cells as well as a reserve pool that can maintain muscle regenerative activity in response to future challenges.

“This work demonstrates, in concept, that stem cell therapy could be beneficial for degenerative muscle diseases,” Wagers said.

Wagers also said the study will lead to other studies in the near-term that will identify pathways that regulate these muscle stem cells in order to figure out ways to boost the normal regenerative potential of these cells.  These could include drug therapies or genomic approaches, she said.  In the long-term, the idea will be to replicate these findings in humans.

“This is still very basic science, but I think we’re going to be able to move forward in a lot of directions.  It opens up many exciting avenues,” she said.

The Wagers Lab at Joslin studies both hematopoietic stem cells, which constantly maintain and can fully regenerate the entire blood system, as well as skeletal muscle stem cells, involved in skeletal muscle growth and repair.  The work is aimed particularly at defining novel mechanisms that regulate the migration, expansion, and regenerative potential of these two distinct adult stem cells.

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.

How does a stem cell decide what specialized identity to adopt – or simply to remain a stem cell? A new study suggests that the conventional view, which assumes that cells are “instructed” to progress along prescribed signaling pathways, is too simplistic. Instead, it supports the idea that cells differentiate through the collective behavior of multiple genes in a network that ultimately leads to just a few endpoints – just as a marble on a hilltop can travel a nearly infinite number of downward paths, only to arrive in the same valley.

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When exposed to a growth factor, a blood stem cell, represented by a blue marble, falls into a new “attractor state,” depicted as a valley in a landscape, to become a red blood cell. Different influences, such as differentiation factors, can lead stem cells to the same attractor state, but each cell can take very different paths though the landscape to get there (just as a marble might take a different path each time it rolls down a hill).Credit: Graham Paterson, Children’s Hospital Boston

The findings, published in the May 22 issue of Nature, give a glimpse into how that collective behavior works, and show that cell populations maintain a built-in variability that nature can harness for change under the right conditions. The findings also help explain why the process of differentiating stem cells into specific lineages in the laboratory has been highly inefficient.

Led by Sui Huang, MD, PhD, a Visiting Associate Professor in the Children’s Hospital Boston Vascular Biology Program (now also on the faculty of the University of Calgary), and Hannah Chang, an MD/PhD student in Children’s Vascular Biology Program, the researchers examined how blood stem cells “decide” to become white blood cell progenitors or red blood cell progenitors.

They began by examining populations of seemingly identical blood stem cells, and found that a cell marker of “stemness,” a protein called Sca-1, was actually present in highly variable amounts from cell to cell – in fact, they found a 1,000-fold range. One might think that low Sca-1 cells are simply those cells that have spontaneously differentiated. However, when Huang and Chang divided the cells expressing low, medium and high levels of Sca-1 and cultured them, each descendent cell population recapitulated the same broad range of Sca-1 levels over nine days or more, regardless of what levels they started with.

“We then asked, are these cells also biologically different?” says Huang, the paper’s senior author. “And it turned out they were dramatically different in differentiation.”

The green balls represent blood stem cells in a stable “basin” on the landscape, where they remain stem cells. Each position on the landscape that the balls occupy corresponds to a gene expression state and can be assigned an “energy.” An increase in the balls’ energy or movement within the basin enhances the likelihood that a ball will escape from the basin, but does not bias it towards a particular fate (in this case, red or white blood cells). Only a change in the landscape induced by a differentiation factor may tip the balance toward another stable state, causing the stem cells to “roll down the valleys” and differentiate to either red or white blood cells.Credit: Courtesy Sui Huang, MD, PhD, Children’s Hospital Boston and University of Calgary

Blood stem cells with low levels of Sca-1 differentiated into red blood cell progenitors seven times more often than cells high in Sca-1 when exposed to erythropoietin, a growth factor that promotes red blood cell production. Conversely, when stem cells were exposed to granulocyte–macrophage colony-stimulating factor, which stimulates white blood cell formation, those that were highest in Sca-1 were the most likely to become white cells. Yet, in both experiments, all three groups of cells retained characteristics of stem cells.

Huang and Chang then looked at the proteins GATA1 and PU.1, transcription factors that normally favor differentiation into red and white blood cells, respectively. Blood stem cells that were low in Sca-1 (and most prone to become red blood cells) had much more GATA1 than did the high- and medium-Sca-1 cells. Stem cells high in Sca-1 (and least prone to become red blood cells) had the highest levels of PU.1.

