Tuesday, 9 September 2014

In Directing Stem Cells, Study Shows Context Matters

In Directing Stem Cells, Study Shows Context Matters
Tuesday, 09 September 2014

Figuring out how blank slate stem cells decide which kind of cell they want to be when they grow up — a muscle cell, a bone cell, a neuron — has been no small task for science.

When blank slate stem cells are exposed to a soft
as opposed to a hard surface on which to grow,
they begin to transform themselves into neurons,
the large, complex cells of the central nervous
system. Absent any soluble factors to direct cell
differentiation, surface matters, according to
new research from the lab of University of
Wisconsin-Madison chemist and biochemist
Laura Kiessling. Credit: Kiessling Lab/UW-
Madison.
Human pluripotent stem cells, the undifferentiated cells that have the potential to become any of the 220 types of cells in the body, are influenced in the lab dish by the cocktail of chemical factors and proteins upon which they are grown and nurtured. Depending on the combination of factors used in a culture, the cells can be coaxed to become specific types of cells.

Now, in a new study published today, Sept. 8, in the Proceedings of the National Academy of Sciences, a team of researchers from the University of Wisconsin-Madison has added a new wrinkle to the cell differentiation equation, showing that the stiffness of the surfaces on which stem cells are grown can exert a profound influence on cell fate.

"To derive lineages, people use soluble growth factors to get the cells to differentiate," explains Laura Kiessling, a UW-Madison professor of chemistry and biochemistry and stem cell expert.

Past work, she notes, hinted that the qualities of the surface on which a cell lands could exert an influence on cell fate, but the idea was never fully explored in the context of human pluripotent stem cell differentiation.

In the lab, stem cells are grown in plastic dishes coated with a gel that contains as many as 1,800 different proteins. Different factors can be introduced to obtain certain types of cells. But even in the absence of introduced chemical or protein cues, the cells are always working to differentiate — but in seemingly random, undirected ways.

The Wisconsin group, directed by Kiessling and led by chemistry graduate student Samira Musah, decided to test the idea that the hardness of a surface can make a difference. After all, in a living body, cells seek different niches with different qualities and transform themselves accordingly.

"Many cell types grow on a surface. If a cell is near bone, the environment has certain features," says Kiessling, whose group — collaborating with UW-Madison colleagues Sean Palecek, Qiang Chang and William Murphy — has been working to produce precise, chemically defined surfaces on which to grow stem cells.

"A cell will react differently if it lands near soft tissue like the brain."

To fully explore the idea that surface matters to a stem cell, Kiessling's group created gels of different hardness to mimic muscle, liver and brain tissues. The study sought to test whether the surface alone, absent any added soluble factors to influence cell fate decisions, can have an effect on differentiation.

Results, according to Kiessling, showed that a soft, brain tissue-like surface, independent of any soluble factors, was catalyst enough to direct cells to become neurons, the large elaborate cells that make up the central nervous system. Stiffer surfaces favoured the stem cell state.

"We didn't change anything but switch from a hard surface to a soft surface," Kiessling says.

"They all started looking like neurons. It was stunning to me that the surface had such a profound effect."

In the case of the soft, brain-like surface, the Wisconsin researchers believe that the mechanical properties of a surface are influencing a protein called YAP. YAP can be found in the cytoplasm but also the nucleus of a cell, and when it is in the nucleus, YAP regulates gene expression. According to the study results, YAP is excluded from the nucleus on the soft gels, and its depletion there helps drive the stem cells onto a brain cell developmental pathway.

The finding, that the simple mechanical properties of a surface can play a big role in helping stem cells decide what to be, promises to help scientists better define the experimental conditions to direct stem cell fate. It may also ultimately inform the methods that will be used for producing large quantities of cells for therapeutic use and other applications such as the high-throughput screening of chemicals for brain toxicity or therapeutics.

Contact: Laura Kiessling

Reference:
Substratum-induced differentiation of human pluripotent stem cells reveals the coactivator YAP is a potent regulator of neuronal specification
Samira Musah, Paul J. Wrighton, Yefim Zaltsman, Xiaofen Zhong, Stefan Zorn, Matthew B. Parlato, Cheston Hsiao, Sean P. Palecek, Qiang Chang, William L. Murphy and Laura L. Kiessling
PNAS 2014, September 8, doi: 10.1073/pnas.1415330111
.........


