Making New Neurons When You Need Them


Western societies are aging societies, and the incidence of dementias, Alzheimer’s disease, and other diseases of the aged are on the rise. Treatments for these conditions are largely supportive, but being able to make new neurons to replace the ones that have died is almost certainly where it’s at.

At INSERM and CEA in Marseille, France, researchers have shown that chemicals that block the activity of a growth factor called TGF-beta improves the generation of new neurons in aged mice. These findings have spurred new investigations into compounds that can enable new neuron production in order to mitigate the symptoms of neurodegenerative diseases. Such treatments could also restore the cognitive abilities of those who have suffered neuron loss as a result of radiation therapy or a stroke.

The brain forms new neurons regularly to maintain our cognitive abilities, but aging or radiation therapy to treat tumors can greatly perturb this function. Radiation therapy is the adjunctive therapy of choice for brain tumors in children and adults.

Various studies suggest that the reduction in our cache of neurons contributes to cognitive decline. For example, exposure of mice to 15 Grays of radiation is accompanied by disruption to the olfactory memory and reduction in neuron production. A similar event occurs as a result of aging, but in human patients undergoing radiation treatment, cognitive decline is accelerated and seems to result from the death of neurons.

How then, can we preserve the cache of neurons in our brains? The first step is to determine the factors responsible for the decline is neuron production. In contrast to contemporary theory, neither heavy doses of radiation nor aging causes completely destruction of the neural stem cells that can replenish neurons. Even after doses of radiation and aging, neuron stem cell activity remains highly localized in the subventricular zone (a paired brain structure located in the outer walls of the lateral ventricles), but they do not work properly.

Subventricular Zone
Subventricular Zone

Experiments at the INSERM and CEA strongly suggest that in response to aging and high doses of radiation, the brain makes high levels of a signaling molecule called TGF-beta, and this signaling molecule pushes neural stem cell populations into dormancy. This dormancy also increases the susceptibility of neural stem cells into apoptosis.

Marc-Andre Mouthon, one of the main authors of this research, explained his results in this manner: “Our study concluded that although neurogenesis is reduced in aging and after a high dose of radiation, many stem cells survive for several months, retaining their ‘stem’ characteristics.”

Part two of this project showed that blocking TGFbeta with drugs restored the production of new neurons in aging or irradiated mice.

Thus targeted therapies that block TGFbeta in the brains of older patients or cancer patients who have undergone high dose radiation for a brain tumor might reduce the impact of brain lesions caused by such events in elderly patients who show distinct signs of cognitive decline.

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Adult Stem Cells Isolated From Human Intestines


A laboratory at the University of North Carolina at Chapel Hill has, for the first time, isolated adult stem cells from human intestinal tissue. This achievement should provide a much-needed resource for stem cells researchers to examine the nuances of stem cell biology. Also, these new stem cells should provide stem cell researchers a new tool to treat inflammatory bowel diseases or to mitigate the side effects of chemotherapy and radiation, which often damage the gut.

Scott T. Magness, assistant professor in the departments of physiology at UNC, Chapel Hill, said, “Not having these cells to study has been a significant roadblock to research. Until now, we have not had the technology to isolate and study these stem cells – now we have the tools to start solving many of these problems.”

The study represents a leap forward for a field that for many years has had to resort to conducting experiments with mouse stem cells. While significant progress has been made using mouse models, differences in stem cell biology between mice and humans have kept researchers from investigating new therapeutics for human afflictions.

Adam Grace, a graduate student in Magness’ lab, and one of the first authors of this publication, noted, “While the information we get from mice is good foundational mechanistic data to explain how this tissue works, there are some opportunities that we might not be able to pursue until we do similar experiments with human tissue”

This study from the Magness laboratory was the first in the United States to isolate and grow single intestinal stem cells from mice. Therefore, Magness and his colleagues already had experience with the isolation and manipulation of intestinal stem cells. In their quest to isolate human intestinal stem cells, Magness and his colleagues also procured human small intestinal tissue for their experiments that had been discarded after gastric bypass surgery at UNC.

