microRNA Regulates Pluripotency in Human Embryonic Stem Cells

Scientists shed light on inner workings of human embryonic stem cells
Thursday, 30 April 2009

Scientists at UC Santa Barbara have made a significant discovery in understanding the way human embryonic stem cells function.

They explain nature's way of controlling whether these cells will renew, or will transform to become part of an ear, a liver, or any other part of the human body. The study is reported in the May 1 issue of the journal Cell.

The scientists say the finding bodes well for cancer research, since tumour stem cells are the engines responsible for the growth of tumours. The discovery is also expected to help with other diseases and injuries. The study describes nature's negative feedback loop in cell biology.

"We have found an element in the cell that controls 'pluripotency,' that is the ability of the human embryonic stem cell to differentiate or become almost any cell in the body," said senior author Kenneth S. Kosik, professor in the Department of Molecular, Cellular & Developmental Biology. Kosik is also co-director and Harriman Chair in Neuroscience Research of UCSB's Neuroscience Research Institute.

"The beauty and elegance of stem cells is that they have these dual properties," said Kosik.

"On the one hand, they can proliferate –– they can divide and renew. On the other hand, they can also transform themselves into any tissue in the body, any type of cell in the body."

The research team includes James Thomson, who provided an important proof to the research effort. Thomson, an adjunct professor at UCSB, is considered the "father of human embryonic stem cell biology." Thomson pioneered work in the isolation and culture of non-human primate and human embryonic stem cells. These cells provide researchers with unprecedented access to the cellular components of the human body, with applications in basic research, drug discovery, and transplantation medicine.

With regard to human embryonic stem cells, Kosik explained that for some time he and his team have been studying a set of control genes called microRNAs.

"To really understand microRNAs, the first step is to remember the central dogma of biology –– DNA is the template for RNA and RNA is translated to protein. But microRNAs stop at the RNA step and never go on to make a protein.”

"The heart of the matter is that before this paper, we knew if you want to maintain a pluripotent state and allow self-renewal of embryonic stem cells, you have to sustain levels of transcription factors, including Oct4, Sox2 and Klf4," said Kosik.

"We also knew that stem cells transition to a differentiated state when you decrease those factors. Now we know how that happens a little better."

Transcription factors are genes that control other genes. On the other hand, microRNAs are single-stranded RNA molecules that control the activity of other genes. When microRNAs in the genome are transcribed from DNA, they target complementary messenger RNAs (mRNAs), which serve as the templates for proteins, to either encourage their degradation or prevent their translation into functional proteins. In general, one gene can be repressed by multiple microRNAs and one microRNA can repress multiple genes, the researchers explained. In a wide variety of developmental processes, microRNAs fine tune or restrict cellular identities by targeting important transcription factors or key pathways.

Kosik's team found that levels of miR-145 change dramatically when human embryonic stem cells differentiate into other cell types. miR-145 was of particular interest because it had been predicted to target Oct4, Klf4 and Sox2. The new research shows that a microRNA –– a single-stranded RNA whose function is to decrease gene expression –– lowers the activity of three key ingredients, Oct4, Sox2 and Klf4, in the recipe for embryonic stem cells. The discovery may have implications for improving the efficiency of methods designed to reprogram differentiated cells into embryonic stem cell-like cells.

As few as three or four genes can make cells pluripotent. Those three factors are perhaps best known as three of four ingredients originally shown to transform adult human skin cells into "induced pluripotent stem cells" (iPS cells), which behave in nearly every respect like true embryonic stem cells. That four-ingredient recipe has since been pared down to one, Oct4, in the case of neural stem cells.

"We know what these genes are," Kosik said. That information was used recently for one of the most astounding breakthroughs of biology of the last couple of years –– the discovery of induced pluripotent skin cells.

"You can take a cell, a skin cell, or possibly any cell of the body, and revert it back to a stem cell," Kosik said.

"The way it's done, is that you take the transcription factors that are required for the pluripotent state, and you get them to express themselves in the skin cells; that's how you can restore the embryonic stem cell state. You clone a gene, you put it into what's called a vector, which means you put it into a little bit of housing that allows those genes to get into a cell, then you shoot them into a stem cell. Next, when those genes –– those very critical pluripotent cell genes –– get turned on, the skin cell starts to change; it goes back to the embryonic pluripotent stem cell state."

The researchers explained that a rise in miR-145 prevents human embryonic stem cells' self-renewal and lowers the activity of genes that lend stem cells the capacity to produce other cell types. It also sends the cells on a path toward differentiation. In contrast, when miR-145 is lost, the embryonic stem cells are prevented from differentiating as the concentrations of transcription factors rise.

They also show that the control between miR-145 and the "reprogramming factors" goes both ways. The promoter for miR-145 is bound and repressed by a transcription factor known as OCT4, they found.

"It's a beautiful double negative feedback loop," Kosik said.

"They control each other. That is the essence of this work."

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Human embryonic stem cells are poised between a proliferative state with the potential to become any cell in the body and a differentiated state with a more limited ability to proliferate. To maintain this delicate balance embryonic stem cells express a set of factors, including OCT4, SOX2, and KLF4, to control multiple genes that sustain the proliferative pluripotent state. A tiny RNA called miR-145 can repress these genes, and in turn, one of the transcription factors, OCT4, can repress miR-145. Thus, a double negative feedback loop sets the delicate balance.

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Because there is typically less "wiggle room" in the levels of microRNA compared to mRNA, further studies are needed to quantify more precisely the copy numbers of miR-145 and its targets, to figure out exactly how this layer of control really works, Kosik said.

The findings in embryonic stem cells might also have importance for cancer.

"There are sets of microRNA that are widely up- or down regulated in cancers," he said, noting that several studies have specifically linked low miR-145 levels to various forms of cancer.

"Tumour stem cells are the engines of tumours. If miR-145 is sustaining or maintaining a differentiated state, loss of that may have something to do with malignant transformation."

Kosik credits the lion's share of this discovery to first author Na Xu, a postdoctoral fellow who is also supported by the California Institute for Regenerative Medicine (CIRM).

"Na Xu deserves enormous credit for this work," said Kosik.

"She performed nearly every experiment in the paper and was the major contributor to the ideas in the paper."

Meanwhile, Thales Papagiannakopoulos, a graduate student working in the Kosik lab, was very generous in helping Na Xu with one of the experiments. He helped with one of several proofs that showed that the targets of miR-145 are the three transcription factors that are being reported, explained Kosik.

Thomson provided one of several proofs for the control point of miR-145 expression, said Kosik.

Reference:
MicroRNA-145 Regulates OCT4, SOX2, and KLF4 and Represses Pluripotency in Human Embryonic Stem Cells
Na Xu, Thales Papagiannakopoulos, Guangjin Pan, James A. Thomson and Kenneth S. Kosik
Cell, 30 April 2009, doi:10.1016/j.cell.2009.02.038
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Making Heart Cells from Stem Cells

Gladstone scientists identify key sequence of transcription factors driving heart cell creation
Tuesday, 28 April 2009

Scientists at the Gladstone Institutes of Cardiovascular Disease have identified for the first time key genetic factors that drive the process of generating new heart cells. The discovery, reported in the current issue of the journal Nature, provides important new directions on how stem cells may be used to repair damaged hearts.

For decades, scientists were unable to identify a single factor that could turn non-muscle cells into beating heart cells. Using a clever approach, the research team led by Benoit Bruneau, Ph.D., found that a combination of three genes could do the trick. This is the first time any combination of factors has been found to activate cardiac differentiation in mammalian cells or tissues.

"The heart has very little regenerative capacity after it has been damaged," said Dr. Bruneau.

"With heart disease the leading cause of death in the Western world, this is a significant first step in understanding how we might create new cells to repair a damaged heart."

Two of the three genes encode proteins called transcription factors, which are master regulators that bind to DNA and determine which genes get activated or shut off. The two transcription factors, GATA4 and TBX5, cause human heart disease when mutated and cooperate with each other to control other genes. When Dr. Bruneau and postdoctoral fellow Jun K. Takeuchi added different combinations of transcription factors to mouse cells, these two seemed important for pushing cells into heart cells — but they were not enough.

"When we finally identified the key factor that could work with GATA4 and TBX5 to turn cells into beating heart cells, it was somewhat of a surprise to us," said Dr. Bruneau.

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This represents how the cardiogenic factors turn on heart genes. The transcription factors, Tbx5 and Gata4, cannot access the DNA unless Baf60c is present. When all three are introduced, Baf60c helps open up the closed chromatin, and lets Tbx5 and Gata4 work together to turn on the heart genes. Credit: Benoit Bruneau, The Gladstone Institute of Cardiovascular Disease.

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The surprising factor was a cardiac-specific protein called BAF60c, which helps determine whether transcription factors like GATA4 and TBX5 can even gain access to the DNA regions they were supposed to turn on or off.

"Our previous studies had shown that chromatin remodelling complexes were important," said Dr. Bruneau.

"Mice with lower levels of these complexes have severe heart defects and defective cardiac differentiation. These observations prompted us to look at Baf60c in heart differentiation."

The effect was dramatic. Addition of the three factors directed differentiation of mouse mesoderm, which normally has the potential to make bone, blood, muscle, heart, and other tissues, specifically into cardiac muscle cells (cardiomyocytes) that beat rhythmically, just like normal heart cells. In fact, even cells that normally contribute to the placenta could be induced to transform into beating cardiomyocytes.

"Together, these factors give us a potent mechanism to control cellular differentiation," said Dr. Bruneau.

"This knowledge may help us to understand how to reprogram new cardiomyocytes for therapeutic purposes."

About the Gladstone Institutes:
The J. David Gladstone Institutes, an independent, non-profit biomedical research organization, affiliated with the University of California, San Francisco, is dedicated to the health and welfare of humankind through research into the causes and prevention of some of the world's most devastating diseases. Gladstone is comprised of the Gladstone Institute of Cardiovascular Disease, the Gladstone Institute of Virology and Immunology and the Gladstone Institute of Neurological Disease.

Reference:
Directed transdifferentiation of mouse mesoderm to heart tissue by defined factors
Jun K. Takeuchi & Benoit G. Bruneau
Nature advance online publication 26 April 2009, doi:10.1038/nature08039
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What makes a cow a cow?

Genome sequence sheds light on ruminant evolution
Friday, 24 April 2009

Researchers report today in the journal Science that they have sequenced the bovine genome, for the first time revealing the genetic features that distinguish cattle from humans and other mammals.

The six-year effort involved an international consortium of researchers and is the first full genome sequence of any ruminant species. Ruminants are distinctive in that they have a four-chambered stomach that – with the aid of a multitude of resident microbes – allows them to digest low quality forage such as grass.

The bovine genome consists of at least 22,000 protein-coding genes and is more similar to that of humans than to the genomes of mice or rats, the researchers report. However, the cattle genome appears to have been significantly reorganized since its lineage diverged from those of other mammals, said University of Illinois animal sciences Professor Harris Lewin, whose lab created the high-resolution physical map of the bovine chromosomes that was used to align the sequence. Lewin, who directs the Institute for Genomic Biology, also led two teams of researchers on the sequencing project and is the author of a Perspective article in Science on the bovine genome sequence and an accompanying study by the Bovine Genome and Analysis Consortium.

"Among the mammals, cattle have one of the more highly rearranged genomes," Lewin said.

