Bush Embryonic Stem Cell Lines Different from Newly Derived Cell Lines

Bush Embryonic Stem Cell Lines Different from Newly Derived Cell Lines
Friday, 02 December 2011

Established human embryonic cell lines, including those approved for federal research funding under former President George W. Bush, are different than newly derived human embryonic stem cell lines, according to a study by UCLA stem cell researchers.

The finding, by scientists with the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, points to the importance of continuing to derive new stem cell lines so researchers can better understand pluripotency, the ability of these cells to make every cell in the human body, said study senior author Amander Clark, an assistant professor of molecular, cell and developmental biology in Life Sciences.

"It is critical to find out the characteristics that result in the highest quality pluripotent stem cell lines that we can make," Clark said.

"It is possible that we have not set the bar high enough yet for embryonic stem cells or induced pluripotent stem cells. We now know that established lines are different from newly derived lines and now we have to find out how important that is."

The study appears Nov. 30, 2011 in the early online edition of the peer-reviewed journal Human Molecular Genetics.

The study looked at the first six human embryonic stem cell lines developed by Clark's research team at UCLA from 2009 to 2011, which have since been accepted by the National Institute of Health's embryonic stem cell registry, founded by executive order in March 2009. Acceptance into the registry allows the UCLA lines to be used in federally funded research projects.

In her study, Clark decided to examine X chromosome inactivation and the mechanisms by which female stem cells turn off one X chromosome during development because it is a large physical marker that is easy to visualize in individual cells. Clark wanted to compare this specific molecular signature in established embryonic stem cell lines versus what occurs during the derivation of new embryonic stem cell lines from human blastocysts.

The established lines examined in the study were from a group of stem cell lines derived prior to 2001. The field has known for many years that the majority of established lines, Clark said, had already undergone X chromosome inactivation, and her work confirmed this finding. However, with the progression of time, Clark found that the molecular signature no longer reflected the normal process of X chromosome inactivation.

The X chromosome normally is inactivated by non-coding RNA and a special form of chromatin in female cells. In abnormal states, such as those found in the older, established human embryonic stem cells, the X chromosome is inactive, but this process is not regulated by the non-coding RNA and the chromatin is different.

"The classic signature is gone, so something else is regulating X chromosome inactivation in the established cell lines," Clark said.

"It will be important not only to find out what that is, but also to discover what else is changing in the nucleus that we cannot see regardless of whether the cell line is male or female."

The new cell lines generated by Clark's research team were derived from human embryos that were donated to the Broad Stem Cell Research Center by couples who had previously undergone in vitro fertilization to overcome infertility. The couples no longer planned to store or use their frozen embryos for reproductive purposes and had declined to donate the embryos to others for reproductive use.

The human embryos were transferred from the fertility clinic to the derivation lab at UCLA in frozen vials. They were then thawed by Clark's research team, and at six to seven days of development the embryos, or blastocysts, contained a cluster of cells called the inner cell mass. The inner cell mass is the source of new embryonic stem cell lines.

Clark's lab examined the human embryonic stem cell lines three to four weeks after growth from the inner cell mass and found that both X chromosomes were still active in many cells, making them more like the cells from the original inner cell mass.

Slowly, with time in culture and cryopreservation – how the lines are ultimately stored – one X chromosome is inactivated and the cell lines become identical to the older, established lines, including abnormal X chromosome inactivation, Clark said.

The question, Clark said, is whether the first cells to grow out from the inner cell mass are of a higher quality, and therefore the ones researchers should be aspiring to use for research and potentially therapeutically.

"It may prove to be important to stabilize these cells at that very young state, one that's closest in identity to the inner cell mass," Clark said.

"And then we can ask whether these cells give the best quality when differentiated into clinical cell types."

Keeping both X chromosomes active will also be important in modeling diseases such as Rett syndrome.

Going forward, Clark will study human embryonic stem cells in three states, lines in which the X chromosome is inactivated by normal means, lines in which the chromosome is inactivated abnormally and lines in which both X chromosomes remain active. Clark will seek to understand the differentiation potential of each of the three states.

"Our data highlights the importance of maintaining human embryonic stem cell derivation efforts. Gold standard human embryonic stem cell lines should be the benchmark for all human pluripotent stem cell research," the study states.

"Developing new experimental approaches aimed at sustaining human pluripotent nuclei in an epigenetic state closer in identity to the day six or seven day human blastocyst is to work towards a more robust gold standard."