But most important, the differences in Sca-1, GATA1 and PU.1 levels across the three cell groups became less pronounced over time, as did the variability in the cells’ propensity to differentiate, suggesting that the differences are transient.

In a final step, Huang and Chang used microarrays to look at the cells’ entire genome. Again, they found tremendous variability within the apparently uniform cell population: more than 3,900 genes were differentially expressed (turned “on” or “off”) between the low- and high-Sca-1 cells. And again, this variability was dynamic: the differences diminished over time, with gene activity in both the low- and high-Sca-1 cells becoming more like that in the middle group.

Together, the findings make the case that a slow fluctuation or cycling of gene activity tends to maintain cells in a stable state, while also priming them to differentiate when conditions are right.

“Even if cells are officially genetically identical and belong to the same clone, individual members of that population are quite different at any given time,” says Huang. “This heterogeneity has usually been seen as random ‘measurement noise,’ and, more recently, as ‘gene expression noise.’ But it turns out to be very important, and is the basis for stem cells’ multipotency – their ability to differentiate into multiple lineages.”

“Nature has created an incredibly elegant and simple way of creating variability, and maintaining it at a steady level, enabling cells to respond to changes in their environment in a systematic, controlled way,” adds Chang, first author on the paper.

Practically speaking, the work suggests that stem cell biologists may need to change their approach to differentiating stem cells in the laboratory for therapeutic applications.

(A) The concentration Sca-1 protein, a marker of “stemness,” varies greatly in a population of stem cells, though the most common concentration is toward the middle of the range. (B) If the population of stem cells is divided into three groups (low, medium and high Sca-1 level), and those cells are allowed to divide and grow, (C) each group of descendents will reproduce the original range of Sca-1 concentrations. This suggests that populations of stem cells, though genetically identical, have an innate variability that may provide the basis for stem-cell differentiation. This variability could be tapped to increase the efficiency of stem-cell differentiation for therapeutic purposes.Credit: Graham Paterson, Children’s Hospital Boston

“So far the process has been highly inefficient – only 10 to 50 percent of cells respond to the hormone or whatever is given to make them differentiate,” Huang says. “That is because of the cells’ inherent heterogeneity. People have been finding more and more sophisticated stimulator cocktails, but we could make the process more efficient by harnessing the heterogeneity and identifying cells that are already highly poised to differentiate.”

Chang has already done follow-up experiments showing that stem cell differentiation can be made dramatically more efficient by choosing the right subpopulation of stem cells and stimulating them promptly, while they are most apt to differentiate. “I’m not doing anything complicated – just using what nature already has,” she says.

But the findings also challenge biologists to change how they think about biological processes. The work supports the idea of biological systems moving toward a stable “attractor state,” a concept borrowed from physics. In this case, blood stem cells tend to remain blood stem cells, yet they experience inherent fluctuations in gene activity and protein production that can sometimes be enough to tip the balance and cause them to fall into other attractor states – namely, red or white blood cell progenitors. Specific growth factors can tip the balance, but these factors are part of an overall landscape that guides cells toward different destinies. A marble going downhill will eventually end up in a valley, but which valley it falls into depends on the shape of the landscape.

“Growth or differentiation factors merely increases the probability that a cell will grow or differentiate,” says Donald Ingber, MD, PhD, a co-author on the paper who, with Huang, served as Chang’s mentor on the project. “Cell differentiation is an ensemble property, a collective behavior, inherent in the system’s architecture and set of regulatory interactions.”

A previous study by Huang established, for the first time, that a given cell can exhibit a very different pattern of gene activity from its neighbor, taking a very different path through the landscape, yet end up in the same valley. He and his colleagues exposed precursor cells to two completely different drugs (DMSO and retinoic acid) and closely monitored the cells’ gene expression. Both groups of cells eventually differentiated to become neutrophils (a type of white blood cell), but the molecular paths they took and their patterns of gene expression were completely different until day seven, when they finally converged.

The landscape analogy and collective “decision-making” are concepts unfamiliar to biologists, who have tended to focus on single genes acting in linear pathways. This made the work initially difficult to publish, notes Huang. “It’s hard for biologists to move from thinking about single pathways to thinking about a landscape, which is the mathematical manifestation of the entirety of all the possible pathways,” he says. “A single pathway is not a good way to understand a whole process. Our goal has been to understand the driving force behind it.”