"They seem to have more translocations and inversions (of chromosome fragments) than other mammals, such as cats and even pigs, which are closely related to cattle.”

"The human is actually a very conserved genome as compared to the ancestral genome of all placental mammals, when you look at its overall organization."

The sequence of the cow's 29 pairs of chromosomes and its X chromosome (the Y chromosome was not studied) also provides new insights into bovine evolution and the unique traits that make cattle useful to humans, Lewin said.

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The first cow genome to be sequenced was that of a Hereford cow named L1 Dominette, shown here with her calf. Credit: Photo courtesy of USDA Agricultural Research Service Research Geneticist Michael D. MacNeil.

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For example, Illinois animal sciences research professor Denis Larkin conducted an analysis of the chromosome regions that are prone to breakage when a cell replicates its genome in preparation for the creation of sperm and egg cells. He showed that in the cattle genome these breakpoint regions are rich in repetitive sequences and segmental duplications and include species-specific variations in genes associated with lactation and immune response.

A previous study from Lewin's lab published this month in Genome Research showed that the breakpoint regions of many species' chromosomes are rich in duplicated genes and that the functions of genes found in these regions differ significantly from those occurring elsewhere in the chromosomes.

These repeats and segmental duplications occur by means of many different mechanisms, one of which involves sporadic and repeated insertions of short bits of genetic material, called retroposons, into the genome.

"The cow genome has many types of repeats that accumulate over time," Lewin said.

"And one of the things that we found is that the new ones are blasting into where the old ones are in the breakpoint regions and breaking them apart. That's the first time that that's been seen."

"The repeats do a lot of things," he said.

"They can change the regulation of the genes. They can make the chromosomes unstable and make them more likely to recombine with other pieces of chromosomes inappropriately."

Lewin calls the breakpoint regions "hotspots of evolution in the genome."

Another analysis led by Lewin, a study of metabolic genes performed by Seongwon Seo, a postdoctoral fellow in Lewin's lab and now a professor at Chungnam National University in South Korea, found that five of the 1,032 genes devoted to metabolic functions in humans are missing from the cattle genome or have radically diverged. This suggests that cattle have some unique metabolic pathways, Lewin said.

These differences in metabolism, along with changes in genes devoted to reproduction, lactation and immunity are a big part of "what makes a cow a cow," Lewin said.

For example, one of the changed genes, histatherin, produces a protein in cow's milk that has anti-microbial properties. The researchers also found multiple copies of a gene for an important milk protein, casein, in a breakpoint region of one of the chromosomes.

“Having the genome sequence is now the window to understanding how these changes occurred, how ruminants ended up with a four-chambered stomach instead of one, how the cow’s immune system operates and how it is able to secrete large amounts of protein in its milk,” Lewin said.

References:
The Genome Sequence of Taurine Cattle: A Window to Ruminant Biology and Evolution
The Bovine Genome Sequencing and Analysis Consortium, Christine G. Elsik, Ross L. Tellam, Kim C. Worley
Science 24 April 2009, Vol. 324. no. 5926, pp. 522 – 528, DOI: 10.1126/science.1169588
Genome-Wide Survey of SNP Variation Uncovers the Genetic Structure of Cattle Breeds
The Bovine HapMap Consortium
Science 24 April 2009, Vol. 324. no. 5926, pp. 528 – 532, DOI: 10.1126/science.1167936

Along with these Science papers, researchers published 20 companion reports describing more detailed analyses of the domestic cattle genome sequence in journals from the open access publisher BioMed Central. All of the articles can be freely accessed at www.biomedcentral.com/series/bovine.

See also:
Completed cattle genome could improve beef and dairy production
EurekAlert - Friday, 24 April 2009
Completed bovine genome sequence opens door to better cattle production
EurekAlert - Friday, 24 April 2009
Cattle genome sequencing milestone promises health benefits
EurekAlert - Friday, 24 April 2009
International science consortium publishes analysis of domestic cattle genome sequence
EurekAlert - Friday, 24 April 2009
Bovine genome provides clues to possible new developments
EurekAlert - Friday, 24 April 2009
Sequencing the cow's genetic code – a new agricultural era dawns
EurekAlert - Friday, 24 April 2009
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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/ and
http://www.geocities.com/giantfideli/index.html

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Safer Therapeutic Stem Cells Generated from Adult Cells

Scientists completely avoid problems of genetic manipulation by instead using chemical programming
Thursday, 23 April 2009

The new technique solves one of the most challenging safety hurdles associated with personalized stem cell-based medicine because for the first time it enables scientists to make stem cells in the laboratory from adult cells without genetically altering them. This discovery has the potential to spark the development of many new types of therapies for humans, for diseases that range from Type 1 diabetes to Parkinson's disease.

The study was published in an advance, online issue of the journal Cell Stem Cell on April 23, 2009.

"We are very excited about this breakthrough in generating embryonic-like cells from fibroblasts [cells that gives rise to connective tissue] without using any genetic material," says Scripps Research Institute Associate Professor Sheng Ding, who led the research.

"Scientists have been dreaming about this for years."

Normally, cells develop from stem cells into a myriad of increasingly more specialized cell types during early development and throughout a lifetime. In humans and other mammals, these developmental events are irreversible. This means that when tissues are damaged or cells are lost, there is usually no source from which to replenish them. Having a source of the most primitive stem cells available would be useful in many medical situations because these cells are "pluripotent," having the ability to become any of the body's cell types — potentially providing doctors with the ability to repair damaged tissues throughout the body.

However bright this promise is, the use of stem cells in medicine has faced many hurdles. One strategy has been to work towards a therapy where doctors could take a patient's own adult cells and "reprogram" them into stem cells. This not only avoids potential ethical problems associated with the use of human embryonic stem cells, it also addresses concerns about compatibility and immune rejection that plague therapies such as organ transplantation.

A few years ago, a team of researchers in Japan made a breakthrough in this general approach by converting mouse skin cells into mouse stem cells. The Japanese team accomplished this remarkable transformation by inserting a set of four genes into these skin cells. While the study was a powerful proof-of-principle, the therapeutic potential of genetically reprogrammed cells is limited because of safety issues. One obvious problem is that the four required genes and their associated foreign DNA sequences permanently reside in the cells when transplanted. Moreover, the specific genes in question are problematic because, in living tissue, they are linked to the development of cancerous tumours.

Many scientists have been trying to find safer ways to generate stem cells from adult cells – developing methods that require fewer genes, or techniques that can put genes in and then take them out. However, to date all of these have still harboured significant safety concerns due to the nature of the genetic manipulations. Ding and his team previously reported the discovery of drug-like small molecules to replace some of those genes, but have also hoped to go even further and find ways to reprogram adult cells into stem cells without using any genes or genetic manipulations at all.

The team of scientists accomplished this extraordinarily challenging feat by engineering and using recombinant proteins, which is proteins made from the recombination of fragments of DNA from different organisms. Many different recombinant proteins have been therapeutically and routinely used to treat human diseases. Instead of inserting the four genes into the cells they wanted to reprogram, the scientists added the purified engineered proteins and experimented with the chemically defined conditions without any genetic materials involved until they found the exact mix that allowed them to gradually reprogram the cells.

The scientists found that those reprogrammed embryonic-like cells (dubbed "protein-induced pluripotent stem cells" or "piPS cells") from fibroblasts behave indistinguishably from classic embryonic stem cells in their molecular and functional features, including differentiation into various cell types, such as beating cardiac muscle cells, neurons, and pancreatic cells.

Reference:
Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins
Hongyan Zhou, Shili Wu, Jin Young Joo , Saiyong Zhu, Dong Wook Han, Tongxiang Lin, Sunia Trauger, Geoffery Bien, Susan Yao, Yong Zhu, Gary Siuzdak, Hans R. Schöler, Lingxun Duan, and Sheng Ding
Cell Stem Cell, 23 April 2009, doi:10.1016/j.stem.2009.04.005
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ZenMaster

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What's wrong with cloning humans?

Panayiotis Zavos again claims he has cloned human embryos
Wednesday, 22 April 2009

A maverick fertility doctor, Dr. Panayiotis Zavos, claims he has cloned human embryos and implanted them into women. It is not the first time he does this.

See also:
Fertility expert: 'I can clone a human being'
The Independent - Wednesday, 22 April 2009
What's wrong with cloning humans?
The Guardian - Wednesday, 22 April 2009

See previous articles on human cloning on CellNEWS:
Therapeutic Cloning Gets a Boost with New Research Findings
China Restrict Clinical Tests of Stem Cells Obtained by Therapeutic Cloning
Chinese Researchers Make Cloned Human Blastocysts
UN-GA Ban on All Human Cloning to be Reconsidered
Hybrid Embryos Created in Newcastle
Human Cloning Achieved
Dolly Professor Abandons Human Cloning Attempts
UN Analysis on Human Cloning
Human Therapeutic Cloning at a Standstill
Was Hwang’s Stem Cells Parthenogenetic?
Seoul National University Report on Dr. Hwang Woo Suk’s Cloning Work
Hwang's Stem Cell Cloning Fabricated
Hwang's Team: Step Towards Therapeutic Cloning
Antinori Say’s Three Cloned Babies Born
Zavos Cloning Attempt Has Failed
Zavos: Implant of Cloned Human Embryo
Can Human Cloning be Made Safe?
Antinori’s Cloning Consortium: II. Repeated Cloning Claim
Dr. Panos Zavos: No implant of cloned human embryo’s yet
'A Clone Would Be Uglier, Sicker and Dimmer'
Dr. Panos Zavos: Ready to Implant Human Clone
Clonaid Says It Will Offer Proof of Cloned Babies
Antinori’s Cloning Consortium: I. The Scientific connections
The Cloning Circus Continues
Dr. Panos Zavos: Where is the proof of a "baby"?
Dr. Panos Zavos Question Clone Claim
The Race Is On: First Cloned Baby 'EVE' Is Born
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ZenMaster

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China Breaks Ground on Regenerative Medicine Centre

China Breaks Ground on Largest Comprehensive Stem Cell Storage and Processing Facility
Wednesday, 22 April 2009

Jiangsu government's China Medical City (CMC) and Shenzhen Beike Biotechnology Co. Ltd. broke ground on the 20,000 square-meter Stem Cell Regenerative Medicine Industrial Project of National Bio-Industry Base (NBPD). This facility will house China's first comprehensive regenerative medicine technology centre and its largest international stem cell bank.

The NBPD facility is part of multi-stage project that consists of industry partnerships aimed at providing a central research zone for China's regenerative medicine and bio-medical industry. Groups associated with this biotechnology incubation joint-project include those from Stanford University, the University of Texas Houston Medical Center, Fudan University, Huazhong Science and Technology University, Nanjing University Medical School, Jiangsu University, Shanghai Jiao Tong University and Jiangsu Provincial People's Hospital.

Mr. Chen Zhu, China's Minister of Health, sent a letter of support that was read at April's ground-breaking ceremony. In his letter he stated that biotechnology is China's fastest developing technology, and stem cell research promises to improve the quality of life for people everywhere. The NBPD program was created to improve the understanding of stem cell technology and facilitate China's development as a world leader in the biotechnology industry.

Located in the heart of Taizhou and occupying more than 20 square kilometres, the CMC district enjoys investment privileges as well as full support from the local, provincial and national governments. CMC is quickly becoming a world centre for biotechnology due to the geographical concentration of medical services and manufacturing, effectively increasing research efficiency and streamlining production.

