Functioning brain tissue grown in 3-D structure

CALB and L7 are Purkinje-cell specific late markers are shown. GRID2 is a marker for a Purkinje-specific glutamate receptor. LHX5 is a marker for early Purkinje cells.

Researchers at the RIKEN Center for Developmental Biology in Japan have succeeded in inducing human embryonic stem cells to self-organize into a three-dimensional structure similar to the cerebellum, providing tantalizing clues in the quest to recreate neural structures in the laboratory. One of the primary goals of stem-cell research is to be able to replace damaged body parts with tissues grown from undifferentiated stem cells. For the nervous system, this is a particular challenge because not only do specific neurons need to be generated, but they must also be coaxed into connecting to each other in very specific ways

RIKEN researchers have taken up this challenge, and the work published in Cell Reports details how sequentially applying several signaling molecules to three-dimensional cultures of human embryotic stem cells prompts the cells to differentiate into functioning cerebellar neurons that self-organize to form the proper dorsal/ventral patterning and multi-layer structure found in the natural developing cerebellum.

Expanding from their previous studies with mice, the researchers first established that under specific conditions, culturing human embryonic stem cells with fibroblast growth factor 2 (FGF2) leads to neural differentiation particular to the midbrain/hindbrain region -- the location of the cerebellum -- within three weeks, and the expression of markers for the cerebellar plate neuroepithelium -- the part of the developing nervous system specific for the cerebellum -- within five. These cells also showed early markers that are specific to developing Purkinje cells, granule cells, or deep cerebellar projection neurons -- all types of neurons only found in the cerebellum.

The researchers then looked for mature cerebellar neurons. They found that cells treated with FGF2 expressed late Purkinje-cell markers and developed structures characteristic of those cells. Electrophysiological recordings of these cells after culture for about 15 weeks revealed proper responses to currents and to inhibition of receptors needed for normal cerebellar signaling, indicating that function had developed along with structure. Some FGF2-treated cells also expressed markers for the rhombic lip -- the structure from which granule cells develop and migrate, and a marker specific to migrating granule precursors by week seven. Moreover, cells were seen to migrate and extend fibers that bent to form the T-shape characteristic of granule cell parallel fibers.

Where these neurons form and how they locate in relation to each other is critical in the developing brain. Early in cerebellar development, particular cell types come to be distributed unevenly from top to bottom, creating a dorsal-ventral separation. Researchers tested several factors, and found that adding FGF19 around day 14 to the FGF2-treated cells caused several flat oval neuroepithelium to form by day 35, expressing dorsal-specific markers on the outside and ventral-specific markers on the inside. By adding stromal cell-derived factor 1 (SDF1) between days 28-35, they were able to generate a continuous neuroepithelial structure with dorsal-ventral polarity.

SDF1 also induced two other important structural changes. The dorsal region spontaneously developed three layers along the dorsal-ventral axis: the ventricular zone, a Purkinje-cell precursor zone, and a rhombic lip zone. At one end of the neuroepithelium, a region developed that was positive for markers of progenitors of granule and deep cerebellar nuclei projection neurons and negative for Purkinje-cell markers, and whose origins could be traced to the rhombic lip zone of the cerebellar plate.

Lead author Keiko Muguruma says that, "the principles of self-organization that we have demonstrated here are important for the future of developmental biology." She added that, "attempts to generate the cerebellum from human iPS cells have already met with some success, and these patient-derived cerebellar neurons and tissues will be useful for modeling cerebellar diseases such as spinocerebellar ataxia."

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Telomere extension turns back aging clock in cultured human cells, study finds

A new procedure can quickly and efficiently increase the length of human telomeres, the protective caps on the ends of chromosomes that are linked to aging and disease, according to scientists at the Stanford University School of Medicine.

Treated cells behave as if they are much younger than untreated cells, multiplying with abandon in the laboratory dish rather than stagnating or dying.

The procedure, which involves the use of a modified type of RNA, will improve the ability of researchers to generate large numbers of cells for study or drug development, the scientists say. Skin cells with telomeres lengthened by the procedure were able to divide up to 40 more times than untreated cells. The research may point to new ways to treat diseases caused by shortened telomeres.

Telomeres are the protective caps on the ends of the strands of DNA called chromosomes, which house our genomes. In young humans, telomeres are about 8,000-10,000 nucleotides long. They shorten with each cell division, however, and when they reach a critical length the cell stops dividing or dies. This internal "clock" makes it difficult to keep most cells growing in a laboratory for more than a few cell doublings.

'Turning back the internal clock'

"Now we have found a way to lengthen human telomeres by as much as 1,000 nucleotides, turning back the internal clock in these cells by the equivalent of many years of human life," said Helen Blau, PhD, professor of microbiology and immunology at Stanford and director of the university's Baxter Laboratory for Stem Cell Biology. "This greatly increases the number of cells available for studies such as drug testing or disease modeling."

A paper describing the research was published today in the FASEB Journal. Blau, who also holds the Donald E. and Delia B. Baxter Professorship, is the senior author. Postdoctoral scholar John Ramunas, PhD, of Stanford shares lead authorship with Eduard Yakubov, PhD, of the Houston Methodist Research Institute.

The researchers used modified messenger RNA to extend the telomeres. RNA carries instructions from genes in the DNA to the cell's protein-making factories. The RNA used in this experiment contained the coding sequence for TERT, the active component of a naturally occurring enzyme called telomerase. Telomerase is expressed by stem cells, including those that give rise to sperm and egg cells, to ensure that the telomeres of these cells stay in tip-top shape for the next generation. Most other types of cells, however, express very low levels of telomerase.

