Efficiency of method could lead to better disease study and future stem cell cures
Friday, 07 March 2008
UC Irvine researchers have discovered a dramatically improved method for genetically manipulating human embryonic stem cells, making it easier for scientists to study and potentially treat thousands of disorders ranging from Huntington’s disease to muscular dystrophy and diabetes.
The technique for the first time blends two existing cell-handling methods to improve cell survival rates and increase the efficiency of inserting DNA into cells. The new approach is up to 100 times more efficient than current methods at producing human embryonic stem cells with desired genetic alterations.
“The ability to generate large quantities of cells with altered genes opens the door to new research into many devastating disorders,” said Peter Donovan, professor of biological chemistry and developmental and cell biology at UCI, and co-director of the UCI Sue and Bill Gross Stem Cell Research Center.
“Not only will it allow us to study diseases more in-depth, it also could be a key step in the successful development of future stem cell therapies.”
This study appears online this week in the journal Stem Cells.
Donovan and Leslie Lock, assistant adjunct professor of biological chemistry and developmental and cell biology at UCI, previously identified proteins called growth factors that help keep cells alive. Growth factors are like switches that tell cells how to behave, for example to stay alive, divide or remain a stem cell. Without a signal to stay alive, the cells die.
The UCI scientists – Donovan, Lock and Kristi Hohenstein, a stem cell scientist in Donovan’s lab – used those growth factors in the current study to keep cells alive, then they used a technique called nucleofection to insert DNA into the cells. Nucleofection uses electrical pulses to punch tiny holes in the outer layer of a cell through which DNA can enter the cell.
With this technique, scientists can introduce into cells DNA that makes proteins that glow green under a special light. The green colour allows them to track cell movement once the cells are transplanted into an animal model, making it easier for researchers to identify the cells during safety studies of potential stem cell therapies.
Scientists today primarily use chemicals to get DNA into cells, but that method inadvertently can kill the cells and is inefficient at transferring genetic information. For every one genetically altered cell generated using the chemical method, the new growth factor/nucleofection method produces between 10 and 100 successfully modified cells, UCI scientists estimate.
With the publication of this study, the new method now may be used by stem cell scientists worldwide to improve the efficiency of genetically modifying human embryonic stem cells.
“Before our technique, genetic modification of human embryonic stem cells largely was inefficient,” Hohenstein said.
“This is a stepping stone for bigger things to come.”
Scientists can use the technique to develop populations of cells with abnormalities that lead to disease. They can then study those cells to learn more about the disorder and how it is caused. Scientists also possibly could use the technique to correct the disorder in stem cells, then use the healthy cells in a treatment.
The method potentially could help treat monogenic diseases, which result from modifications in a single gene occurring in all cells of the body. Though relatively rare, these diseases affect millions of people worldwide. Scientists currently estimate that more 10,000 human diseases are monogenic, according to the World Health Organization. Examples include Huntington’s disease, sickle cell anaemia, cystic fibrosis and haemophilia.
UCI is at the forefront of stem cell research. The Sue and Bill Gross Stem Cell Research Center promotes basic and clinical research training in the field of stem cell biology. More than 60 UCI scientists use stem cells in their studies. These scientists study spinal cord injuries, brain injuries and central nervous system diseases such as multiple sclerosis, Alzheimer’s and Huntington’s. They also study muscular dystrophy, diabetes, cancer and other disorders.
April Pyle of UCLA and Jing Yi Chern of Johns Hopkins University also worked on the genetic modification study, which was funded by the National Institutes of Health.
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ZenMaster
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Sunday, 9 March 2008
New Stem Cell Technique Improves Genetic Alteration
Molecular Alliance That Sustains Embryonic Stem Cell State
Allied proteins known as transcription factors
Tuesday, 04 March 2008
One of the four ingredients in the genetic recipe that scientists in Japan and the US followed last year to persuade human skin cells to revert to an embryonic stem cell state, is dispensable in ES cells, thanks to the presence of a molecular alliance between a specific group of key proteins known as transcription factors, a research team led by the Genome Institute of Singapore (GIS) under the Agency for Science, Technology and Research (A*STAR) reports in the current issue of Nature Cell Biology.
The reprogramming factor - Klf4, one of the transcription factors that determine whether a cell's genes are active or silent - has at least two other sibling molecules that will substitute Klf4 to maintain a pluripotent embryonic stem (ES) cell state, the GIS-led team said.
Klf4 (also known as gut-enriched Krüppel-like factor or Gklf) belongs to the Krüppel-like factor (Klf) family of transcription factors that regulate numerous biological processes including proliferation, differentiation, development and apoptosis, or programmed cell death.
Since reprogramming mature cells to the ES state may provide a ready source of tissue for biomedical research and clinical treatment of diseases such as Parkinson's and diabetes, several laboratories, including GIS, are trying to better understand and finely tune the reprogramming process.
The team looks for clues for what these reprogramming ingredients are doing in ES cells.
"Klf4 has been a mysterious player among the four reprogramming factors. As taking out Klf4 in ES cells did not have any apparent effects, it is difficult to reconcile why such a potent reprogramming factor has no role in ES cell biology," said GIS scientist Ng Huck Hui, Ph.D., who headed the research team. Other members of the team include researchers from the National University of Singapore and University of Illinois at Urbana-Champaign.