Jiangsu's Minister of Health Guo Qinghua explained Beike's role in the NBPD project saying:

"Beike is Jiangsu's leading stem cell biotechnology company and can easily take on the task of building the NBPD. This project is set to become China's largest stem cell research centre." Shenzhen Beike Biotechnology was selected as CMC's partner to build, develop and operate the NBPD project.

The new stem cell processing facility includes four centres. The first is the Stem Cell Technology Transfer Center where leading scientists can collaborate and transfer their research to the clinic. The second is the Stem Cell Bank, which will be the largest stem cell bank in Asia with the capacity to store one million samples and facilities for a commercialized iPS bank. The third is the Testing Center, accredited to test the purity, safety, potency and stability of stem cell products. The fourth is the Clinical Technology Service Center, which interfaces the stem cell processing base with hospitals, distribution paths, and offers clinical support services to analyze the outcomes of the stem cell treatments.

Dr. Sean Hu, chairman and CEO of Beike Biotech, praised China Medical City's foresight to develop fertile ground for stem cell technology development both from a regulatory and a funding standpoint.

Dr. Hu states:

"In the 1970's the US government provided the intellectual property support and venture capital companies offered the funding that allowed the US to leapfrog past Europe to become the leader in biotechnology and pharmaceuticals. China's Medical City is now doing the same and we are starting to see the results of these efforts. Beike feels honoured to be able to help China Medical City fulfil its mission of making China the world leader in clinical stem cell technology."

In June 2008, Beike Biotechnology Co. Ltd. opened a 1,800 square-meter stem cell bank in Taizhou, capable of storing 100,000 samples, marking the first phase in the NBPD development process. The stem cell bank currently contracts with major hospitals to store stem cell material and provide finished stem-cell products to patients throughout China and abroad.

About China Medical City in Taizhou:
Jiangsu Province is considered the number one location for China's medical industry based on revenue generated over the past 5 years. The city of Taizhou in Jiangsu is not only the hometown of China's President Hu Jintao but is considered the fastest growing medical industry location in Jiangsu, with over 35% annual growth in that time. Established by the Chinese Government in 2005 and consisting of 20-25 square kilometres in the heart of Taizhou City, China Medical City is fully supported by China's local and national governments. CMC is emerging as a strong leader in China's efforts to develop a streamlined pharmaceutical and medical materials industry that concentrates all medical services and support in one location. Businesses located in CMC carry out a range of manufacturing and support services including research and development, creation and processing of medical materials, distribution, comprehensive healthcare delivery solutions and patent filing support.

About Beike Biotechnology Co., Ltd.:
Beike Biotechnology focuses on stem cell research and clinical application. It currently offers a full line of stem cell products derived from umbilical cord, cord blood, peripheral blood and bone marrow. The proprietary technology used in clinical production of Beike's stem cell products was developed in cooperation with the leading universities in China.

See also:
Stem Cell Forum in China Demonstrate Cutting Edge Research
CellNEWS - Friday, 25 July 2008

Source: Beike Biotech Company Co., Ltd..
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ZenMaster

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Proposed NIH Stem Cell Guidelines Dismay Leading Stanford Researcher

Irving Weissman think therapeutic cloning should be allowed
Monday, 20 April 2009

The director of stem cell research at the Stanford University School of Medicine says he is troubled by draft guidelines issued today by the National Institutes of Health that would prohibit federal funding for research on stem cell lines created through a technique sometimes referred to as “therapeutic cloning” or somatic cell nuclear transfer.

Irving Weissman, MD, director of Stanford’s Institute for Stem Cell Biology and Regenerative Medicine, said the SCNT technique is one way to create disease-specific human embryonic stem cell lines on which to conduct research and test therapies. He also took issue with the assertion that the NIH consulted existing guidelines from the National Academy of Sciences and the International Society for Stem Cell Research — both of which sanction the use of SCNT-derived cell lines — in coming up with its draft recommendations.

“Instead of facts, the NIH placed its own version of ethics in place of the president’s clear proclamation,” said Weissman, the Virginia & D.K. Ludwig Professor for Clinical Investigation in Cancer Research.

“As head of the National Academy of Sciences' panel that unanimously endorsed research using SCNT, and as a drafter of the guidelines for the International Society for Stem Cell Research, I know that this suggested ban on federal funding of SCNT-derived human embryonic stem cell lines is against our policies and against President Obama’s March 9 comments. The NIH has not served its president well.”

On March 9, President Barack Obama signed an executive order removing previous restrictions on the use of federal funds for research on any human embryonic stem cell line derived after Aug. 9, 2001. He used the ceremony to remark that it is important to ensure “that scientific data is never distorted or concealed to serve a political agenda — and that we make scientific decisions based on facts, not ideology.”

In announcing the draft guidelines, acting NIH director Raynard Kington, MD, PhD, justified the restriction in part by saying that there is a lack of scientific consensus as to the necessity of funding lines derived by SCNT and that, although the technique has been used to create many embryonic stem cell lines in animals, such human embryonic stem cell lines have not yet been documented.

“We believe there is strong, broad public and scientific support for the use of federal funds for research on cell lines from embryos derived through in vitro fertilization for reproductive purposes that would not otherwise be used,” said Kington, noting that similar legislation had twice passed both the House and Senate only to be vetoed by former President George W. Bush.

“We do not see similar broad support for using federal funding for research on cell lines from other sources.”

The somatic cell nuclear transfer technique involves removing the nucleus from an egg cell and replacing it with a nucleus from a different cell in order to create an embryonic stem cell line genetically identical to the donor nucleus. In the case of a donor who suffers from a condition like Parkinson’s disease, the SCNT process would yield an embryonic stem cell line that could be used to test specific therapies for that patient.

If the draft guidelines are adopted, they would underscore the continued need for the California Institute for Regenerative Medicine, which has funded grants to several scientists working to create specific human embryonic stem cell lines for research purposes. The institute was established in 2005 by Proposition 71 to counteract the effect of President Bush’s limits on federal funding of such research.

“Methods like SCNT were specifically sanctioned by Prop. 71,” said Geoff Lomax, PhD, the senior officer to the state institute’s Standards Working Group, which was instituted to develop ethical guidelines for the use of embryos in CIRM-funded research.

“These potential restrictions on the range of research materials available for federal funding ensure that CIRM will continue to play a unique role in the world of stem cell research.”

“For certain types of research, CIRM could remain very important,” concurred Renee Reijo-Pera, PhD, director of Stanford’s Center for Human Embryonic Stem Cell Research and Education. Reijo-Pera said she had expected the NIH guidelines to be somewhat conservative, particularly where SCNT is concerned.

“I am happy that these are draft guidelines,” said Weissman, who noted that the NIH did not solicit input from either the National Academy of Sciences or the International Society for Stem Cell Research during the consensus process.

“I’d like to remind the NIH of the principles enunciated by the president on March 9. Research in this area is moving very fast, and it’s not possible to say whether advances will come from work on adult-derived iPS cells or from embryonic stem cells created by nuclear transfer. Policy needs to be developed as the field develops, rather than precluding something based on ideology.”

The proposed NIH guidelines will be available for public comment for 30 days, and the final guidelines will be released by the agency on or before July 7. Comments can be mailed, or submitted electronically after the guidelines are published in the Federal Register by April 24.

About Stanford University School of Medicine:
The Stanford University School of Medicine consistently ranks among the nation’s top 10 medical schools, integrating research, medical education, patient care and community service. The medical school is part of Stanford Medicine, which includes Stanford Hospital & Clinics and Lucile Packard Children’s Hospital.

Source: Stanford University School of Medicine.
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Researchers Collaborate on Stem Cell Therapy for ALS

A team in Utah is collaborating on a stem cell therapy to fight amyotrophic lateral sclerosis, also known as Lou Gehrig’s disease.
Monday, 20 April 2009

A team of researchers from the University of Utah, Salt Lake city-based Q therapeutics Inc., and the John Hopkins University School of Medicine is collaborating on a stem cell therapy to fight amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease.

With $5 million dollars in funding from the National Institutes of Health (NIH), Linda Kelley, Ph.D., director of the University of Utah’s Cell Therapy Facility, James Campanelli, Ph.D., of University of Utah spin-out Q Therapeutics, Inc., and Utah native Nicholas Maragakis, M.D., of The Johns Hopkins University School of Medicine, have teamed up to bring the cell-based therapy to the point of human clinical trials to treat this deadly disease. The four-year NIH grant will enable critical manufacturing and testing requirements necessary to gain U.S. Food and Drug Administration approval for human clinical trials.

Kelley, principal investigator on the grant and professor of internal medicine at the University of Utah School of Medicine, said the project is a collaboration in the truest sense.

“While the University will be home to the grant, the stem-cell technology that Q Therapeutics brings to the table and the clinical expertise of Dr. Maragakis are essential to the project. We are pleased to help bring this groundbreaking therapy toward human use,” Kelley said.

“Our collaboration is a terrific example of how public-private partnerships can make innovative therapeutic products a reality.”

Jack Brittain, University vice president for technology venture development, said:

“The translational research that this funding supports – beyond basic research, but not yet in clinical trials – has been traditionally very difficult to fund. This award validates the approach being taken here at the University of Utah toward emerging technologies, such as regenerative medicine. This kind of collaboration between the University and its commercial spin-out companies is something we strive for and enthusiastically support.”

ALS is a progressive neurodegenerative disease that kills certain nerve cells in the brain and spinal cord. As these cells degenerate, they lose the ability to send impulses that control muscle movement for speech, breathing, limb movement, and other functions, with death from respiratory failure typically occurring from two to five years after diagnosis. ALS affects roughly 30,000 people in this country.

The cell-based ALS therapeutic originates from research at the University of Utah by Mahendra Rao, M.D., Ph.D., a co-founder of Salt Lake City-based Q Therapeutics, Inc..

“Q Therapeutics is delighted to be working with the University of Utah Cell Therapy Facility and Dr. Maragakis on this groundbreaking project,” said Campanelli, senior director of research and development for Q Therapeutics.

“The Cell Therapy Facility is one-of-a-kind in the Intermountain West. We are fortunate to be able to work so closely with Dr. Kelley and her team. The close proximity of our two groups has allowed us to readily address manufacturing and processing issues that would have been a challenge to overcome had we needed to go outside Utah.”

In bringing together cell therapy and neurology, the collaboration focuses on two of seven life science industry sectors identified by the State of Utah for long-term development.

“Given the current economic climate, this type of grassroots effort is critical to both near-term job preservation and long-term development of Utah’s life sciences industry,” said Jason Perry, executive director of the Governor’s Office of Economic Development.

“This project is perfectly aligned with the state’s targeted economic cluster for the Life Sciences and is a model for public and private collaboration.”

Maragakis, a Salt Lake City native and graduate of the University of Utah School of Medicine, added:

“This is an important milestone in the development of therapeutics to treat those who suffer with ALS. Given the lack of good treatment alternatives for this fatal disease, this project could lead to a first-in-class therapy that significantly alters the course of disease for many ALS patients.”

Maragakis and his team of researchers at Johns Hopkins recently published results of their work in ALS in Nature Neuroscience, showing that a specific type of brain stem cell therapy can be effective in an animal model of ALS.