For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/

Monday, 18 August 2014

Suspect Gene Corrupts Neural Connections

'Diseases of synapses' demonstrated in a dish

Monday, 18 August 2014

Researchers have long suspected that major mental disorders are genetically-rooted diseases of synapses – the connections between neurons. Now, investigators supported in part by the National Institutes of Health have demonstrated in patients' cells how a rare mutation in a suspect gene disrupts the turning on and off of dozens of other genes underlying these connections.

Synapses – sites of intercellular communications
– are revealed in a mature iPSC cortex neuron
derived from a participant in the study. Immune-
based staining shows synapse markers (red,
green) and the cell's nucleus (blue). Credit:
Hongjun Song, Ph.D., Johns Hopkins University.
"Our results illustrate how genetic risk, abnormal brain development and synapse dysfunction can corrupt brain circuitry at the cellular level in complex psychiatric disorders," explained Hongjun Song, Ph.D., of Johns Hopkins University, Baltimore, a grantee of the NIH's National Institute of Mental Health (NIMH), a founder of the study.

Song and colleagues, from universities in the United States, China, and Japan, report on their discovery in the journal Nature, August 17, 2014.

"The approach used in this study serves as a model for linking genetic clues to brain development," said NIMH director Thomas R. Insel, M.D..

Most major mental disorders, such as schizophrenia, are thought to be caused by a complex interplay of multiple genes and environmental factors. However, studying rare cases of a single disease-linked gene that runs in a family can provide shortcuts to discovery. Decades ago, researchers traced a high prevalence of schizophrenia and other major mental disorders – which often overlap genetically – in a Scottish clan to mutations in the gene DISC1 (Disrupted In Schizophrenia-1). But until now, most of what's known about cellular effects of such DISC1 mutations has come from studies in the rodent brain.

To learn how human neurons are affected, Song's team used a disease-in-a-dish technology called induced pluripotent stem cells (iPSCs). A patient's skin cells are first induced to revert to stem cells. Stem cells play a critical role in development of the organism by transforming into the entire range of specialized cells which make up an adult. In this experiment, these particular "reverted" stem cells were coaxed to differentiate into neurons, which could be studied developing and interacting in a petri dish. This makes it possible to pinpoint, for example, how a particular patient's mutation might impair synapses. Song and colleagues studied iPSCs from four members of an American family affected by DISC1-linked schizophrenia and genetically related mental disorders.

Strikingly, iPSC-induced neurons, of a type found in front brain areas implicated in psychosis, expressed 80 percent less of the protein made by the DISC1 gene in family members with the mutation, compared to members without the mutation. These mutant neurons showed deficient cellular machinery for communicating with other neurons at synapses.

The researchers traced these deficits to errant expression of genes known to be involved in synaptic transmission, brain development, and key extensions of neurons where synapses are located. Among these abnormally expressed genes were 89 previously linked to schizophrenia, bipolar disorder, depression, and other major mental disorders. This was surprising, as DISC1's role as a hub that regulates expression of many genes implicated in mental disorders had not previously been appreciated, say the researchers.

The clincher came when researchers experimentally produced the synapse deficits by genetically engineering the DISC1 mutation into otherwise normal iPSC neurons – and, conversely, corrected the synapse deficits in DISC1 mutant iPSC neurons by genetically engineering a fully functional DISC1 gene into them. This established that the DISC1 mutation, was, indeed the cause of the deficits.

The results suggest a common disease mechanism in major mental illnesses that integrates genetic risk, aberrant neurodevelopment, and synapse dysfunction. The overall approach may hold promise for testing potential treatments to correct synaptic deficits, say the researchers.

Contact: Jules Asher

Reference:
Synaptic dysregulation in a human iPS cell model of major mental disorders 
Wen Z, Nguyen HN, Guo Z, Lalli MA, Wang X, Su Y, Kim N-S, Yoon K-J, Shin J, Zhang C, Makri G, Nauen D, Yu H, Guzman E, Chiang C-H, Yoritomo N, Kaibuchi K, Zou J, Christian KM, Cheng L, Ross CA, Margolis RL, Chen G, Kosik KS, Song H, Ming G
Nature, Aug. 17, 2014, doi:10.1038/nature13716
.........