Source: University of California - Los Angeles Health Sciences

Contact: Kim Irwin
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Singapore Scientists Lead Human Embryonic Stem Cell Study

Singapore Scientists Lead Human Embryonic Stem Cell Study
Friday, 02 December 2011

Researchers from A*STAR Singapore took lead roles in a study that identified a portion of the genome mutated during long-term culture of human embryonic stem cells (hESCs). The study was a worldwide collaboration, led by Drs Peter Andrews of the University of Sheffield (UK), Paul Robson of the Genome Institute of Singapore (GIS), Steve Oh of Singapore's Bioprocessing Technology Institute (BTI), and Barbara Knowles and others in the international stem cell community. The GIS, IMB and BTI are research institutes under the umbrella of the Agency for Science, Technology and Research, (A*STAR), Singapore.

Involving 125 ethnically diverse hESC lines originating from 38 laboratories globally, and now identified to represent multiple ethnic groups from different parts of the globe, the study is the largest to be conducted on the genetic stability of cultured hESCs. The findings are published today in the journal Nature Biotechnology.

Research into the variability of hESCs is very important as these cells may lead to future cell therapy and regenerative medicine. During long-term culture, however, these cells can acquire genetic changes (mutations), some of which could compromise the cells' utility for regenerative medicine. It is believed that mutations that arise and endure over long-term culture provide a selective advantage for the cells, such as a greater propensity for self-renewal.

The study re-emphasized that many chromosome changes occur repeatedly, resulting in increased copies in specific areas of the genome. Interestingly, through molecular karyotyping performed in Dr Robson's laboratory at the GIS, about 20% of the karyotypically normal cell lines exhibited subkaryotypic amplifications of a specific region in chromosome 20. This is also one of the karyotypically defined areas of change. The minimal region common to these cells contains three ES-cell expressed genes, and one of them, BCL2L1, is a strong candidate for driving hESC culture adaptation. The data generated in this study will be useful for understanding the frequency and types of genetic changes affecting cultured hESCs, an important issue in evaluating the cells for potential therapeutic applications.

Dr Paul Robson, Senior Group Leader of the Developmental Cellomics Laboratory, GIS, said:
"Not only does this work provide important information for evaluating human embryonic stem cell genetic integrity, it also highlights the general utility of these cells in understanding human biology and disease. This same region has recently been identified to repeatedly occur in numerous human cancer cell types, this likely indicative of similar selection pressures at play in stem cells and cancer cells. Interestingly, we found the propensity for mutation at this location is associated with a relatively recent chromosomal rearrangement that occurred in the last common ancestor of the human, chimp, and gorilla thus pointing to the value of having a comparative perspective for understanding human biology."

Dr Barbara Knowles, Principle Investigator at IMB added:
"This is a prodigious piece of community work comparing the genome of cell lines from around the world that were sampled after they had been grown in cell culture for a short period of time to samples from the same cell lines taken after they had been in culture for a longer period of time. Scientists at GIS used these globally obtained samples to pinpoint an area of the genome that contains a gene(s) that affects the cell's ability to control its own growth."

Dr Steve Oh, Principal Scientist at BTI said:
"This study took over three years to complete and is a great testimony of the international stem cell community working persistently together as a force for good. A special thanks goes to Prof Peter Andrews for his leadership! The fact that of the 125 cell lines tested, over 65% of them exhibited normal karyotypes in long term culture bodes well for the use of human embryonic stem cells for cell therapy in the future."

Source: Agency for Science, Technology and Research (A*STAR), Singapore

Contact: Winnie Serah Lim

Reference:

Screening a large, ethnically diverse population of human embryonic stem cells identifies a chromosome 20 minimal amplicon that confers a growth advantage
The International Stem Cell Initiative
Nature Biotechnology, 27 November, 2011, doi:10.1038/nbt.2051
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Neurons Grown from Skin Cells May Hold Clues to Autism

Rare syndrome's workings could help explain how brain wiring goes awry
Sunday, 27 November 2011

Potential clues to how autism miswires the brain are emerging from a study of a rare, purely genetic form of the disorders that affects fewer than 20 people worldwide. Using cutting-edge "disease-in a-dish" technology, researchers funded by the National Institutes of Health have grown patients' skin cells into neurons to discover what goes wrong in the brain in Timothy Syndrome. Affected children often show symptoms of autism spectrum disorders along with a constellation of physical problems.

Representative iPSC-derived neurons from

Timothy syndrome patient (bottom) shows

increased numbers of neurons that produce

the chemical messengers norepinephrine

and dopamine, compared to those from a

control subject (top). Credit: Ricardo

Dolmetsch, Ph.D., Stanford University.

Abnormalities included changes in the composition of cells in the cortex, the largest brain structure in humans, and of neurons that secrete two key chemical messengers. Neurons that make long-distance connections between the brain's hemispheres tended to be in short supply.

