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Researchers for the first time are able to reprogram cells from sick patients. Though hurdles remain, such cells could be used to help screen drugs to treat the crippling disease.

Scientists have created the first personalized stem cells for patients with a genetic disease by rewinding their skin cells to an embryonic state, according to a study published Thursday in the online edition of Science.

The researchers then converted some of those stem cells into the two kinds of brain cells that cause their crippling disease, amyotrophic lateral sclerosis, commonly known as Lou Gehrig's disease.


Stem cell experts said they were delighted -- though not surprised -- to see proof that the reprogramming technique worked on human cells from sick patients.

Previously, human versions of the so-called induced pluripotent stem cells had only been made from skin samples provided by healthy subjects.

"It is quite amazing and an important step that should allow the development of experimental and therapeutic interventions for this disease," said Kathrin Plath, a researcher at the Broad Center of Regenerative Medicine and Stem Cell Research at UCLA, who was not involved in the study.

The new cells were derived from 3-millimeter patches of skin removed from the arm of an 82-year-old woman and her 89-year-old sister, who share a rare genetic mutation that causes about 2% of ALS cases.

The scientists from Harvard University and Columbia University focused on the rare form of ALS in part to test whether cells from elderly patients could be reprogrammed, said biologist Kevin Eggan of the Harvard Stem Cell Institute.

"This opens the door to being able to make patient-specific stem cell lines from diseases which affect people very late in life, like Parkinson's disease or Alzheimer's disease," said Eggan, the study's senior author.

The team followed a cellular reprogramming recipe pioneered in Japan that has swept through stem cell research labs around the world in the last year. The scientists isolated fibroblast cells from the sisters' skin biopsies and infected them with viruses, prompting the cells to express four dormant genes -- Klf4, Sox2, Oct4 and c-Myc -- that are active during early embryonic development.

The scientists produced eight stable cell lines, and they studied three of them from the 82-year-old woman, whose ALS symptoms were more advanced.

The cells expressed the same markers as embryonic stem cells and were able to grow into all the body's main tissue types.

When the scientists exposed the cells to certain small molecules, the jumbles of tissue began to differentiate into motor neurons, the cells that regulate voluntary muscle movement.

They also found evidence of glial cells, a crucial component of the central nervous system.

ALS is caused by the degeneration of motor neurons, but until now scientists have had no way to take samples from patients and study them in the lab, said Christopher Henderson, a professor of pathology, neurology and neuroscience at Columbia and coauthor of the study. Now he anticipates growing unlimited supplies of motor neurons using the reprogrammed stem cells.

"This is an extremely important resource," said Lucie Bruijn, science director of the ALS Assn. in Calabasas Hills, which was not involved in the study. "It gives you a tool to start screening drugs."

Researchers at Harvard and Columbia are already working to create motor neurons that are genetically matched to healthy people so that they can compare them with the ones derived from ALS patients, Eggan said.

Eventually, the stem cells might be used to create fresh motor neurons that could replace the diseased cells in ALS patients.

But many significant hurdles remain, including finding a way to reprogram the skin cells without relying on viruses or embryonic genes that cause mutations that can lead to cancer.

The research was funded in part by Project A.L.S. and the New York Stem Cell Foundation.

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Scientists have created stem cells from an ALS patient using a new reprogramming method.

Stem cells derived from the skin of an 82-year-old patient with amyotrophic lateral sclerosis (ALS) could provide a novel model for studying the degenerative motor disease and for screening new treatment drugs; eventually, it could pave the way for cell-replacement therapies. The findings, published today online in Science, were made possible by new techniques to reprogram adult cells to become pluripotent--able to become any type of cell in the body.

Researchers have long wanted to make stem cells from actual patients to better understand the diseases from which they suffer. "Because the cells harbor genes that led to the disease in that patient, we might be able to use them in the laboratory to understand certain aspects of disease," says Kevin Eggan, a stem-cell scientist at the Harvard Stem Cell Institute, who led part of the research.

To create the stem cells, researchers used a novel technique, recently developed by scientists in Japan, that doesn't require human eggs or the creation or destruction of embryos, and thus bypasses major ethical and technical hurdles that have plagued the field of embryonic stem-cell research. Eggan's team exposed the patient's skin cells to four genetic factors found in the developing embryo. The procedure turned back the clock on the cells, triggering them to look and behave like embryonic stem cells.

While scientists had already used these reprogramming techniques to create stem cells from skin cells, this is the first time that these cells--called induced pluripotent stem cells, or IPS cells--have been generated from a patient. The ability to do so is key to creating models for studying complex genetic diseases, such as Alzheimer's. The findings also confirm that it's possible to use reprogramming techniques in older people and in those with a serious disease. "It was unclear if the fact that the patient had been sick for many years would interfere with our ability to reprogram [the cells]," says Eggan.