About the University of Utah Cell Therapy Facility:
Established in 1990, the University of Utah’s Cell Therapy Facility (CTF) has grown from a two-person laboratory at the University Hospital to a 18,000-square-foot cell processing and manufacturing facility that employs 40 scientists and staff. CTF provides cell processing and manufacturing services for University of Utah researchers as well as commercial entities in the cell therapy field. To date, CTF has supported two successful Investigational New Drug (IND) filings with the FDA for cell-based therapeutics. It currently supports three pre-IND cell therapeutics and has 15 contracts with commercial entities for a variety of cell processing and manufacturing services.

About Q Therapeutics, Inc.:
Q Therapeutics, Inc. is an emerging biopharmaceutical company, venture-backed and privately held, developing products to treat debilitating diseases of the central nervous system. The Company has exclusive rights to 17 patents arising out of work done by Mahendra Rao, M.D., Ph.D., at the University of Utah and NIH, as well as rights to pending patents from Steven Goldman, M.D., Ph.D. and the Cornell Medical Foundation. The company’s first product, Q-Cells®, is a cell-based therapeutic intended to restore or preserve normal function of neurons by providing essential support functions that occur in healthy central nervous system tissues. Q-Cells® may be applicable to a wide range of demyelinating diseases, including multiple sclerosis, transverse myelitis, cerebral palsy, and white matter stroke, as well as other neurodegenerative diseases such as ALS (Lou Gehrig’s Disease), traumatic spinal cord injury, Parkinson’s and Alzheimer’s Disease. Initial clinical targets are transverse myelitis, a rapidly paralyzing, inflammatory demyelinating spinal cord injury related to MS; and ALS, with a first IND filing targeted in 2010. Q’s pipeline includes other neural cell products for treating diseases including peripheral neuropathies, as well as use of its proprietary cells for new drug discovery.
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Human Stem Cells Promote Healing of Diabetic Ulcers

Foetal stem cells are therapeutically effective
Monday, 20 April 2009

Treatment of chronic wounds is a continuing clinical problem and socio-economic burden with diabetic foot ulcers alone costing the NHS £300 million a year. Scientists in Bristol have found that human foetal stem cells can effectively be used to treat back leg ischemic ulcers in a model of type 1 diabetes.

The researchers also found the culture in which the stem cells had been grown mimicked the wound-healing ability of the cells, suggesting that they could be used as a "factory" of wound-healing substances. Alternatively, the active ingredients in the culture, once identified, could be used instead; this would avoid the ethical concerns of using human foetal stem cells.

In humans, diabetic patients with ischemic foot ulcers have the worst outcome of all chronic skin wounds, with higher amputation and mortality rates than patients carrying non-ischemic ulcers. Topical gels containing single growth factors have recently been used with some success in non-ischemic ulcers, but have been unsuccessful in ischemic ulcers, which are also resistant to other conventional treatment. Ischemia results when the blood supply to a tissue is greatly reduced or stopped - this can occur in diabetes since it can also cause impaired blood flow in patients.

The healing activity of stem cells is recognised for their ability to separate into the various component cells of injured tissues, as well as to discharge growth factors that may encourage the formation of new blood vessels in the patient.

Paolo Madeddu, Professor of Experimental Cardiovascular Medicine and colleagues at the Bristol Heart Institute, previously used stem cells in models of back leg ischemia, showing that foetal stem cells could be more therapeutically effective than adult stem cells.

Foetal stem cells possess a better ability to multiply and to graft onto host tissue, and to separate into other cell types to replace those in the damaged tissue. The group led by Bristol University's Professor Madeddu have found that foetal stem cells accelerate the closure of ischemic diabetic ulcers, while stem cells from blood of adult donors are ineffective.

Professor Madeddu, commenting on the research, said:

"This is the first study to demonstrate the healing capacity of local therapy with CD133+ stem cells in a model of diabetic ischemic foot ulcer. The foetus-derived cells would be difficult to obtain for therapeutic applications. However, the finding that conditioned culture is also effective in stimulating wound healing may have important implications for the cure of the ischemic complications of diabetes."

"Foetal CD133+ cells might be used in the future as a "factory" of therapeutic substances. Alternatively, synthetic replica of the conditioned medium could be produced to obviate ethical concerns surrounding the direct use of foetal stem cells."

Karen Addington, Chief Executive of Juvenile Diabetes Research Foundation (JDRF), added:

"Chronic wounds and diabetic foot ulcers are serious long-term complications of type 1 diabetes. Because of the difficulties involved in managing type 1 diabetes, people living with the condition are at an increased risk of requiring a non-traumatic limb amputation. Although more work needs to be done before we can begin to think about potential new treatments, this research represents a useful way to help identify new strategies for dealing with type 1 diabetes."

The researchers discovered that a particular type of stem cell – CD133+ cells (derived from human foetal aorta) promoted blood vessel formation in order to salvage the diabetic limb. Three days following the graft consisting of collagen plus CD133+ cells, hardly any CD133+ cells were detected in the ischemic diabetic ulcer – indicating that transplanted cells had done their task in the very first days after transplantation possibly by boosting the generation of new vessels through an indirect mechanism.

They found that the CD133+ cells released large amount of growth factors and cytokines endowed of pro-angiogenic and pro-survival potential. To confirm the importance of these released factors, Professor Madeddu and colleagues have grown the CD133+ cells in vitro, and then used the "conditioned" culture to reproduce the effects on wound healing and angiogenesis. These additional experiments confirmed that wound healing and angiogenesis are equally benefited by either giving stem cells or the stem cells' released product.

In the attempt to explain which component of the healing cocktail were really important, they withdrew likely candidates one by one by blocking antibodies. Interestingly, they found that the vascular endothelial growth factor A (VEFG-A) and some interleukins were the crucial factors accounting for the healing effect of transplanted stem cells.

Importantly, VEGF-A was recognized to be the responsible for reactivation of foetal genes, belonging to the Wingless gene family, in the wounded tissue. Withdrawal of wingless gene products also prohibited the beneficial action of conditioned medium on the wound closure and reparative angiogenesis.

This discovery provides a new perspective in the use of foetal stem cells. It is known that wounds heal so well in foetuses that no scar can be visible at birth. It is therefore possible that, when foetal stem cells are transplanted onto diabetic ulcers, they reactivate a foetal program in the recipient to allow those adult ulcers to repair as efficiently as foetal wounds do.

Reference:
Human CD133+ Progenitor Cells Promote the Healing of Diabetic Ischemic Ulcers by Paracrine Stimulation of Angiogenesis and Activation of Wnt Signaling
Lucíola S Barcelos, PhD; Cécile Duplaa, PhD; Nicolle Kränkel, PhD; Gallia Graiani, PhD; Gloria Invernici, PhD; Rajesh Katare, PhD; Mauro Siragusa, MS; Marco Meloni, PhD; Ilaria Campesi, MS; Manuela Monica, MS; Andreas Simm, PhD; Paola Campagnolo, MS; Giuseppe Mangialardi, MD; Lara Stevanato, PhD; Giulio Alessandri, PhD; Costanza Emanueli, PhD; & Paolo Madeddu, MD
Circulation Research, Volume 104, 2 April 2009, doi: 10.1161/CIRCRESAHA.108.192138
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Clue to How Stem Cells Form

Emory study yields evidence of processes that erases epigenetic signals
Friday, 17 April 2009

An Emory University study shows some of the first direct evidence of a process required for epigenetic reprogramming between generations – a finding that could shed more light on the mechanisms of fertilization, stem-cell formation and cloning. The journal Cell published the results of the study on the nematode C. elegans in its April 17 issue.

"We believe that we have demonstrated one of the processes that erases the information in a fertilized egg, so that the offspring can begin life with a clean slate," says David Katz, lead author of the study. Katz is a post-doctoral fellow in the lab of William Kelly, associate professor of biology at Emory and a co-author of the study.

"One of the most fundamental mysteries in biology is how a sperm and egg create a new organism. By looking at the process at the molecular level, we're gaining understanding of this basic question of life," Katz says.

When a sperm cell fertilizes an egg cell, the specialized programming of each parent cell must be erased, in order to form a zygote that can give rise to a new organism. The process by which these two differentiated cells return to a developmental ground state in the zygote – the ultimate stem cell – is little understood.

'An amazing phenotype'
The Emory researchers wanted to test the theory that removal of a particular histone protein modification involved in the packaging of DNA – dimethylation of histone H3 on lysine 4 – is involved in reprogramming the germ line.

They compared successive generations of a normal strain of C. elegans – a microscopic worm commonly used for studying cell differentiation – with a mutant strain. The mutants lacked an enzyme (KDM1) that test-tube experiments have previously shown appears to play an "erasing" role – demethylating histones to remove information from the packaging of DNA.

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This is KDM1 enzyme localisation in a dissected worm, C. elegans, gonad. Credit: David Katz.

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In the normal strain of the worms, the histone modification the Emory researchers had targeted was not passed on to the next generation, but in the mutant strain the modification continued through 30 generations, and each generation became progressively less fertile.

"That's an amazing phenotype," Katz says.

"The organism gradually lost its ability to reproduce. We have shown that when this enzyme is missing, the worms can inherit the histone modification – not only from cell to cell, but from generation to generation."

When the researchers re-inserted the missing enzyme into the sterile generations of mutant worms, they were able to reverse the process: the worms no longer inherited the histone modification, and they regained fertility.

Showing inheritance of epigenetic event
For years, it has been accepted that histone proteins help coil six-foot strands of DNA into tight balls, compact enough to fit inside the nucleus of a cell. Histone modifications have also been known to correlate with gene expression. More recently, researchers have theorized that a chemical change in the histone packaging of DNA, known as an epigenetic event, can be passed on – just as genes themselves can be inherited.

"This study is one of the first demonstrations in a living organism that this theory may be true – that every generation can be affected by an epigenetic event," Kelly says.

"Our work provides some of the best, direct evidence that chemical modifications in the packaging of DNA can be inherited from cell to cell," Katz added.

"That indicates that these chemical modifications are not just involved in packaging – they contain information."

Groundwork for stem-cell therapies
A better understanding of the role of histones, and the enzymes involved in their modification, could lead to therapies for everything from cancer to infertility.

"Stem-cell therapies are an incredibly promising technology for treating any problem that has to do with defective cells," Katz says.

"We're hoping that our work will help this technology to develop."

Katz and his colleagues are now building on the results of the study, to see if a lack of the erasing enzyme shows a similar effect in mice.

About Emory University:
Emory University is known for its demanding academics, outstanding undergraduate experience, highly ranked professional schools and state-of-the-art research facilities. Perennially ranked as one of the country's top 20 national universities by U.S. News & World Report, Emory encompasses nine academic divisions as well as the Carlos Museum, The Carter Center, the Yerkes National Primate Research Center and Emory Healthcare, Georgia's largest and most comprehensive health care system.

Reference:
A C. elegans LSD1 Demethylase Contributes to Germline Immortality by Reprogramming Epigenetic Memory
David J. Katz, T. Matthew Edwards, Valerie Reinke and William G. Kelly
Cell, Volume 137, Issue 2, 308-320, 17 April 2009
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DECIPHER-ing human disease

Database provides a key to unlock the causes of illnesses
Thursday, 16 April 2009

Yesterday - five years after the inception of the DECIPHER database - researchers have published a report that reveals the developing role of the database in revolutionising both clinical practice and genetic research.

The report explores the growing benefits of DECIPHER for researchers, clinicians and patients - highlighting how the data, provided by around 100 centres and shared openly worldwide, can benefit all three groups.