To develop their technique, Magness and others simply tried to recapitulate the technique they had developed in used to isolate mouse intestines to isolate stem cells from human intestinal stem cells. They used cell surface molecules found on in the membranes of mouse intestinal stem cells. These proteins, CD24 and CD44, were also found on the surfaces of human intestinal stem cells. Therefore, the antibodies that had been used to isolate mouse intestinal stem cells worked quite well to isolate human intestinal stem cells. Magness and his co-workers attached fluorescent tags to the stem cells and then isolated by means of fluorescence-activated cell sorting.

This technique worked so well, that Magness and his colleagues were able to not only isolated human intestinal stem cells, but also distinct types of intestinal stem cells. These two types of intestinal stem cells are either active stem cells or quiescent stem cells that are held in reserve. This is a fascinating finding, since the reserve cells can replenish the stem cell population after radiation, chemotherapy, or injury.

“Now that we have been able to do this, the next step is to carefully characterize these populations to assess their potential, said Magness. He continued: “Can we expand these cells outside the body to potentially provide a cell source for therapy? Can we use these for tissue regeneration? Or to take it to the extreme, can we genetically modify these cells to cure inborn disorders or inflammatory bowel disease? Those are some questions that we are going to explore in the future.”

Certainly more papers are forthcoming on this fascinating and important topic.

The Surprising Ability of Blood Stem Cells to Respond to Emergencies


A research team from Marseille, France has revealed an unexpected role for hematopoietic stem cells (the cells that make blood cells): not only do these cells continuously renew our blood cells, but in emergencies these cells can make white blood cells on demand. that help the body deal with inflammation and infection. This stem cell-based activity could be utilized to protect against infection in patients who are undergoing a bone marrow transplant.

The research team that discovered this previously unknown property of hematopoietic stem cells were from INSERM, CNRS and MDC led by Michael Sieweke of the Centre d’Immunologie de Marseille Luminy and the Max Delbruck Centre for Molecular Medicine, Berlin-Buch.

Cells in our blood feed, clean, and defend our tissues, but their lifespan is limited. The life expectancy of a red blood cell rarely exceeds three months, our platelets die after ten days and the vast majority of our white blood cells survive only a few days.

Therefore, our bodies must produce replacements for these dying cells in a timely manner and in the right quantities and proportions. Blood cells replacement is the domain of the hematopoietic stem cells, which are nested in the bone marrow; that soft tissue inside long bones of the chest, spine, pelvis, upper leg and shoulder. Bone marrow produces and releases billions of new cells into out blood every day. To do this, hematopoietic stem cells must not only divide but their progeny must also differentiate into specialized cells, such as white blood cells, red blood cells, platelets, and so on.

For several years, researchers have been interested in how the process of differentiation and specialization is triggered in stem cell progeny. Sieweke and his colleagues discovered in previous work that hematopoietic stem cell progeny are not preprogrammed to assume a particular cell fate, but respond to environmental cues that direct them to become one cell type or another.

Nevertheless, it is still unclear how stem cells respond during emergencies? How are hematopoietic stem cells able to meet the demand for white blood cells during an infection? Recently, the answer was considered clear: the stem cells neither sensed nor responded to the signals sent to induce their progeny to differentiate into particular cell types. They merely proliferated and their progeny responded to the available signals and differentiated into the necessary cell fates. However, Sieweke’s research team has found that rather than being insensitive to these inductive signals meant for their progeny, hematopoietic stem cells perceive these environmental signals and, in response to them, manufacture the cells that are most appropriate for the danger faced by the individual.

Dr. Sandrine Sarrazin, INSERM researcher and co-author of the publication, said, “We have discovered that a biological molecule produced in large quantities by the body during infection or inflammation directly shows stem cells the path to take.”

Sieweke added, “Now that we have identified this signal, it may be possible in the future to accelerate the production of these cells in patients facing the risk of acute infection.” He continued: “This is the case for 50,000 patients worldwide each year who are totally defenseless against infections just after bone marrow transplantation. Thanks to M-CSF [monocyte-colony stimulating factor], it may be possible to stimulate the production of useful cells while avoiding to produce those that can inadvertently attack the body of these patients. They could therefore protect against infections while their immune system is being reconstituted.”