Dr. Richard Flavell (Yale University) and colleagues identify the c-Cbl protein as a critical repressor of hematopoietic stem cell (HSC) self-renewal in the April 15th issue of G&D,.  In addition to establishing a key role for protein ubiquitylation in HSC development, this finding posits c-Cbl as a potential target in research into stem cell engineering as well as cell-based leukemia treatments.

Dr. Flavell describes the work as elucidating “a novel dimension in our understanding the self-renewal of Hematopoietic stem cells.”

Like all stem cell populations, HSC reply upon asymmetric cell division to generate two different daughter cells: one future stem cell, and another cell that will further differentiate into a more specialized cell type.  Thus, a balance is struck between the production of new cell types and the renewal of the stem cell pool.  However, imbalances between HSC self-renewal and differentiation can lead to hematologic malignancies like leukemia.

Dr. Flavell’s group discovered that the E3 ubiquitin ligase, c-Cbl, suppresses HSC self-renewal.  The researchers generated transgenic mice deficient in c-Cbl, and demonstrated that these c-Cbl-mutant mice display an increased number of HSCs.

Lead author, Dr. Chozhavendan Rathinam, is confident that “our findings may facilitate the expansion and manipulation of hematopoietic stem cells for tissue engineering and stem cell based therapies.”

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

A team of researchers at Yale School of Medicine have identified, characterized and cloned ovarian cancer stem cells and have shown that these stem cells may be the source of ovarian cancer’s recurrence and its resistance to chemotherapy.

“These results bring us closer to more effective and targeted treatment for epithelial ovarian cancer, one of the most lethal forms of cancer,” said Gil Mor, M.D., associate professor in the Department of Obstetrics, Gynecology & Reproductive Sciences at Yale School of Medicine.

Mor presented his findings recently at the annual meeting of the American Association for Cancer Research (AACR) Meeting in San Diego, California.

Cancerous tumors are made up of cells that are both cancerous and non-cancerous.  Within cancerous cells, there is a further subclass referred to as cancer stem cells, which can replicate indefinitely.

“Present chemotherapy modalities eliminate the bulk of the tumor cells, but cannot eliminate a core of these cancer stem cells that have a high capacity for renewal,” said Mor, who is also a member of the Yale Cancer Center.  “Identification of these cells, as we have done here, is the first step in the development of therapeutic modalities.”

Mor and colleagues isolated cells from 80 human samples of either peritoneal fluid or solid tumors.  The cancer stem cells that were identified were positive for traditional cancer stem cell markers including CD44 and MyD88.  These cells also showed a high capacity for repair and self-renewal.

The isolated cells formed tumors 100 percent of the time.  Within those tumors, 10 percent of the cells were positive for cancer stem cell marker CD44, while 90 percent were CD44 negative.

Mor and his team were able to isolate and clone the ovarian cancer stem cells.  They found that these cells were highly resistant to conventional chemotherapy while the non-cancer stem cells responded to treatment.  “Isolating and cloning these cells will lead to development of new treatments to target and eliminate the cancer stem cells and hopefully prevent recurrence,” said Mor.

New cellular therapies benefits came to light as a result of powerful PET and SPECT imaging in a recent study reported in the April issue of the Journal of Nuclear Medicine. Researchers in Germany were able to observe the repair action of circulating progenitor cells (CPCs), immature blood-derived cells capable of developing into adult stem cells, as they successfully preserved healthy heart tissue and corrected blood flow imbalance within the heart.

Twenty-six patients took part in the randomized, placebo-controlled and double-blinded study. Following the recanalization of blocked coronary arteries (the surgical reopening or formation of new paths for blood flow), one group received an infusion of progenitor cells. FDG PET and 99mTc-tetrofosmine-SPECT were then used to image relative changes in myocardial perfusion (blood flow through the middle and thickest part of the heart) and glucose metabolism.

The results were compared with a control group that had undergone recanalization but did not receive CPCs. In the CPC group, normalization of glucose metabolism and coronary blood flow was seen in nearly 50 percent of the repaired artery segments.

“PET and SPECT are the only techniques capable of validating the metabolic changes we needed to observe in the heart once we had administered the progenitor cells,” said Kai Kendziorra, M.D., a specialist in Nuclear Medicine at the University of Leipzig in Leipzig, Germany. “The results shown by these imaging modalities provide the evidence needed to expand the use of CPC treatment.”

Earlier research has shown that when a patient’s progenitor cells are activated by growth factors, the result is increased cell division, which is vital to the tissue repair process. In this study, progenitor cells developed from circulating blood were also found to be capable of repairing dysfunctional—yet viable—myocardial tissue, a condition referred to as “hibernating myocardium.”