DECIPHER - the Database of Chromosomal Imbalance and Phenotype in Humans using Ensembl Resources - is hosted at the Wellcome Trust Sanger Institute. It was established in 2004 to catalogue sub-microscopic structural duplications, deletions and rearrangements in the genome - called copy number variants (CNVs) - and to uncover their possible role in disease.

"The first comprehensive map of human copy number variation was produced just three years ago, changing our understanding of human genetics" explains Nigel Carter, a lead member of the DECIPHER team from the Wellcome Trust Sanger Institute.

"Since then, over 10,000 CNVs have been found, covering about 5 per cent of the human genome. This rate of advance has been remarkable: using new technologies, we are able to uncover the smaller, elusive variants at a 50 fold-higher resolution. But the pivotal role that DECIPHER plays is in looking at how these variants affect human health."

The problem researchers face is that while many CNVs initially appear to have no visible effect on individual health, others appear to have minor effects, and some are harmful. What DECIPHER helps clinicians to do is to evaluate CNVs and determine whether or not they are linked to the patient's problems. In some cases, the findings are novel or have been observed only a handful of times before. With consent from the patient, data can be shared worldwide and clusters of people with overlapping genetic rearrangements can be identified.

By looking at genetic information first in an unbiased and less subjective manner, recurrent genetic changes can be found, researchers can then seek matching symptoms. This reverses the traditional practice of identification where researchers would move from individuals with shared symptoms back to a chromosomal cause and is particularly helpful for conditions such as learning disability and congenital disorders which have a large number of different genetic causes.

"We need new ways to uncover those rearrangements that cause human disease. But we must also be wary of dismissing CNVs if they appear to have no physical effect," says Charles Lee, an Associate Professor at Harvard Medical School and a Clinical Cytogeneticist at Brigham and Women's Hospital in Boston, USA.

"For example, there may be variants that only affect people with a specific genetic makeup; or sometimes specific combinations of variants may result in pathology."

The report provides case studies in which DECIPHER played a pivotal role. In one example a four-year-old girl with symptoms of developmental delay and poor eye contact had a novel genetic finding and remained without a clear diagnosis. However, two new cases with similar structural variants were submitted to the database one year later, to provide the elusive diagnosis. The case studies exemplify increasing value of the database as clinicians add case information.

"DECIPHER is particularly useful when we look at patients with developmental delay, learning disability, dysmorphic features or congenital abnormalities, where, using genomic array technology, we can assign a diagnosis in 15 per cent of previously undiagnosed cases," explains Helen Firth, Consultant Clinical Geneticist at Addenbrookes Hospital and lead author on the study.

"This improvement is dependent on a fantastic level of collaboration. More than 2000 patient cases have been contributed to the DECIPHER database since its inception: its diagnostic power strengthens as new cases are added"

DECIPHER is built upon the Ensembl genome browser. It is the only open-access, web-based interactive database of its type, although data from other databases are available. The report's authors suggest that while combination of all data in one resource would be ideal, providing access to the data in one genome browser is a realistic and practical method of harnessing the combined power of the datasets.

Sharing data between researchers is increasingly important. As the role of CNVs in human disease is better understood, so resources such as DECIPHER will gain momentum that will drive significant health benefits and improvements to genetic counselling.

Reference:
DECIPHER: Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources
Helen V. Firth, Shola M. Richards, A. Paul Bevan, Stephen Clayton, Manuel Corpas, Diana Rajan, Steven Van Vooren, Yves Moreau, Roger M. Pettett, Nigel P. Carter
American Journal of Human Genetics, (2009), doi: 10.1016/j.ajhg.2009.03.010
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Gene Therapy for Muscular Dystrophy Shows Promise

Safety hurdle also cleared
Thursday, 16 April 2009

Researchers have cleared a safety hurdle in efforts to develop a gene therapy for a form of muscular dystrophy that disables patients by gradually weakening muscles near the hips and shoulders.

Described as the first gene therapy trial in muscular dystrophy demonstrating promising findings, researchers from the University of Florida (UF), Nationwide Children's Hospital in Columbus, Ohio, and The Ohio State University report how they safely transferred a gene to produce a protein necessary for healthy muscle fibre growth into three teenagers with limb-girdle muscular dystrophy.

The findings, which have relevance to genetic disorders beyond muscular dystrophy as well as conditions in which muscles atrophy, were published online today in the Annals of Neurology.

"We think this is an important milestone in establishing the successful use of gene therapy in muscular dystrophy," said Jerry Mendell, MD, director of the Center for Gene Therapy in The Research Institute at Nationwide Children's Hospital and the lead author of the study. Mendell is also a professor of Paediatrics and Pathology at The Ohio State University College of Medicine.

"This trial sets the stage for moving forward with treatment for this group of diseases and we are very pleased with these promising initial results. In subsequent steps we plan to deliver the gene through the circulation in hopes of reaching multiple muscles. We also want to extend the trials over longer time periods to be sure of the body's reaction."

Limb-girdle muscular dystrophy actually describes more than 19 disorders that occur because patients have a faulty alpha-sarcoglycan gene. In each of the disorders, the muscle fails to produce a protein essential for muscle fibres to thrive. It can occur in children or adults, and it causes their muscles to get weaker throughout their lifetimes. The trial evaluated the safety of a modified adeno-associated virus — an apparently harmless virus known as AAV that already exists in most people — as a vector to deliver the alpha-SG gene to muscle tissue.

"The safety data is accumulating because this is the same type of vector that we and other research groups have successfully used in gene therapy trials for other diseases," said Barry Byrne, MD, a UF paediatric cardiologist who is a member of the UF Genetics Institute and director of the Powell Gene Therapy Center.

"In this effort, although proof of safety was the main endpoint, the added benefit was that this was an effective gene transfer. Even though we were dealing with a small area of muscle, the effect was long-lasting, and that has never been observed before."

Research subjects received a dose of the gene on one side of the body and saline on the opposite side. Neither the researchers nor the patients knew which of the foot muscles received the actual treatment until the end of the experiment. The volunteers were evaluated at set intervals through 180 days, and therapy effectiveness was measured by assessing alpha-SG protein expression in the muscle, which was four to five times higher than in the muscles that received only the saline. The volunteers encountered no adverse health events, and the transferred genes continued to produce the needed protein for at least six months after treatment.

In addition, scientists actually saw that muscle-fibre size increased in the treated areas, suggesting that it may be possible to combat the so-called "dystrophic process" that causes muscles to waste away during the course of the disease. Beyond muscular dystrophy, the discovery shows muscle tissue can be an effective avenue to deliver therapeutic genes for a variety of muscle disorders, including some that are resistant to treatment, such as inclusion body myositis, and in conditions where muscle is atrophied, such as in cancer and aging.

"These exciting results demonstrate the feasibility of gene therapy to treat limb-girdle muscular dystrophy," said Jane Larkindale, portfolio director with Muscular Dystrophy Association Venture Philanthropy, a program that moves basic research into treatment development.

"The lack of adverse events seen in this trial not only supports gene therapy for this disease, but it also supports such therapies for many other diseases."

Reference:
A phase I/IItrial of MYO-029 in adult subjects with muscular dystrophy
Kathryn R. Wagner, James L. Fleckenstein, Anthony A. Amato, Richard J. Barohn, Katharine Bushby, Diana M. Escolar, Kevin M. Flanigan, Alan Pestronk, Rabi Tawil, Gil I. Wolfe, Michelle Eagle, Julaine M. Florence, Wendy M. King, Shree Pandya, Volker Straub, Paul Juneau, Kathleen Meyers, Cristina Csimma, Tracey Araujo, Robert Allen, Stephanie A. Parsons, John M. Wozney, Edward R. LaVallie, Jerry R. Mendell
Annals of Neurology, Volume 63, Issue 5, Date: May 2008, Pages: 561-571
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Patients Own Stem Cells Help Stroke Victim

UT Houston researchers will enrol 10 patients in phase I trial
Thursday, 16 April 2009

For the first time in the United States, a stroke patient has been intravenously injected with his own bone marrow stem cells as part of a research trial at the University of Texas Medical School at Houston.

Roland "Bud" Henrich, 61, was transferred to Memorial Hermann – Texas Medical Center on March 25 after suffering a stroke while working on his farm in Liberty. He arrived too late to receive tissue plasminogen activator (tPA), the only treatment for ischemic strokes. He became the first patient in the trial.

The Phase I safety trial, funded with a pilot grant from The National Institutes of Health and support from the Notsew Orm Sands Foundation, will enrol nine more patients who have suffered a stroke and can be treated with the stem cell procedure within 24 to 72 hours of initial symptoms.

Stroke occurs when a blockage or a rupture in an artery, depriving brain tissue of oxygen, interrupts blood flow to the brain. It is the third-leading cause of death behind heart disease and cancer. According to the American Stroke Association, nearly 800,000 Americans suffer a stroke each year – one every 40 seconds. On average, someone dies of stroke every three to four minutes.

"It's still very early in this safety study, but this could be an exciting new therapeutic approach for people who have just suffered a stroke," said Sean Savitz, M.D., assistant professor of neurology at the medical school and the study's lead investigator.

"Animal studies have shown that when you administer stem cells after stroke, the cells enhance the healing. We know that stem cells have some kind of guidance system and migrate to the area of injury. They're not making new brain cells but they may be enhancing the repair processes and reducing inflammatory damage."

Savitz said animal studies have shown that the healing effects of stem cells can occur as early as a week but cautioned it is too early to attribute Henrich's improvement to the stem cell treatment.

"I'm hoping he will get better and it will be because of the cells, but it's just hope at this point," Savitz said.

The stem cells were harvested from the bone marrow in the iliac crest of his leg, then separated and returned to Henrich several hours later. Because they are his own stem cells, rejection is not expected to be an issue.

When he arrived at the hospital, Henrich could not speak and had significant weakness on his right side. When he was released after nearly two weeks of hospitalization and rehabilitation, he was able to walk and climb stairs unassisted and said his first words.

His wife, Reba Henrich, said she believes the stem cells have helped. He has spoken a few times with a single word or a phrase since his return home. "Too crowded," he told her at a megastore as they shopped for Easter gifts for their grandchildren and "senior" meal he told a waitress at a local restaurant. He also has fed the cows by himself, she said. They are hopeful he will eventually be able to return to his job as a painter.

"This study is the critical first step in translating laboratory work with stem cells into benefit for patients. If effective, this treatment could be helpful to a huge segment of stroke patients to reduce their disability," said James C. Grotta, M.D., Roy M. and Phyllis Gough Huffington Distinguished Professor of Neurology and chair of the Department of Neurology at the medical school.

"We are fortunate here at UT Houston and the Texas Medical Center to have the resources needed to carry out this work, and to have attracted someone of Dr. Savitz's calibre to lead this study."
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Novel Technique to Sequence Human Genome

Physicists at Brown University have developed a novel procedure to map a person’s genome
Thursday, 16 April 2009

Since the human genome was sequenced six years ago, the cost of producing a high-quality genome sequence has dropped precipitously. More recently, the National Institutes of Health called for cutting the cost to $1,000 or less, which may enable sequencing as part of routine medical care.

The obstacles to reaching that goal have been primarily technological: Scientists have struggled to figure out how to accurately read the 3 billion base pairs – the amount of DNA found in humans and other mammals – without time-consuming, inefficient methods.