The researchers prodded the stem cells to differentiate into motor neurons by exposing them to another series of chemicals. Motor neurons are the primary cell type destroyed in ALS, a progressive neurodegenerative disease. While animal models of the disease exist, they can't capture the complexity of human biology.

The new research allows scientists to generate an endless supply of motor neurons that are genetically identical to those of the cell donor, which should allow them to study the molecular events that trigger the disease. "Now we can see if they behave in a manner that mimics the disease," says Chris Henderson, codirector of the Motor Neuron Center at Columbia University, in New York, who led part of the research. "For example, do they tend to die and degenerate in the culture dish? If so, we can try to understand more about the mechanism of degeneration." Scientists also hope to use the cells to screen for new drugs that protect against neurodegeneration in ALS.

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Research team creates human ALS motor neurons
NEW YORK - A team of researchers from the Harvard Stem Cell Institute (HSCI) and Columbia University, in a collaboration catalyzed by the Project A.L.S./Jenifer Estess Laboratory for Stem Cell Research, has demonstrated that pluripotent stem cells generated from a patient with ALS (amyotrophic lateral sclerosis) can be directed to differentiate into motor neurons—the very brain cells destroyed by ALS. The results of the team's study appear in today's online issue of Science. This is the first published report to show that disease-specific stem cells may be derived from an individual patient.

In the study, led by Kevin Eggan, of the Harvard Stem Cell Institute, skin cells taken from a patient with a familial form of ALS were induced to become pluripotent stem cells. Scientists then differentiated the pluripotent cells into motor neurons and glia (support cells in the brain) that featured an ALS genotype.

"This is a seminal discovery," said Valerie Estess, director of research for Project A.L.S. "The ability to derive ALS motor neurons through a simple skin biopsy opens the doors to improved drug discovery. For the first time, researchers will be able to look at ALS cells under a microscope and see why they die. If we can figure out how a person's motor neurons die, we will figure out how to save motor neurons."

Starting in 1999, Project A.L.S. recruited leading scientists and clinicians to define the potential role of stem cells in understanding and treating ALS, the fatal neurodegenerative disease, also known as Lou Gehrig's disease. Project A.L.S.-funded scientists began by transplanting stem cells directly into mice with ALS, with limited success. More recent experiments have shown that stem cells may be more valuable as tools to understand the disease process and create mini-representations of disease—or assays--for the purpose of drug screening.

"For the first time, we have the opportunity to examine cellular and molecular defects in motor neurons and glial cells derived from patients with ALS. And we can now begin drug screens on disease-specific classes of human motor neurons," said Thomas Jessell, a Howard Hughes Investigator at Columbia University, and Project A.L.S. advisor. "Through the work of the Jenifer Estess Laboratory for Stem Cell Research we now can glimpse the new age of ALS research, an age of progress and promise."

Co-author on the paper, Christopher Henderson, who is co-director of the Columbia University Center for Motor Neuron Biology and Disease, and senior scientific advisor to the Project A.L.S. Laboratory, said: "It has been a privilege to collaborate with Kevin Eggan and his team and to contribute to this critical step forward. We will continue to work hand-in-hand with Harvard researchers and Project A.L.S. to exploit the potential of these cells for drug screening".

Three years ago, Project A.L.S. asked Dr. Eggan, a stem cell expert, and Chris Henderson, Hynek Wichterle, as authorities on motor neuron biology and drug screening at Columbia University, to work together to understand ALS, one of our most complicated and devastating neurological disorders. Today's publication marks the first major breakthrough of this collaboration.

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Scientists say they have proved the viability of a new way to study diseases using a patient's own cells. Yesterday, a team from Harvard and Columbia universities announced that they have generated a population of motor neurons--nerve cells that control muscle movement--from the skin cells of an 82-year-old woman with amyotrophic lateral sclerosis (ALS). Such cells provide a way to take ALS studies "out of the patient and into the petri dish," Harvard biologist Kevin Eggan said at a press conference.
For many illnesses, researchers would like to study the diseased cells from a patient in the lab; their ultimate hope is that they can also fix those cells by modifying them genetically and then inject them back into the patient. Until recently, scientists believed the way to do this would be through therapeutic cloning, a controversial technique--still unproven for humans--that involves putting the nucleus from a body cell into an enucleated egg. The resulting cells can then be coaxed to differentiate into any bodily tissue type.