The GIS research team found that when Klf4 was depleted, Klf2 and Klf5 took over Klf4's role. To understand the molecular basis of the Klf4 redundancy, the scientists studied the DNA binding and transcription activation properties of the three Klfs and found that the profiles of the three Klfs were strikingly similar.
"Most important, the data showed that the other Klfs were bound to the target sites when one of them was depleted." said Dr. Ng.
"These Krüppel-like factors form a very powerful alliance that work together on regulating common targets. The impact of losing one of them is masked by the other two sibling molecules."
For example, Klfs were found to regulate the Nanog gene and other key genes that must be active for ES cells to be pluripotent, or capable of differentiating into virtually any type of cells. Nanog gene is one of the key pluripotency genes in ES cells.
"We suggest that Nanog and other genes are key effectors for the biological functions of the Klfs in ES cells," Dr. Ng said.
"Together, our study provides new insight into how the core Klf circuitry integrates into the Nanog transcriptional network to specify gene expression unique to ES cells. The way these factors network with key genes in ES cells suggest a way of how Klf4 (along with the other three reprogramming factors) can jump-start the ES cell gene expression engine in adult cells," he noted.
Although these three Klfs are involved in diverse biological roles, their redundant roles have not been previously appreciated.
"Dr. Ng and his colleagues at the Genome Institute of Singapore again have unravelled another intricacy of what makes a stem cell," said Edison Liu, M.D., Executive Director of GIS.
"This work brings us closer to a detailed understanding of the genetic components of stemness."
Alan Colman, Ph.D., internationally recognized leader in stem cell research, said, "Klf4 is a transcription factor that came to prominence recently because it was one of four factors used to reprogram somatic cells back to the pluripotent state seen in embryonic stem cells.”
"The mystery of the role of Klf4 has been revealed in studies by Huck Hui and colleagues," added Colman, Executive Director of the Singapore Stem Cell Consortium, which like GIS, is part of Singapore's A*STAR.
"They show for the first time that Klf4 itself is not needed for the maintenance of the pluripotent state in ES cells; however, this is because the cells have a number of other Klf-like transcription factors that can substitute for Klf4."
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ZenMaster
For more on stem cells and cloning, go to CellNEWS at
http://www.geocities.com/giantfideli/index.html
Role of Tiny RNAs In Controlling Stem Cell Fate
Understanding these key regulatory factors is critical for potential therapeutic use of stem cells
Thursday, 06 March 2008
Researchers at the Gladstone Institute of Cardiovascular Disease (GICD) and the University of California, San Francisco have identified for the first time how tiny genetic factors called microRNAs may influence the differentiation of pluripotent embryonic stem (ES) cells into cardiac muscle. As reported in the journal Cell Stem Cell, scientists in the lab of GICD Director, Deepak Srivastava, MD, demonstrated that two microRNAs, miR-1 and miR-133, which have been associated with muscle development, not only encourage heart muscle formation, but also actively suppress genes that could turn the ES cells into undesired cells like neurons or bone.
“Understanding how pluripotent stem cells can be used in therapy requires that we understand the myriad processes and factors that influence cell fate,” said Dr. Srivastava.
“This work shows that microRNAs can function both in directing how ES cells change into specific cells — as well as preventing these cells from developing into unwanted cell types. ”
The differentiation of ES cells into heart cells or any other type of adult cell is a very complicated process involving many factors. MicroRNAs, or miRNAs, seem to act as rheostats or “dimmer switches” to fine-tune levels of important proteins in cells. More than 450 human miRNAs have been described and each is predicted to regulate tens if not hundreds of proteins that may determine cellular differentiation.
While many ES cell-specific miRNAs have been identified, the role of individual miRNAs in ES cell differentiation had not previously been determined. The Gladstone team showed that miRNAs can control how pluripotent stem cells determine their fate, or “cell lineage” – in this case as cardiac muscle cells.
Specifically, they found that miR-1 and miR-133 are active at the early stages of heart cell formation, when an ES cell is first “deciding” to become mesoderm, one of the three basic tissue layers in mammals and other organisms. Activity of either miR-1 or miR-133 in ES cells caused genes that encourage mesoderm formation to be turned on. Equally important, they caused other genes that would have told the cell to become ectoderm or endoderm to turn off. For example, expression of a specific factor called Delta-like 1 was repressed by miR-1. Removal of this factor from cells by other methods also caused the cells to begin transforming into heart cells.
“Our findings provide insight into the fine regulation of cells and genes that is needed for a heart to form,” said Kathy Ivey, PhD, a California Institute of Regenerative Medicine (CIRM) postdoctoral fellow and lead author on the study.
“By better understanding this complicated system, in the future, we may be able to identify ways to treat or prevent childhood and adult diseases that affect the heart.”
The Gladstone team included Alecia Muth, Joshua Arnold, Jason Fish, Edward Hsaio and Bruce Conklin. They were joined by USCF’s Frank King, Ru-Fang Yeh and Harold S. Bernstein. The research was supported by the National Institutes of Health, the California Institute of Regenerative Medicine and the Lynda and Stewart Resnick Foundation.
Reference:
MicroRNA Regulation of Cell Lineages in Mouse and Human Embryonic Stem Cells
Cell Stem Cell, Vol 2, 219-229, 06 March 2008
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ZenMaster
For more on stem cells and cloning, go to CellNEWS at
http://www.geocities.com/giantfideli/index.html
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