Physicists at Brown University may have an answer. They introduce a novel procedure to vastly slow the DNA's movement through openings that are used to read the code. In the journal Nanotechnology, the physicists report the first experiment to move DNA through a solid-state nanopore using magnets. The approach is promising because it allows multiple segments of a DNA strand to be threaded simultaneously through numerous tiny pores and for each fragment to move slowly enough through the opening so that the base pairs can be accurately read.

"When it comes to sequencing anyone's genome, you need to do it cheaply, and you need to do it quickly," explained Xinsheng Sean Ling, professor of physics, who joined the Brown faculty in 1996.

"This is a step in that direction."

The idea of reading DNA by threading strands through tiny openings is not new. Scientists have shown that an applied electric field can drive the DNA molecules through a nanopore, a tiny hole in a membrane. But in those experiments, the base pairs moved too quickly through the openings for the code to be read accurately. So, while a large electric field is needed to draw the DNA molecules into the pore, Ling explained, the same field moves the DNA too quickly, a classic scientific Catch-22.

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In this illustration, a DNA strand is leashed to a magnetized, iron-oxide bead, with a magnet hovering over it. As the bead moves toward the magnet, the DNA strand passes through nano-sized opening slowly enough that its base pairs can be read. Credit: Hongbo Peng/IBM Research.

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The trick is to figure out how to slow the strands' movement through the opening so the base pairs (A, T, C, and G) can be read. To solve that, Ling and Hongbo Peng, the lead author who performed the work as a graduate student at Brown and who now works at IBM, attached the DNA strand to a bead using a streptavidin-biotin bond. Like previous researchers, they used an electric field to drive the DNA strand toward the pore. But while the strand could pass through the pore, the bead, with a 2.8-micron diameter, was too large for the pore, which has a diameter of only 10 nanometres. So the bead was stuck in the hole with the attached DNA strand suspended on the other side of the membrane.

The Brown researchers then used magnets — they call them "magnetic tweezers" — to draw the iron-oxide bead away from the pore. As the bead moves toward the magnets, the attached DNA strand moves through the pore — slowly enough so that the base pairs can be read.

The scientists named their process "reverse DNA translocation" because, as Ling explained, "the DNA is essentially caught in a tug-of-war. And the speed of translocation will be controlled not solely by the electric field but by striking some balance between the magnetic and the electric fields. From there, we can tune it to dictate the speed."

The scientists report their technique reduces the average speed of the DNA strand's passage by more than 2,000-fold.

"It can be slower even. There is no limit," Ling said.

A similar experiment has been done using optical tweezers, Ling said, but it involves only one DNA strand at a time. The Brown method sends multiple strands through the nanopores simultaneously.

"It is scalable," Ling said.

The researchers expect to test their technique in experiments using bacterial DNA.
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A New Method for Bone-marrow-derived Liver Stem Cells Isolation

Researchers designed a culture system to isolate, proliferate and differentiate liver stem cells directly from bone marrow cells.
Thursday, 16 April 2009

Great interest has been aroused in the identification and isolation of liver stem cells from bone marrow cells. Several subsets of bone marrow cells have been found to have the potential to differentiate into hepatocytes, however, sorting based on immunological methods is difficult because of the complicated surface markers of the stem cells; furthermore, no report of successful passage has been published.

A research article to be published on April 7, 2009 in the World Journal of Gastroenterology addresses this question. The research team led by Dr. Yun-Feng Cai and his colleagues from the Affiliated Foshan Hospital and the Second Affiliated Hospital of Sun Yat-sen University established a carefully designed culture system to isolate, proliferate and differentiate liver stem cells directly from bone marrow cells, and they were able to achieve six passages of the stem cells. The results suggest that BDLSCs can be purified and passaged (proliferated).

The selecting culture system that contains cholestatic serum can purify BDLSCs directly from bone marrow cells, which provides an easy method to separate stem cells, by avoiding complicated immunological manipulation. The successful passage of the stem cells further verifies the proliferating ability of the cells, although the passage is limited, and further research will provide more experience.

In this study, the authors used their original method to retrieve the cells, which are possibly BDLSCs. Then, they used fluorescence-activated cell sorting to determine the cells' characteristics before and after differentiation. This is an interesting and potentially important study, which suggests that bone-marrow-derived cells can be stimulated to expand and then differentiate into hepatocyte-like cells, which can possibly be used to treat liver disease.

Reference:
Passage of bone marrow-derived liver stem cells with a proliferating culture system
Yun-Feng Cai, Ji-Sheng Chen, Shu-Ying Su, Zuo-Jun Zhen, Huan-Wei Chen
World J Gastroenterol 2009, 15(13): 1630-1635
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Stem Cells Promising for Type 1 Diabetes

Stem cell transplantation helps patients with diabetes become insulin free
Wednesday, 15 April 2009

The majority of patients with type 1 diabetes who underwent a certain type of stem cell transplantation became insulin free, several for more than three years, with good glycemic control, and also increased C-peptide levels, an indirect measure of beta-cell function, according to a study in the April 15 issue of JAMA, a theme issue on diabetes.

Richard K. Burt, M.D., of the Northwestern University Feinberg School of Medicine, Chicago, presented the findings of the study at a JAMA media briefing.

Clinical evidence indicates that there is an inverse association between beta-cell (a type of cell in the pancreas that secretes insulin) preservation and function and chronic complications of type 1 diabetes mellitus (DM). The higher the C-peptide levels (a by-product of insulin production, made up of amino acids), the lower the incidence of some types of complications of type 1 DM. A previous study found that autologous nonmyeloablative hematopoietic stem cell transplantation (HSCT) in 15 patients with newly diagnosed type 1 DM resulted in the majority of patients becoming insulin free during the follow-up, which averaged about 19 months.

"However, it was suggested that subsequent insulin independence was a prolonged honeymoon period due to dietary and exercise changes associated with close post-transplant medical observation," the authors write, and it was not known if this change was because of an improvement in beta-cell preservation.

HSCT, which uses a patient's own blood stem cells, involves the removal and treatment of the stem cells, and their return to the patient by intravenous injection.

Dr. Burt and colleagues conducted a study to determine if post-transplant insulin independence was due to improved beta-cell function by monitoring the C-peptide levels of 23 patients who underwent stem cell transplantation. The patients, with type 1 DM, were ages 13-31 years.

Of the 23 patients, 20 experienced time free from insulin (12 continuously and 8 transiently). Patients remained continuously insulin free for an average time of 31 months (range, 14-52 months). One patient had more than 4 years with no exogenous (produced outside the body) insulin use, 4 patients for at least 3 years, 3 patients for at least 2 years, and 4 patients for at least 1 year. Eight patients relapsed and resumed insulin use at low doses. The majority of patients achieved good glycemic control.

In the continuously insulin-free group, average area under the curve (AUC; a type of measurement) of C-peptide levels before transplantation (225.0 ng/mL per 2 hours) showed a significant increase at 24 months after transplantation (785.4 ng/mL per 2 hours) and at 36 months after transplantation (728.1 ng/mL per 2 hours). In the transient insulin–independent group, average AUC of C-peptide levels also increased from 148.9 ng/mL per 2 hours pre-transplantation to 546.8 ng/mL per 2 hours at 36 months, which was sustained at 48 months. In this group, 2 patients regained insulin independence after treatment with the antihyperglycemic drug sitagliptin, which was associated with an increase in C-peptide levels.

Two patients developed pneumonia in the hospital, 3 patients developed late endocrine dysfunction, and 9 patients developed oligospermia (sperm deficiency). There were no deaths.

"In conclusion, autologous nonmyeloablative HSCT was able to induce prolonged and significant increases of C-peptide levels associated with absence of or reduction of daily insulin doses in a small group of patients with type 1 DM," the researchers write.

"At the present time, autologous nonmyeloablative HSCT remains the only treatment capable of reversing type 1 DM in humans. Randomized controlled trials and further biological studies are necessary to confirm the role of this treatment in changing the natural history of type 1 DM."

Reference:
C-Peptide Levels and Insulin Independence Following Autologous Nonmyeloablative Hematopoietic Stem Cell Transplantation in Newly Diagnosed Type 1 Diabetes Mellitus
Carlos E. B. Couri, Maria C. B. Oliveira, Ana B. P. L. Stracieri, Daniela A. Moraes, Fabiano Pieroni, George M. N. Barros, Maria Isabel A. Madeira, Kelen C. R. Malmegrim, Maria C. Foss-Freitas, Belinda P. Simões, Edson Z. Martinez, Milton C. Foss, Richard K. Burt, Júlio C. Voltarelli,
JAMA. 2009;301(15):1573-1579

This release is also available in Chinese:
干细胞移植可帮助I型糖尿病患者摆脱长期的胰岛素依赖性并改善其β细胞的功能

据4月15日刊JAMA上发表的一项研究披露（本期为有关糖尿病的专刊），对那些接受了某种类型干细胞移植的I型糖尿病患者来说，其大部分变得不再需要打胰岛素。有些人在超过3年的时间中获得对血糖的良好控制，其C-肽水平（这是对β细胞功能的一种间接的测定方法）也有所增加。
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Way to Jumpstart Bones Healing Process

In-body stem cell therapy has enormous potential for bone injuries
Wednesday, 15 April 2009

Rarely will physicians use the word "miraculous" when discussing patient recoveries. But that's the very phrase orthopaedic physicians and scientists are using in upstate New York to describe their emerging stem cell research that could have a profound impact on the treatment of bone injuries. Results from preliminary work being released today show patients confined to wheelchairs were able to walk or live independently again because their broken bones finally healed.

At the heart of the research is the drug teriparatide, or Forteo, which was approved by the FDA in 2002 for the treatment of osteoporosis. Astute observations led a team of clinicians and researchers to uncover how this drug can also boost our bodies' bone stem cell production to the point that adults' bones appear to have the ability to heal at a rate typically seen when they were young kids.

Baseline research presented in February at the Orthopaedic Research Society meeting revealed that of 145 patients who had an unhealed bone fracture – half of them for six months or longer – 93 percent showed significant healing and pain control after being on teriparatide for only eight to 12 weeks. These findings were enough to convince the National Institutes of Health to fund a clinical trial underway in Rochester, and if the preliminary data are any indication, researchers may have discovered a new, in-the-body stem cell therapy that can jumpstart the body's natural healing process in bones.

The clinical implication is significant, as orthopaedists can soon have a new tool at their disposable to deal with many common, painful bone ailments. These include the tens of thousands of painful fractures for which there is no treatment (pelvic fractures, vertebral compression fractures, and clavicle fractures), fractures that won't heal, fractures in patients that are either too sick to have surgery or chose not to have surgery, and even reduce the size of an incision in some surgeries.

Aging Bones Heal Slower
Of the estimated six million fractures in the United States each year, approximately five percent will have slow or incomplete healing. According to J. Edward Puzas, Ph.D., who heads up orthopaedic bone research at the University of Rochester Medical Center and is the principal investigator of the clinical trial, a large portion of non-healing fractures tend to occur in older adults.

"In many people, as they get older, their skeleton loses the ability to heal fractures and repair itself," Puzas said.

"With careful application of teriparatide, we believe we've found a way to turn back the clock on fracture healing through a simple, in-body stem cell therapy."

Those especially hard hit are the nearly 60,000 Americans suffering from pelvic fractures, where bracing and immobilization are not an option for an injury that leaves people immobile and in pain before the bone fuses.