To reach their conclusions the team had to measure the change of state in each cell. This was a terrifically difficult challenge since the stem cells in question are very rare in the bone marrow: only one cell in 10,000 in the bone marrow of a mouse. Furthermore, the hematopoietic stem cells are, by appearance, indistinguishable from their progeny, the hematopoietic progenitor cells. Therefore, this experiment was tedious and difficult, but it proved that M-CSF could instruct single hematopoietic stem cells to differentiate into the monocyte lineage.

The clinical use of M-CSF will hopefully follow in the near future, but for now, this is certainly an exciting finding that may lead to clinical trials and applications in the future.

The Transformation of Ordinary Skin Cells into Functional Brain Cells


A paper in Nature Biotechnology from research groups at Case Western Reserve School of Medicine describes a technique that directly converts skin cells to the specific type of brain cells that suffer destruction in patients with multiple sclerosis, cerebral palsy, and other so-called myelin disorders. This particular breakthrough now enables “on demand” production of those cells that wrap or “myelinate” the axons of neurons.

Myelin is a sheath that wraps the extension of neurons called the axons. Neurons are the conductive cells that initiate and propagate nerve impulses. Neurons contain cell extensions known as axons that connect with other neurons. The nerve impulse runs from the base of the cell body of the neurons, down the axon, to the neuron to which it is connected. An insulating myelin sheath that surrounds the axon increases the speed at which nerve impulses move down the axon. When this myelin sheath is damaged, nerve impulse conduction goes awry as does nerve function. For example, patients with multiple sclerosis (MS), cerebral palsy (CP), and rare genetic disorders called leukodystrophies, myelinating cells are destroyed are not replaced.

neuron

The new technique discussed in this Nature Biotechnology paper, directly converts skin cells called fibroblasts, which are rather abundant in the skin and most organs, into oligodendrocytes, the type of cell that constructs the myelin sheath in the central nervous system.

Oligodendrocyte

“Its ‘cellular alchemy,'” explains Paul Tesar, PhD, assistant professor of genetics and genome sciences at Case Western Reserve School of Medicine and senior author of the study. “We are taking a readily accessible and abundant cell and completely switching its identity to become a highly valuable cell for therapy.”

Tesar and his group used a technique called “cellular reprogramming,” to manipulate the levels of three naturally occurring proteins to induce the fibroblasts to differentiate into the cellular precursors to oligodendrocytes (called oligodendrocyte progenitor cells, or OPCs).

OPCs

Led by Case Western Reserve researchers and co-first authors Fadi Najm and Angela Lager, Tesar’s research team rapidly generated billions of these induced OPCs (called iOPCs). They demonstrated that iOPCs could regenerate new myelin coatings around nerves after being transplanted to mice—a result that offers hope the technique might be used to treat human myelin disorders.

Demyelinating diseases damage the oligodendrocytes and cause loss of the insulating myelin coating. A cure for these diseases requires replacement of the myelin coating by replacement oligodendrocytes.

Until now, OPCs and oligodendrocytes could only be obtained from fetal tissue or pluripotent stem cells. These techniques have been valuable, but have distinct limitations.

“The myelin repair field has been hampered by an inability to rapidly generate safe and effective sources of functional oligodendrocytes,” explains co-author and myelin expert Robert Miller, PhD, professor of neurosciences at the Case Western Reserve School of Medicine and the university’s vice president for research. “The new technique may overcome all of these issues by providing a rapid and streamlined way to directly generate functional myelin producing cells.”

Even though this initial study used mouse cells, the next critical next step is to demonstrate feasibility and safety of human cells in a laboratory setting. If successful, the technique could have widespread therapeutic application to human myelin disorders.

“The progression of stem cell biology is providing opportunities for clinical translation that a decade ago would not have been possible,” says Stanton Gerson, MD, professor of Medicine-Hematology/Oncology at the School of Medicine and director of the National Center for Regenerative Medicine and the UH Case Medical Center Seidman Cancer Center. “It is a real breakthrough.”

The Nooks and Crannies in Bone Marrow that Nurture Stem Cells


Stems cells in our bodies often require a specific environment to maximize their survival and efficiency. These specialized locations that nurture stem cells is called a stem cell niche. Finding the right niche for a stem cell population can go a long way toward growing more stem cells in culture and increasing their potency.