Most patients with Timothy Syndrome meet diagnostic criteria for an autism spectrum disorder. Yet, unlike most cases of autism, Timothy syndrome is known to be caused by a single genetic mutation.

"Studying the consequences of a single mutation, compared to multiple genes with small effects, vastly simplifies the task of pinpointing causal mechanisms," explained Ricardo Dolmetsch, Ph.D., of Stanford University, a National Institute of Mental Health (NIMH) grantee who led the study. His work was partially funded by a NIH Director's Pioneer Award.

Dolmetsch, and colleagues, report on their findings Nov. 27, 2011 in the journal Nature Medicine.

"Unlike animal research, the cutting-edge technology employed in this study makes it possible to pinpoint molecular defects in a patient's own brain cells," said NIMH Director Thomas R. Insel, M.D..

"It also offers a way to screen more rapidly for medications that act on the disordered process."

Prior to the current study, researchers knew that Timothy syndrome is caused by a tiny glitch in the gene that code for a calcium channel protein in cell membranes. The mutation results in too much calcium entering cells, causing a tell-tale set of abnormalities throughout the body. Proper functioning of the calcium channel is known to be particularly critical for proper heart rhythm – many patients die in childhood of arrhythmias – but its role in brain cells was less well understood.

To learn more, Dolmetsch and colleagues used a new technology called induced pluripotent stem cells (iPSCs). They first converted skin cells from Timothy Syndrome patients into stem cells and then coaxed these to differentiate into neurons.

"Remarkable reproducibility" observed across multiple iPSC lines and individuals confirmed that the technique can reveal defects in neuronal differentiation – such as whether cells assume the correct identity as the brain gets wired-up in early development, said the researchers. Compared to those from controls, fewer neurons from Timothy Syndrome patients became neurons of the lower layers of the cortex and more became upper layer neurons. The lower layer cells that remained were more likely to be the kind that project to areas below the cortex. In contrast, there were fewer-than-normal neurons equipped to form a structure, called the corpus callosum, which makes possible communications between the left and right hemispheres.

Forebrain of a mouse genetically engineered

to express the mutated gene that causes

Timothy syndrome (TS) shows fewer neurons

contributing to a brain structure responsible

for long-distance communications between

the left and right hemispheres, called the

corpus callosum, compared to the same

structure in a control animal (Ctrl). Human

iPSCs from TS patients showed a similar

reduction. Credit: Ricardo Dolmetsch, Ph.D.,

Stanford University.

Many of these defects were also seen in parallel studies of mice with the same genetic mutation found in Timothy syndrome patients. This supports the link between the mutation and the developmental abnormalities.

Several genes previously implicated in autism were among hundreds found to be expressed abnormally in Timothy Syndrome neurons. Excess cellular calcium levels also caused an overproduction of neurons that make key chemical messengers. Timothy Syndrome neurons secreted 3.5 times more norepinephrine and 2.3 times more dopamine than control neurons. Addition of a drug that blocks the calcium channel reversed the abnormalities in cultured neurons, reducing the proportion of catecholamine-secreting cells by 68 percent.

The findings in Timothy Syndrome patient iPSCs follow those in Rett Syndrome, another single gene disorder that often includes autism-like symptoms. About a year ago, Alysson Muotri, Ph.D., and colleagues at University of California, San Diego, reported deficits in the protrusions of neurons, called spines, which help form connections, or synapses. The Dolmetsch team's discovery of earlier (neuronal fate) and later (altered connectivity) defects suggest that disorders on the autism spectrum affect multiple stages in early brain development.

"Most of these abnormalities are consistent with other emerging evidence that ASDs arise from defects in connectivity between cortex areas and show decreased size of the corpus callosum," said Dolmetsch.

"Our study reveals how these might be traceable to specific mechanisms set in motion by poor regulation of cellular calcium. It also demonstrates that neurons derived from iPSCs can be used to identify the cellular basis of a neurodevelopmental disorder."

The mechanisms identified in this study may become potential targets for developing new therapies for Timothy Syndrome and may also provide insights into the neural basis of deficits in other forms of autism, said Dolmetsch.

Source: NIH/National Institute of Mental Health

Contact: Jules Asher

Reference:

Using iPS cell-derived neurons to uncover cellular phenotypes associated with Timothy Syndrome
Pasca SP, Portmann T, Voineagu I, Yazawa M, Shcheglovitov O, Pasca AM, Cord B, Palmer TD, Chikahisa S, Seiji N, Bernstein JA, Hallmayer J, Geschwind DH, Dolmetsch RE.
Nature Medicine, November 27, 2011, doi:10.1038/nm.2576
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ZenMaster

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

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