"It takes three to four months for a typical pelvic fracture to heal. But during those three months, patients can be in excruciating pain, because there are no medical devices or other treatments that can provide relief to the patient," said Susan V. Bukata, M.D., medical director of the Center for Bone Health at the University of Rochester Medical Center.

"Imagine if we can give patients a way to cut the time of their pain and immobility in half? That's what teriparatide did in our initial research."

Bukata said much more was at stake then just comfort and pain relief. Patients who would ordinarily be confined to nursing homes or require additional medical attention because of non-healing fractures might be able to live an independent life. Bukata and Puzas estimate that if this drug saved just one week in a nursing home, it would pay for itself – and beyond.

"Many people don't realize that pelvic fracture carries with them the same mortality as hip fractures – in one year, approximately one-quarter of all older women with pelvic fractures will die from complications," Bukata said.

"And during that year of recovery, a patient typically puts a greater strain on our health care system, not to mention their pain and suffering."

Translational Research at Work
The impetus for the research began in Bukata's clinic, where she saw painful bone fractures in osteoporotic patients quickly heal within a few months of taking teriparatide. At the time, Bukata also served on a research team at the University's Center for Musculoskeletal Research, and she began to advocate that the team direct its efforts in an entirely new direction based on the results she was seeing with patients who were taking teriparatide.

"I had patients with severe osteoporosis, in tremendous pain from multiple fractures throughout their spine and pelvis, who I would put on teriparatide," said Bukata.

"When they would come back for their follow-up visits three months later, it was amazing to see not just the significant healing in their fractures, but to realize they were pain-free – a new and welcome experience for many of these patients."

Puzas and Bukata developed a plan to focus attention in both the lab and clinic to understand if her observations were a fluke or if there was an underlying scientific process producing such life-changing results for patients.

"While we had come to understand how teriparatide builds bone more robustly than the body can on its own, up to that point, we had no clue how the drug would or could help with fracture healing," Puzas said.

Bukata began prescribing teriparatide to patients with non-healing fractures, and was amazed at her findings: 93 percent showed significant healing and pain control after being on teriparatide for only eight to 12 weeks. And in the lab, Puzas began to understand how teriparatide stimulates bone stem cells into action.

Closing the Gap
When a fracture occurs, a bone becomes unstable and can move back and forth creating a painful phenomenon known as micro-motion. As the bone begins healing it must progress through specific, well-defined stages. First, osteoclasts – cells that can break down bone – clean up any fragments or debris produced during the break. Next, a layer of cartilage – called a callus – forms around the fracture that ultimately calcifies, preventing the bony ends from moving, providing relief from the significant pain brought on by micro-motion.

Only after the callus is calcified do the bone forming cells – osteoblasts – begin their work. They replace the cartilage with true bone, and eventually reform the fracture to match the shape and structure of the bone into what it was before the break.

According to Puzas, teriparatide significantly speeds up fracture healing by changing the behaviour and number of the cartilage and the bone stem cells involved in the process.

"Teriparatide dramatically stimulates the bone's stem cells into action," Puzas said.

"As a result, the callus forms quicker and stronger. Osteoblasts form more bone and the micro-motion associated with the fracture is more rapidly eliminated. All of this activity explains why people with non-healing fractures can now return to normal function sooner."

"The decreased healing time is significant, especially when fractures are in hard-to-heal areas like the pelvis and the spine, where you can't easily immobilize the bone – and stop the pain," Bukata added.

"Typically, a pelvic fracture will take months to heal, and people are in extreme pain for the first eight to 12 weeks. This time was more than cut in half; we saw complete pain relief, callus formation, and stability of the fracture in people who had fractures that up to that point had not healed."

The new clinical research will study post-menopausal women and men over 50 who come to the Emergency Department at Strong Memorial Hospital with a low-energy pelvic fracture. Patients will be divided into two groups – one offered teriparatide, the other a placebo – and followed for 16 weeks to measure the fracture healing process in a variety of ways: pain levels, microscopic bone growth determined through CT scans and functional testing of bone strength, among others.
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Creating Ideal Neural Cells for Clinical Use

New protocol quickly and efficiently differentiates human ESCs into committed neural precursor cells
Tuesday, 14 April 2009

Investigators at the Burnham Institute for Medical Research (Burnham) have developed a protocol to rapidly differentiate human embryonic stem cells (hESCs) into neural progenitor cells that may be ideal for transplantation. The research, conducted by Alexei Terskikh, Ph.D., and colleagues, outlines a method to create these committed neural precursor cells (C-NPCs) that is replicable, does not produce mutations in the cells and could be useful for clinical applications. The research was published on March 13 in the journal Cell Death and Differentiation.

When the C-NPCs created using the Terskikh protocol were transplanted into mice, they became active neurons and integrated into the cortex and olfactory bulb. The transplanted cells did not generate tumour outgrowth.

"The uniform conversion of embryonic stem cells into neural progenitors is the first step in the development of cell-based therapies for neurodegenerative disorders or spinal injuries," said Dr. Terskikh.

"Many of the methods used to generate neural precursor cells for research in the lab would never work in therapeutic applications. This protocol is very well suited for clinical application because it is robust, controllable and reproducible."

Dr. Terskikh notes that the extensive passaging (moving cells from plate to plate) required by some protocols to expand the numbers of neural precursor cells limits the plasticity of the cells, can introduce mutations and may lead to the expression of oncogenes. The Terskikh protocol avoids this by using efficient conversion of hESCs into primary neuroepithelial cells without the extensive passaging.

The scientists were able to rapidly “neuralize” the hESCs by culturing them in small clusters in a liquid suspension. The cells developed the characteristic "rosettes" seen in neuroepithelial cells. The C-NPCs were then cultured in monolayers. Immunochemical and RT-PCR analysis of the cells demonstrated that they were uniformly C-NPCs. Whole-genome analysis confirmed this finding. Immunostaining and imaging showed that the cells could be differentiated into three distinct types of neural cells. The team then demonstrated that the C-NPCs differentiated into neurons after transplantation into the brains of neonatal mice.

About Burnham Institute for Medical Research
Burnham Institute for Medical Research is dedicated to revealing the fundamental molecular causes of disease and devising the innovative therapies of tomorrow. Burnham, with operations in California and Florida, is one of the fastest-growing research institutes in the country. The Institute ranks among the top-four institutions nationally for NIH grant funding and among the top-25 organizations worldwide for its research impact. Burnham utilizes a unique, collaborative approach to medical research. It has established major research programs in cancer, neurodegeneration, diabetes, infectious and inflammatory and childhood diseases. The Institute is known for its world-class capabilities in stem cell research and drug discovery technologies. Burnham is a non-profit, public benefit corporation.

Reference:
Molecular stages of rapid and uniform neuralization of human embryonic stem cells
R Bajpai, G Coppola, M Kaul, M Talantova, F Cimadamore, M Nilbratt, D H Geschwind, S A Lipton, A V Terskikh
Cell Death and Differentiation, 13 Mar 2009, doi: 10.1038/cdd.2009.18
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microRNAs Turn Adult Cells to Embryonic

UCSF team closer to creating safe embryonic-like stem cells
Monday, 13 April 2009

A team of UCSF researchers has for the first time used tiny molecules called microRNAs to help turn adult mouse cells back to their embryonic state. These reprogrammed cells are pluripotent, meaning that, like embryonic stem cells, they have the capacity to become any cell type in the body.

The findings suggest that scientists will soon be able to replace retroviruses and even genes currently used in laboratory experiments to induce pluripotency in adult cells. This would make potential stem cell-based therapies safer by eliminating the risks posed to humans by these DNA-based methods, including alteration of the genome and risk of cancer.

"Using small molecules such as microRNAs to manipulate cells will play a major role in the future of stem cell biology," says senior author Robert Blelloch, MD, PhD, of the Eli and Edythe Broad Center for Regeneration Medicine and Stem Cell Research at UCSF.

Scientists are interested in reprogramming because it would offer a way to create cells that provide a genetic match for individual patients. A patient's skin cells could be reverted to pluripotent cells in the culture dish and then prompted to differentiate into adult cells, such as those of the heart, lung and brain. These cells could then be transplanted into patients, without the fear of rejection.

The study, reported in the April 12, 2009 advanced online edition of the journal Nature Biotechnology and scheduled for the May 8, 2009 print issue, used a combination of microRNAs and retrovirus-introduced genes to transform fibroblast cells — found throughout the body of mice and humans — into pluripotent cells.

The current finding comes on the heels of a study published by the group in the December 2008 print edition of Nature Genetics that showed that microRNAs, which can be synthesized in the lab, encouraged embryonic stem cells to self-replicate, a finding that has implications for replicating stem cells in the culture dish and exploring stem cells' role in cancers.

Previous methods for creating embryonic stem cell-like cells have relied on the introduction of DNA that encodes four transcription factors, proteins that play a role in the production of genes. The limitation of this method is that three of the four genes that code for these transcription factors — oct4, klf4 and c-myc – are oncogenes, meaning they promote the uncontrolled cell growth characteristic of cancer.

In the current study, led by Robert Judson, a graduate student in the Blelloch lab, the scientists induced pluripotency using a combination of infection and transfection. The infection involved introducing three viruses, each containing a transcription factor known to induce pluripotency. The transcription factor for c-myc was not included. The transfection involved a simple process in which the tiny microRNA molecules were mixed with a lipid, allowing them to pass through the cell membrane. By labelling the fibroblast cells, they showed that the treated cells could be incorporated into a mouse embryo and become every cell type in the adult animal — including germline cells that would produce the next generation of mice.

"These are transient, non-coding molecules that do not incorporate into the genome, but promote self-replication and have the potential to induce pluripotency," Blelloch says.

"They do their thing — turn a somatic cell into an embryonic stem cell-like one — and then they're gone."

"MicroRNAs give us a new tool to manipulate the fate of cells," Blelloch says.

MicroRNAs are snippets of single-stranded RNA that prevent a gene's code from being translated from messenger RNA into protein. They were debuted in 1993, when scientists reported the discovery of a microRNA in the microscopic roundworm C. elegans. Since then, the field has "exploded," says Blelloch, with hundreds of microRNAs discovered in the last eight years across a broad range of species, from plants to animals.

Produced in the nucleus and released into the cytoplasm, microRNAs home in on messenger RNAs that share part of their genetic sequence. When they find them, they latch on, preventing the messenger RNA from being processed by the protein-making machines known as ribosomes. As such, microRNAs are able to ratchet down a cell's production of a given protein.

Currently, Blelloch and his colleagues are working to replace all four transcription factors with microRNAs and conducting experiments that will reveal the mechanism by which these small molecules are able to induce pluripotency. The team will also be looking to determine which microRNAs might be able to turn adult cells directly into particular adult cell types, by-passing the embryonic stem cell-like stage altogether.

"The goal now is to ensure the safety of induced pluripotent stem cells and to differentiate them into cells that can be used to repair damaged tissue and treat disease," he says.

Reference:
Embryonic stem cell–specific microRNAs promote induced pluripotency
Robert L Judson, Joshua E Babiarz, Monica Venere & Robert Blelloch
Nature Biotechnology Published online: 12 April 2009, doi:10.1038/nbt.1535
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Human ESCs Progress Slowly in Myelin’s Direction

Human ESCs Progress Slowly in Myelin’s Direction
Thursday, 09 April 2009

Scientists from the University of Wisconsin, USA, report in the journal Development the successful generation from human embryonic stem cells of a type of cell that can make myelin, a finding that opens up new possibilities for both basic and clinical research.