Kendziorra said he believes that in addition to assisting in monitoring and guiding treatment of heart patients, PET scans may also be helpful in selecting those who would profit the most from CPC administration.

“Early detection of hibernating myocardial tissue via noninvasive imaging modalities such as PET and SPECT will help us to assess a patient’s myocardial metabolism and blood flow,” he said. “Subsequent early coronary recanalization and CPC administration may lead to treatment-specific normalization and reduce the risk of cardiac events over longer periods.”

“For decades, nuclear medicine imaging has contributed functional assessment to the anatomical definition of the presence or absence of disease,” said Alexander J. McEwan, M.D., president of SNM. “Today molecular imaging is on the way to revolutionizing patient care—by integrating information about location, structure, function and biology—leading to a package of non-invasive imaging tools with enormous potential for improving patient care and outcomes.”

Co-authors of “Effect of Progenitor Cells on Myocardial Perfusion and Metabolism in Patients After Recanalizatoin of a Chronically Occluded Coronary Artery” include Henryk Barthel, Osama Sabri and Regine Kluge, Department of Nuclear Medicine; Sandra Erbs and Gerhard Schuler, Heart Center Leipzig GmbH; and Frank Emmrich, Institute of Clinical Immunology and Transfusion Medicine, all with the University of Leipzig, Leipzig, Germany; and Rainer Hambrecht, Department of Nuclear Medicine, University of Leipzig, Leipzig, Germany and Heart Center Bremen, Bremen, Germany.

CHICAGO – Neurons which were grafted into the brain of a patient with Parkinson’s disease fourteen years ago have developed Lewy body pathology, the defining pathology for the disease, according to research by Jeffrey H. Kordower, PhD, and associates and published in the April 6 issue of Nature Medicine.

These findings suggest that Parkinson’s disease is an ongoing process that can affect cells grafted into the brain in the same way the disease affects host dopamine neurons in the substantia nigra of the brain, according to Kordower, who is the lead author of the study and a neuroscientist at Rush University Medical Center.

“These findings give us a bit of pause for the value of cell replacement strategy for Parkinson’s disease,” said Kordower.  “We still need to vigorously investigate this approach among the full armament of surgically-delivered Parkinson’s disease therapies. While it is not clear to us whether the same fate would befall stem cell grafts, the next generation of cell replacement procedures, this study does suggest that grafted cells can be affected by the disease process.”

The collaborative research study described in the article involves Rush, Mt. Sinai School of Medicine, New York, and the University of South Florida, Tampa, In it, individuals with Parkinson’s disease received fetal cell transplants to reverse the loss in the brain of striatal dopamine.

The individual described in this article was a woman with a 22-year history of Parkinson’s disease who underwent transplantation in 1993. After transplantation she experienced improvements in disease symptoms as measured by the Unified Parkinson Disease Rating Scale (UPDRS) and required substantially lower doses of antiparkinsonian medications. Her UPDRS scores remained improved into1997, but by 2004, she experienced progressive worsening of Parkinson’s disease symptoms. She died in 2007 and her brain and that of two other patients in the study were comprehensively processed and analyzed. She had the longest survival after transplantation that had been reported to date among this study’s participants.

Double-blind, sham-controlled studies that followed did not establish clinical benefit although significant improvement was observed in a subpopulation of patients. Post mortem studies of individuals in these studies showed a robust survival of grafted neurons, suggesting that the cells were not affected by Parkinson’s disease as Kordower explains “Because Parkinson’s disease pathology progresses over decades, we think that the individuals did not live long enough for the Parkinson’s disease pathology to develop in the grafted cells.”

Scientists have long debated whether Parkinson’s disease results from an acute insult or event, or whether it is an ongoing pathological process that continues to affect healthy neurons, according to Kordower. This research indicates that mechanisms and molecules responsible for initiating the degenerative process are still present at a late stage and are capable of affecting grafted neurons.  In addition, the processes that destroy dopamine neurons are not restricted to the midbrain.

“The findings also suggest that there may be either a pathogenic factor in the brain that affects dopamine producing neurons or a pathological process that can spread from one cellular system to another,” said Kordower.  “These findings have striking implications for understanding what causes PD and the potential for cell replacement strategies to reverse the motor symptoms.”

The study is available online at http:/www.nature.com/naturemedicine