For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/

Stem Cells Reveal How Schizophrenia-linked Genetic Variation Affects Neurons

Stem Cells Reveal How Schizophrenia-linked Genetic Variation Affects Neurons
Monday, 18 August 2014

A genetic variation linked to schizophrenia, bipolar disorder and severe depression wreaks havoc on connections among neurons in the developing brain, a team of researchers reports. The study, led by Guo-li Ming, M.D., Ph.D., and Hongjun Song, Ph.D., of the Johns Hopkins University School of Medicine and described online Aug. 17 in the journal Nature, used stem cells generated from people with and without mental illness to observe the effects of a rare and pernicious genetic variation on young brain cells. The results add to evidence that several major mental illnesses have common roots in faulty "wiring" during early brain development.

In this image, cell nuclei are shown in blue and
synapses in red and green. Credit: Zhexing Wen-
Johns Hopkins Medicine.
"This was the next best thing to going back in time to see what happened while a person was in the womb to later cause mental illness," says Ming.

"We found the most convincing evidence yet that the answer lies in the synapses that connect brain cells to one another."

Previous evidence for the relationship came from autopsies and from studies suggesting that some genetic variants that affect synapses also increase the chance of mental illness. But those studies could not show a direct cause-and-effect relationship, Ming says.

One difficulty in studying the genetics of common mental illnesses is that they are generally caused by environmental factors in combination with multiple gene variants, any one of which usually could not by itself cause disease. A rare exception is the gene known as disrupted in schizophrenia 1 (DISC1), in which some mutations have a strong effect. Two families have been found in which many members with the DISC1 mutations have mental illness.

Video of human neurons firing. Credit: Zhexing
Wen-Johns Hopkins Medicine.
To find out how a DISC1 variation with a few deleted DNA "letters" affects the developing brain, the research team collected skin cells from a mother and daughter in one of these families who have neither the variation nor mental illness, as well as the father, who has the variation and severe depression, and another daughter, who carries the variation and has schizophrenia. For comparison, they also collected samples from an unrelated healthy person. Postdoctoral fellow Zhexing Wen, Ph.D., coaxed the skin cells to form five lines of stem cells and to mature into very pure populations of synapse-forming neurons.

After growing the neurons in a dish for six weeks, collaborators at Pennsylvania State University measured their electrical activity and found that neurons with the DISC1 variation had about half the number of synapses as those without the variation. To make sure that the differences were really due to the DISC1 variation and not to other genetic differences, graduate student Ha Nam Nguyen spent two years making targeted genetic changes to three of the stem cell lines.

In one of the cell lines with the variation, he swapped out the DISC1 gene for a healthy version. He also inserted the disease-causing variation into one healthy cell line from a family member, as well as the cell line from the unrelated control. Sure enough, the researchers report, the cells without the variation now grew the normal amount of synapses, while those with the inserted mutation had half as many.

"We had our definitive answer to whether this DISC1 variation is responsible for the reduced synapse growth," Ming says.

To find out how DISC1 acts on synapses, the researchers also compared the activity levels of genes in the healthy neurons to those with the variation. To their surprise, the activities of more than 100 genes were different.

"This is the first indication that DISC1 regulates the activity of a large number of genes, many of which are related to synapses," Ming says.

The research team is now looking more closely at other genes that are linked to mental disorders. By better understanding the roots of mental illness, they hope to eventually develop better treatments for it, Ming says.

Contact: Shawna Williams

Reference:
Synaptic dysregulation in a human iPS cell model of major mental disorders 
Wen Z, Nguyen HN, Guo Z, Lalli MA, Wang X, Su Y, Kim N-S, Yoon K-J, Shin J, Zhang C, Makri G, Nauen D, Yu H, Guzman E, Chiang C-H, Yoritomo N, Kaibuchi K, Zou J, Christian KM, Cheng L, Ross CA, Margolis RL, Chen G, Kosik KS, Song H, Ming G
Nature, Aug. 17, 2014, doi:10.1038/nature13716
.........


For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/