The cells the researchers made are called oligodendrocytes, which are responsible for making myelin in the central nervous system. Myelin forms an insulating sheath that surrounds nerve fibres, both protecting them and speeding up the transmission of nerve impulses. Its loss or damage has serious consequences, as is seen in the condition of multiple sclerosis, because without it nerves lose the ability to transmit impulses to each other and to function properly.

Unlike human embryonic stem (ES) cells, it is relatively easy to persuade mouse ES cells to turn into oligodendrocytes; it is often done by exposing these cells to a protein called Sonic Hedgehog, which produces oligodendrocytes in the spinal cord of developing embryos. Now Su-Chun Zhang and his co-workers show in the May issue of Development that treating human ES cells with this same protein also turns them into oligodendrocytes – they just take longer to do it, 14 weeks as opposed to the 2 weeks taken by mouse ES cells. They also report another difference between mouse and human ES cells: a growth factor called Fgf2 that promotes oligodendrocyte development in mouse ES cells actually stalls it in human ES cells.

As Dr Zhang reveals, these findings were quite unexpected.

“This was quite a surprise given that this is exactly how we direct mouse ES cells to become oligodendrocytes. But we have discovered an unexpected twist in the cell’s response to the same external factor”, explained Dr Zhang.

“It nevertheless explains why so many research groups have failed to persuade human neural stem cells to become oligodendrocytes for the past decade.”

As Dr Zhang went on to discuss, these findings are also of clinical importance.

“We are now able to generate a relatively enriched population of oligodendrocyte precursor cells that may be used to repair lost myelin sheaths. These findings also raise awareness of the direct translatability of animal studies to human biology. In this regard, the human oligodendrocytes generated from human ES cells or the generation of disease-induced pluripotent stem cells can provide a useful tool in the future for screening pharmaceuticals directly on human cells.”
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Stem Cell Therapy Makes Cloudy Corneas Clear

Stem Cell Therapy Makes Cloudy Corneas Clear
Thursday, 09 April 2009

Stem cells collected from human corneas restore transparency and don't trigger a rejection response when injected into eyes that are scarred and hazy, according to experiments conducted in mice by researchers at the University of Pittsburgh Schools of Medicine. Their study will be published in the journal Stem Cells and appears online today.

The findings suggest that cell-based therapies might be an effective way to treat human corneal blindness and vision impairment due to the scarring that occurs after infection, trauma and other common eye problems, said senior investigator James L. Funderburgh, Ph.D., associate professor, Department of Ophthalmology. The Pitt corneal stem cells were able to remodel scar-like tissue back to normal.

"Our experiments indicate that after stem cell treatment, mouse eyes that initially had corneal defects looked no different than mouse eyes that had never been damaged," Dr. Funderburgh said.

The ability to grow millions of the cells in the lab could make it possible to create an off-the-shelf product, which would be especially useful in countries that have limited medical and surgical resources but a great burden of eye disease due to infections and trauma.

"Corneal scars are permanent, so the best available solution is corneal transplant," Dr. Funderburgh said.

"Transplants have a high success rate, but they don't last forever. The current popularity of LASIK corrective eye surgery is expected to substantially reduce the availability of donor tissue because the procedure alters the cornea in a way that makes it unsuitable for transplantation."

A few years ago, Dr. Funderburgh and other University of Pittsburgh researchers identified stem cells in a layer of the cornea called the stroma, and they recently showed that even after many rounds of expansion in the lab, these cells continued to produce the biochemical components, or matrix, of the cornea. One such protein is called lumican, which plays a critical role in giving the cornea the correct structure to make it transparent.

Mice that lack the ability to produce lumican develop opaque areas of their corneas comparable to the scar tissue that human eyes form in response to trauma and inflammation, Dr. Funderburgh said. But three months after the lumican-deficient mouse eyes were injected with human adult corneal stem cells, transparency was restored.

The cornea and its stromal stem cells themselves appear to be "immune privileged," meaning they do not trigger a significant immune response even when transplanted across species, as in the Pitt experiments.

"Several kinds of experiments indicated that the human cells were alive and making lumican, and that the tissue had rebuilt properly," Dr. Funderburgh noted.

In the next steps, the researchers intend to use the stem cells to treat lab animals that have corneal scars to see if they, too, can be repaired with stem cells. Under the auspices of UPMC Eye Center's recently established Center for Vision Restoration, they plan also to develop the necessary protocols to enable clinical testing of the cells.
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Stem Cell Therapy Grows New Blood Vessels

Purified bone marrow cells hone in on the area of ischemia
Monday, 06 April 2009

Research led by Dr. David Hess of the Robarts Research Institute at The University of Western Ontario has identified how to use selected stem cells from bone marrow to grow new blood vessels to treat diseases such as peripheral artery disease. It is one of the severe complications often faced by people who have had diabetes for a long time. Reduced blood flow (ischemia) in their limbs can lead to resting pain, trouble with wound healing and in severe cases, amputation. The research is published in Blood.

Hess drew human bone marrow and simultaneously isolated three different types of stem cells that co-ordinate together to form new blood vessels. These are called pro-angiogenic stem cells. They were purified to remove any inflammatory or contaminated cells, and then injected into the circulation of mice, which had one of their leg arteries ligated and removed. The researchers showed how these stem cells have a natural ability to hone in on the area of ischemia to induce blood vessel repair and improve blood flow. Hess says this research is clinically-applicable because they studied the function of human stem cells in immune-deficient mice.

The preclinical data from Hess' research was used by a biopharmaceutical company, Aldagen to receive FDA approval for a multi-centre clinical trial now underway in Houston, Texas, involving 21 patients with end-stage peripheral artery disease.

"We can select the right stem cells from the patient's own bone marrow and put them back in the area of ischemia to allow these cells to coordinate the formation of new blood vessels." says Hess, a professor in physiology and pharmacology at Western's Schulich School of Medicine & Dentistry.

"These principles could be applied not only to ischemic limbs, but to aid in the formation of new blood vessels in ischemic tissue anywhere in the body, for example after a stroke or heart attack."

Reference:
Revascularization of ischemic limbs after transplantation of human bone marrow cells with high aldehyde dehydrogenase activity
Benjamin J. Capoccia, Debra L. Robson, Krysta D. Levac, Dustin J. Maxwell, Sarah A. Hohm, Marian J. Neelamkavil, Gillian I. Bell, Anargyros Xenocostas, Daniel C. Link, David Piwnica-Worms, Jan A. Nolta, and David A. Hess
Blood, published online March 26, 2009; DOI 10.1182/blood-2008-04-154567
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Stem Cell Innovation at Risk in UK

Survey of the Cell Therapy Industry and the Main Products in UK
Monday, 06 April 2009

Despite great hopes for stem cell therapy, major structural and cultural changes within the NHS are needed if it is to succeed in the UK. Currently the chances of getting effective treatments into routine use in the short-term are small and the industry is at serious risk of 'market failure'.

These are the findings of two major studies into the commercialisation and adoption of stem cell therapy carried out by researchers at The

University of Nottingham.

Dr Paul Martin, from the Institute of Science and Society said:

"While the government has identified regenerative medicine as a national priority and the US has lifted its ban on stem cell therapy, urgent public policy action is needed if it is to become a reality. Although cell therapy is now established as an important branch of medicine, innovative firms struggle to make money, putting the UK industry in a very vulnerable position in the short term. Unless the situation changes the industry will contract and the progress needed to develop important cell therapies will be adversely affected."

The research, funded by the Engineering and Physical Sciences Research Council (EPSRC) identified a number of important barriers to knowledge translation. It found that closer collaboration with clinicians was needed along with better funding for clinical studies, greater regulatory certainty and clearer reimbursement policies. There is also a need to develop enabling technologies to lower manufacturing costs.

Commercial activity in cell therapy has grown very significantly since 2002. The industry now involves nearly 200 companies developing primary and secondary cell therapies, plus another 180 banking cord blood. In total the global cell therapy industry currently has sales of over $1 billion a year and a steady number of products are now reaching late stage clinical trials. However, the sector suffers from a high level of company turn over. As a consequence, the industry is dominated by small, young companies lacking the resources to bring products easily and successfully to market and those that do struggle to make sales.

Dr Martin, whose expertise lies in the sociology of emerging medical technologies, said:

"There are major structural barriers within the NHS that make it difficult to translate new scientific knowledge of stem cells into improved patient care. For a clinician to use a cell therapy routinely it needs to meet a number of strict criteria. They are also expensive and many are yet to have proven clinical outcomes."

The reports are the result of a two-year study examining the UK regenerative medicine sector. They have been published ahead of the second National Stem Cell Network's Annual Scientific Conference, which is being held at Oxford University on Monday 6 April 2009 to Wednesday 8 April 2009. The conference attended by leading experts in the field is a celebration of the latest in UK stem cell science.

References:
The Commercial Development of Cell Therapy – Lessons for the Future?
Survey of the Cell Therapy Industry and the Main Products in Use and Development
Paul Martin, Ruth Hawksley and Andrew Turner, April 2009

Barriers to the Commercialisation & Utilisation of Regenerative Medicine in the UK
Emma Rowley and Paul Martin, April 2009
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For more on stem cells and cloning, go to CellNEWS at
http://cellnews-blog.blogspot.com/ and
http://www.geocities.com/giantfideli/index.html

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Human Embryonic Stem Cells Make Complex Oral Tissue

Tissue engineering principles used to produce complex oral-lining tissues from hESCs
Saturday, 04 April 2009

Human embryonic stem cells (hESC) provide a potentially unlimited source of oral mucosal tissues that may revolutionize the treatment of oral diseases. When fully exploited in the future, this source of cells will be able to produce functional tissues to treat a broad variety of oral diseases. However, little is known about how hESC can be developed into complex, multilayer oral tissues that line the gums, cheeks, lips, and other intra-oral sites. However, the use of hES cells for oral application faces numerous obstacles that must be overcome before their therapeutic potential can be realized.

Today, during the 87th General Session of the International Association for Dental Research, investigators from Tufts University in Boston report on their research to optimize the potential of hESC cells to generate complex, functional multilayer tissues, such as the oral mucosa and skin, and to understand how tissue fabrication is controlled and directed.

The Garlick lab has used tissue engineering principles to produce complex oral-lining tissues that mimic many features of their counterparts found in the oral cavity. Making these tissues was a two-step process. With a combination of chemical signals and specialized surfaces on which these cells attach, an hESC cell line (H9) was directed toward two divergent cell populations. The first population comprises the surface layer (epithelial cells) of complex tissues, while the other is found beneath these cells (mesenchymal cells). Following their isolation and characterization, the team incorporated these two distinct cell populations into the two tissue compartments that comprise these tissue types. The populations were then grown at an air-liquid interface to mimic their growth environment in the oral cavity. Within two weeks, tissues developed that shared many features in common with normal tissues that were constructed with mature cells that are the "gold standard" of normal tissue generation in our lab.

For the first time, researchers have established proof of concept that a single, common source of pluripotent hESC could provide the multiple cell types needed to be recombined within different, but interactive, tissue compartments to generate complex, multilayer tissues. In addition to providing oral mucosal tissues for future transplantation, the tissues generated in these studies can now be used to answer questions regarding the stability and safety of hESC-derived cells and tissues by providing information that will predict how they will respond after therapeutic transplantation in the future.
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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/ and
http://www.geocities.com/giantfideli/index.html

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