The ethical problems of using eggs can be circumvented with induced pluripotent stem (iPS) cells, which are adult cells reprogrammed to behave like embryonic stem cells. In 2006, scientists created such cells from mice and rats by introducing a combination of four genes to a culture of skin cells. Then last year, scientists showed that they could do the same thing with human cells. The new study, published online today by Science, shows that iPS cells can be successfully generated even from the skin cells of an elderly, sick person, Eggan says.

ALS involves the progressive degeneration of spinal cord motor neurons, leading to paralysis of limbs and respiration. The team, led by Eggan and Christopher Henderson of Columbia University Medical Center, grew iPS cells by introducing the four genes used in the earlier studies into about 30,000 skin cells from the patient. Among the hundreds of colonies that grew from these cells, the scientists found that a handful had markers for pluripotency. To these iPS cell lines the scientists added molecules known to guide mammalian pluripotent cells into nerve cells. A significant proportion of them showed markers characteristic of motor neurons. Further tests--such as injecting the cells into mouse or chick embryos to see if they establish proper connections--will be needed to see if they are full-fledged neurons.

The woman had a familial form of ALS caused by a mutation in the gene called superoxide dismutase 1, or SOD1, which is responsible for only 2% of ALS cases. Ninety-five percent of ALS cases are "sporadic"--meaning there is no known inherited mutation; many probably result from genetic changes occurring through life interacting with environmental influences. Nonetheless, said Eggan, "I think this approach has incredible promise for studying other forms of ALS." The symptoms of the familial and the sporadic forms of ALS are so similar, he added, that they probably share many common mechanisms.

"It is exciting that they have generated human cells from the patient material," says stem cell researcher Jeffrey Rothstein of Johns Hopkins University in Baltimore, Maryland, who also studies ALS. But he warns that for the cells to be useful in research, they have to be exactly the same as those causing disease in the patient. "You don't want a partial replicate of the motor neuron," Rothstein says. iPS-generated neurons, he says, may be of little use in replicating the fate of motor cells buffeted by a lifetime of drug exposure and other metabolic and environmental influences.

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Induced Pluripotent Stem Cells Generated from Patients with ALS Can Be Differentiated into Motor Neurons

The generation of pluripotent stem cells from an individual patient would enable the large-scale production of the cell-types affected by that patient’s disease. These cells could in turn be used for disease modeling, drug discovery, and eventually autologous cell-replacement therapies. Although recent studies have demonstrated the reprogramming of human fibroblasts to a pluripotent state, it remains unclear whether these induced pluripotent stem (iPS) cells can be produced directly from elderly patients with chronic disease. We have generated iPS cells from an 82-year-old woman diagnosed with a familial form of amyotrophic lateral sclerosis (ALS). These patient-specific iPS cells possess properties of embryonic stem cells and were successfully directed to differentiate into motor neurons, the cell type destroyed in ALS.

http://www.sciencemag.org/cgi/content/abstract/1158799

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据7月31日的《科学》（Science）杂志报道说，研究人员朝着应用诱导多能干细胞（或称“iPS”细胞）来治疗疾病的目标又迈出了重要的一步。研究人员从来自2位罹患肌萎缩性脊髓侧索硬化症（ALS）这种神经退行性病变的老年病人的皮肤采样中制造出了iPS。而且，他们用这些iPS 细胞发展出了看来像是健康的运动神经元细胞。较早的研究显示，iPS 细胞就像是多能胚胎干细胞那样可以演变为多种类型的细胞，而这些iPS 可以从健康的捐赠者的细胞中产生。但是，这种技术是否也适用于来自罹患慢性疾病的老年人则仍然是一个悬而未决的问题。

John Dimos 及其同事开始对来自两位罹患遗传形式的ALS 病患的皮肤细胞进行实验，并通过加入四种基因将这些细胞转变回iPS 细胞，而这四种基因是研究人员先前用来将细胞程序进行重新设定使其演变为iPS 状态的基因。接着，研究人员将来自其中一名病患的iPS 细胞沉浸在多种信号分子中，设法使这些细胞成为看上去像是运动神经元的细胞，而运动神经元是在ALS 中遭损害的细胞。这一做法的终极希望是：用像这样的iPS 来制造在遗传上相匹配的健康细胞，并用其取代病变的细胞。但是，在将这种方法安全地用于人身上之前，仍然还有重大的障碍需要克服。与此同时，病患特异性的iPS 细胞将是研究ALS 样疾病发生机理的重要工具。在大多数的情况下，ALS是遗传与环境因子间复杂的相互作用的结果，这使得在细胞培养中来研究这种疾病变得非常困难。但是，来自有遗传变异（这些变异使得变异基因携带者容易罹患该种疾病）的病人的iPS 细胞则恰好携带着个体病人中与该疾病有关的“众多”的遗传信息。