To that end, a recent discovery has identified the distinct niches that exist in bone marrow for hematopoietic stem cells (HSCs), which form the blood cells in our bodies.

A research team from Washington university School of Medicine in St. Louis has shown that stem niches in bone marrow can be targeted, which may potentially improve bone marrow transplants and cancer chemotherapy. Drugs that support particular niches could encourage stem cells to establish themselves in the bone marrow, which would greatly increase the success rate of bone marrow transplants. Alternatively, tumor cells are known to hide in stem cell niches, and if drugs could disrupt such niches, then the tumor cells would be driven from the niches and become more susceptible to chemotherapeutic agents.

Daniel Link, the Alan A. and Edith L. Wolff Professor of Medicine at Washington University, said, “Our results offer hope for targeting these niches to treat specific cancers or to impress the success of stem cell transplants. Already, we and others are leading clinical trials to evaluate whether it is possible to disrupt these niches in patients with leukemia or multiple myeloma.”

Working in mice, Link and his colleagues deleted a gene called CXCL12, only in “candidate niche stromal cell populations.” CXCL12 which encodes a receptor protein known to be crucial for maintaining HSC function, including retaining HSCs in the bone marrow, controlling  HSCs activity, and repopulating the bone marrow with HSCs after injury.

CXCL12 crystal structure
CXCL12 crystal structure

CXCL12 signaling pathways

In bone marrow, HSCs are surrounded by a whole host of cells, and it is difficult to precisely identify which type of cells serve as the niche cells. These bone marrow cells are known collectively as “stroma,” but there are several different types of cells in stroma. Cells that have been implicated in the HSC niche include endosteal osteoblasts (osteoblasts are bone-making cells and the endosteum in the layer of connective tissue that lines the inner cavity of the bone), perivascular stromal cells (cells that hang out around blood vessels), CXCL12-abundant reticular cells, leptin-receptor-positive stromal cells, and nestin–positive mesenchymal progenitors. Basically, there are a lot of cells in the stroma and figuring out which one is the HSC niche is a big deal.

bone marrow stromal cells

When HSCs divide, they form two cells, one of which replaces the HSC that just divided and a new cells called a hematopoietic progenitor cell (HPC), which can divide and differentiate into either a lymphoid progenitor or a myeloid progenitor. The lymphoid progenitor differentiates into either a B or T lymphocyte and the myeloid progenitor differentiates into a red blood cell, or other types of white blood cells (neutrophil, basophil, macrophage, platelet or eosinophil). As the cells become more differentiated, they lose their capacity to divide.

HSC differentiation

Deleting CXCL12 from mineralizing osteoblasts (bone making cells) did nothing to the HSCs or those cells that form lymphocytes (lymphoid progenitors). Deletion of Cxcl12 from osterix-expressing stromal cells, which include CXCL12-abundant reticular cells and osteoblasts, causes mobilization of hematopoietic progenitor cells (HPCs) from the bone marrow into the bloodstream, and loss of B-lymphoid progenitors, but HSC function is normal. Cxcl12 deletion from blood vessel cells causes a modest loss of long-term repopulating activity. Deletion of Cxcl12 from nestin-negative mesenchymal progenitors causes a marked loss of HSCs, long-term repopulating activity, and lymphoid progenitors. All of these data suggest that osterix-expressing stromal cells comprise a distinct niche that supports B-lymphoid progenitors and retains HPCs in the bone marrow. Also, the expression of CXCL12 from stromal cells in the perivascular region, including endothelial cells and mesenchymal progenitors, supports HSCs.

Link summarized his results this way: “What we found was rather surprising. There’s not just one niche for developing blood cells in the bone marrow. There’s a distinct niche for stem cells, which have the ability to become any blood cell in the body, and a separate niche for infection-fighting cells that are destined to become T cells and B cells.”

These data provide the foundation for future investigations whether disrupting these niches can improve the effectiveness of cancer chemotherapy.