Transient effect an advantage

The newly developed technique has an important advantage over other potential methods: It's temporary. The modified RNA is designed to reduce the cell's immune response to the treatment and allow the TERT-encoding message to stick around a bit longer than an unmodified message would. But it dissipates and is gone within about 48 hours. After that time, the newly lengthened telomeres begin to progressively shorten again with each cell division.

The transient effect is somewhat like tapping the gas pedal in one of a fleet of cars coasting slowly to a stop. The car with the extra surge of energy will go farther than its peers, but it will still come to an eventual halt when its forward momentum is spent. On a biological level, this means the treated cells don't go on to divide indefinitely, which would make them too dangerous to use as a potential therapy in humans because of the risk of cancer.

The researchers found that as few as three applications of the modified RNA over a period of a few days could significantly increase the length of the telomeres in cultured human muscle and skin cells. A 1,000-nucleotide addition represents a more than 10 percent increase in the length of the telomeres. These cells divided many more times in the culture dish than did untreated cells: about 28 more times for the skin cells, and about three more times for the muscle cells.

"We were surprised and pleased that modified TERT mRNA worked, because TERT is highly regulated and must bind to another component of telomerase," said Ramunas. "Previous attempts to deliver mRNA-encoding TERT caused an immune response against telomerase, which could be deleterious. In contrast, our technique is nonimmunogenic. Existing transient methods of extending telomeres act slowly, whereas our method acts over just a few days to reverse telomere shortening that occurs over more than a decade of normal aging. This suggests that a treatment using our method could be brief and infrequent."

Potential uses for therapy

"This new approach paves the way toward preventing or treating diseases of aging," said Blau. "There are also highly debilitating genetic diseases associated with telomere shortening that could benefit from such a potential treatment."

Blau and her colleagues became interested in telomeres when previous work in her lab showed that the muscle stem cells of boys with Duchenne muscular dystrophy had telomeres that were much shorter than those of boys without the disease. This finding not only has implications for understanding how the cells function -- or don't function -- in making new muscle, but it also helps explain the limited ability to grow affected cells in the laboratory for study.

The researchers are now testing their new technique in other types of cells.

"This study is a first step toward the development of telomere extension to improve cell therapies and to possibly treat disorders of accelerated aging in humans," said John Cooke, MD, PhD. Cooke, a co-author of the study, formerly was a professor of cardiovascular medicine at Stanford. He is now chair of cardiovascular sciences at the Houston Methodist Research Institute.

"We're working to understand more about the differences among cell types, and how we can overcome those differences to allow this approach to be more universally useful," said Blau, who also is a member of the Stanford Institute for Stem Cell Biology and Regenerative Medicine.

"One day it may be possible to target muscle stem cells in a patient with Duchenne muscular dystrophy, for example, to extend their telomeres. There are also implications for treating conditions of aging, such as diabetes and heart disease. This has really opened the doors to consider all types of potential uses of this therapy."

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Patient stem cells used to make dementia-in-a-dish; help identify new treatment strategy

Induced pluripotent stem cells (iPSCs) derived from patients with frontotemporal dementia were genetically corrected and converted to cortical neurons. The green staining indicates the cortical marker CTIP2, the red stain is the neuronal marker TUJ1, and the blue stains the nuclei of the cells.

Belgian researchers have identified a new strategy for treating an inherited form of dementia after attempting to turn stem cells derived from patients into the neurons most affected by the disease. In patient-derived stem cells carrying a mutation predisposing them to frontotemporal dementia, which accounts for about half of dementia cases before the age of 60, the scientists found a targetable defect that prevents normal neurodevelopment. These stem cells partially return to normal when the defect is corrected.

The study appears in the December 31st issue of Stem Cell Reports, the official journal of the International Society of Stem Cell Research published by Cell Press.

"Use of induced pluripotent stem cell (iPSC) technology"--which involves taking skin cells from patients and reprogramming them into embryonic-like stem cells capable of turning into other specific cell types relevant for studying a particular disease--"makes it possible to model dementias that affect people later in life," says senior study author Catherine Verfaillie of KU Leuven.

Frontotemporal disorders are the result of damage to neurons in parts of the brain called the frontal and temporal lobes, gradually leading to behavioral symptoms or language and emotional disorders. Mutations in a gene called progranulin (GRN) are commonly associated with frontotemporal dementia, but GRN mutations in mice do not mimic all the features of the human disorder, which has limited progress in the development of effective treatments.

"iPSC models can now be used to better understand dementia, and in particular frontotemporal dementia, and might lead to the development of drugs that can curtail or slow down the degeneration of cortical neurons," Verfaillie says.

Verfaillie and Philip Van Damme of the Leuven Research Institute for Neuroscience and Disease explore this approach in the Stem Cell Reports study by creating iPSCs from three patients carrying a GRN mutation. These immature cells were impaired at turning into mature, specialized cells called cortical neurons--the most affected cell type in frontotemporal dementia.

One of the top defective pathways in the iPSCs was the Wnt signaling pathway, which plays an important role in neuronal development. However, genetic correction or treatment with a compound that inhibits the Wnt signaling pathway restored the ability of the iPSCs to turn into cortical neurons. Taken together, the findings demonstrate that the GRN mutation causes the defect in cortical neuron formation by altering the Wnt signaling pathway.

"Our findings suggest that signaling events required for neurodevelopment may also play major roles in neurodegeneration," Van Damme says. "Targeting such pathways, as for instance the Wnt pathway presented in this study, may result in the creation of novel therapeutic approaches for frontotemporal dementia."

The researchers will now work to better understand what goes wrong in GRN-mutated cells, as well as identify precise molecular targets that could then be used for drug screens.

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