In a phase 2 study at Washington University, led by oncologist Geoffrey Uy, assistant professor of medicine, Link and his team are evaluating whether the drug G-CSF (granulocyte colony stimulating growth factor) can alter the stem cell niche in patients with acute lymphoblastic leukemia and whose disease is resistant to chemotherapy or has recurred. The FDA approved this drug more than 20 years ago to stimulate the production of white blood cells in patients undergoing chemotherapy, who have often weakened immune systems and are prone to infections.

Uy and his colleagues want to evaluate G-CSF if it is given prior to chemotherapy. Patients enrolled at the Siteman Cancer Center will receive G-CSF for five days before starting chemotherapy, and the investigators will determine whether it can disrupt the protective environment of the bone marrow and make cancer cells more sensitive to chemotherapy.

This trial is ongoing, and the results are not yet in, but Link’s work has received a welcome corroboration of his work. A companion paper was published in the same issue of Nature by Sean Morrison, the director of the Children’s Medical Center Research Institute at the University of Texas Southwestern Medical Center in Dallas. Morrison and his team used similar methods as Link and his colleagues and came to very similar conclusions.

Link said, “There’s a lot of interest right now in trying to understand these niches. Both of these studies add new information that will be important as we move forward. Next, we hope to understand how stem cells niches can be manipulated to help patients undergoing stem cell transplants.”

Increasing Reprogramming Efficiency by Turning Off One Gene


The removal of one genetic roadblock could improve the efficiency of adult cell reprogramming by some 10 to 30 fold, according to research by stem cell scientists at the Methodist Hospital Research Institute and two other institutions.

Rongfu Wang, the principal investigator and director of the Center for Inflammation and Epigenetics, said this about his group’s findings: “The discovery six years ago that scientist can convert adult cells into inducible pluripotent stem cells, or iPSCs, bolstered the dream that a patient’s own cells might be reprogrammed to make patient-specific iPSCs for regenerative medicine, modeling human diseases in Petri dishes, and drug screening. But reprogramming efficiency has remained very low, impeding its applications in the clinic.”

Wang and his group identified a protein encoded by a gene called Jmjd3, which is known as KDM6B, acts as an impediment to the reprogramming of adult cells into iPSCs. Jmjd3 is involved in several different biological processes, including the maturation of nerve cells and immune cell differentiation (Popov N, Gil J. Epigenetics. 2010 5(8):685-90).

These findings by Wang’s team are the first time anyone has identified a role for Jmjd3 in the reprogramming process. According to Wang, fibroblasts that lack functional Jmjd3 showed greatly enhanced reprogramming efficiency.

Helen Wang, one of the co-principal authors of this study, said, “Our findings demonstrate a previously unrecognized role of Jmjd3 in cellular reprogramming and provide molecular insight into the mechanisms by which the Jmjd3-PHF20 axis controls this process.’

While teasing apart the roles of Jmjd3 in reprogramming, Wang and his colleagues discovered that this protein regulates cell growth and cellular aging. These are two previously unidentified functions of Jmjd3, and Jmjd3 appears to work primarily by inactivating the protein PHF20. PHF20 is a protein that is required for adult cell reprogramming, and cells that lack PHF20 do not undergo reprogramming to iPSCs.

Rongfu Want explained it like this: “So when it comes to increasing iPSC yields, knocking down Jmjd3 is like hitting two birds with one stone.”

Jmjd3 is almost certainly not the only genetic roadblock to stem cell conversion. Wang noted, “Removal of multiple roadblacks could further enhance the reprogramming efficiency with which researchers can efficiently generate patient-specific iPSCs for clinical applications.”

While this is certainly an exciting finding, there is almost certainly a caveat that comes with it. increased reprogramming efficiency almost certainly brings the potential for increased numbers of mutations. Other studies have shown that iPSC generation is much more efficient if the protein P53 is inhibited, but P53 is the guardian of the genome. It prevents the cell from dividing if there is substantial amounts of DNA damage. Inhibiting P53 activity allows iPSC generation even if the cells have excessive amounts of DNA damage. Therefore, inhibiting those cellular processes that are meant to guard against excessive cell proliferation and growth can lead to greater numbers of mutations. Thus, before Jmjd3 inactivation is used to generate iPSCs for clinical uses, extensive animal testing must be required to ensure that this procedure does make iPSCs even less safe than they already are.