Induced Pluripotent Stem Cells Generated from Patients with ALS

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

John T. Dimos,1* Kit T. Rodolfa,1,2* Kathy K. Niakan,1 Laurin M. Weisenthal,1 Hiroshi Mitsumoto,3,4 Wendy Chung,4,5 Gist F. Croft,4,6 Genevieve Saphier,1 Rudy Leibel,5 Robin Goland,7 Hynek Wichterle,4,6 Christopher E. Henderson,4,6 Kevin Eggan1

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.

1 Harvard Stem Cell Institute, Stowers Medical Institute, Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138, USA.
2 Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
3 Eleanor and Lou Gehrig MDA-ALS Research Center, Neurological Institute, Columbia University Medical Center, New York, NY 10032, USA.
4 Center for Motor Neuron Biology and Disease, Columbia University Medical Center, New York, NY 10032, USA.
5 Division of Molecular Genetics and Naomi Berrie Diabetes Center, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA.
6 Departments of Pathology, Neurology and Neuroscience, Columbia University Medical Center, New York, NY 10032, USA.
7 Department of Medicine and Naomi Berrie Diabetes Center, Columbia University Medical Center, New York, NY 10032, USA.

* These authors contributed equally to this work.


To whom correspondence should be addressed. E-mail: eggan@mcb.harvard.edu

Originally published in Science Express on 31 July 2008
Science 29 August 2008:
Vol. 321. no. 5893, pp. 1218 - 1221
DOI: 10.1126/science.1158799

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

http://www.medicalnewstoday.com/articles/117006.php

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Researchers Report Stem Cell Advance

By Jeffrey Perkel
HealthDay Reporter
September 25, 2008

Researchers report that they have sidestepped a major technical hurdle in the generation of pluripotent stem cells from adult cells.

A team of Boston scientists developed a way to generate induced pluripotent stem cells (iPS) -- which are functionally similar to embryonic stem cells, but which can be produced from adult cells, rather than via the creation or destruction of an embryo -- more safely than ever.

Should the findings, which involved mouse cells, be repeated with humans, they could pave the way for using iPS to delve into the biology of a wide range of genetic diseases. Longer term, they could lead to patient-specific stem-cell therapies.

"I think it's a really important, landmark study," said Kevin Eggan, an assistant professor of Stem Cell and Regenerative Biology and an assistant investigator of the Stowers Medical Institute at Harvard University. He was not involved in the study.

The results were published in the Sept. 25 online edition ofScience.

Shinya Yamanaka, of Kyoto University, Japan, first demonstrated in 2006 that adult mouse cells -- for instance, skin cells -- could be reprogrammed into something akin to an embryonic stem cell by the introduction of four specific genes. According to the lead author of this latest study, Matthias Stadtfeld, that "was like a gigantic, essentially quantum leap for biology." The following year, Yamanaka and James Thomson, of the University of Wisconsin, Madison, demonstrated the same approach could create human iPS cells.

Normally, the four genes -- all of which can induce cancer if left unchecked -- are delivered using retroviruses, which integrate their viral DNA into the cells' chromosomes; the worry is that these random insertions will introduce mutations into the cells that would alter their behavior, thus minimizing the cells' potential usefulness as research tools. Should these cells ever be used to generate tissues that were transplanted into human patients, researchers fear they could inadvertently lead to cancer.

Konrad Hochedlinger, of Massachusetts General Hospital and the Harvard Stem Cell Institute, his postdoctoral fellow Stadtfeld, and their colleagues circumvented this problem by delivering the genes using adenoviruses instead, which do not insert their viral DNA into a cell's chromosomes. iPS cells generated by this new approach appear indistinguishable from other iPS cells, carry some of the molecular hallmarks of embryonic stem cells, and can form multiple cell types when transplanted into mice (that is, they are pluripotent).

"My conclusion is that viral integration is not necessary for reprogramming to a pluripotent state, which is an important step toward safer patient-specific iPS cells, if it can be translated into humans," said Stadtfeld.

Human iPS cells have several potential applications. On a research level, they may be used to study how particular genetic defects lead to disease. Pharmaceutical companies might be able to use these cells to design drugs that alter, circumvent or repair these behaviors. Ultimately, iPS cells could be used clinically to develop patient-specific transplants, for instance, of genetically repaired neurons in patients with neurodegenerative diseases.

Before any of that can happen, however, this new method must be optimized. Only about one in 1 million skin cells actually developed into an iPS by Stadtfeld's method, compared to one in 10,000 using retroviruses. Using liver cells (hepatocytes), the efficiency was about one in 50,000 -- better, but still worse than with retroviruses.

"For this to be translated into humans, we have to find ways to make the process more efficient and properly make it work in cell types which are more easily accessible than hepatocytes, such as skin cells, for example," said Stadtfeld.

Dr. Rudolf Jaenisch, of the Whitehead Institute and Massachusetts Institute of Technology, who studies iPS cells, called the findings "clearly an advance."

"They show that, although very inefficiently, they can get iPS cells, which apparently don't have any genetic alterations," he said.

Though he praised the study, Eggan noted several caveats. First, though adenoviruses do not normally integrate their DNA into the host cell's chromosomes, sometimes they do. "There's always going to be this lingering possibility that it can happen in some cases," he said.

Yet Eggan also said that, given the success of this study, researchers may ultimately find that they can reprogram adult cells into iPS cells without using viruses at all, for instance, with chemicals or by delivering pure RNAs instead.

"All sorts of things like this become more of a reality due to the success of this experiment," he said.

Eggan's second caveat was that, despite their apparent similarity, iPS cells are not embryonic stem cells. It remains an open question as to whether the two are functionally equivalent.

"There are a variety of fundamental issues which haven't been well addressed about the equivalence of iPS and embryonic stem cells," he said, "I cannot say that strongly enough."

For instance, no one has yet been able to grow a mouse from a single iPS cell -- what Eggan calls the "gold standard" assay of embryonic stem cell pluripotency -- and not for lack of trying, he said.

Questions aside, Stadtfeld said he anticipates no problems translating these findings to human cells, aside from the fact that human embryonic stem cell culture is inherently trickier than its murine counterpart.

"I believe it will be a technical hurdle, but I would expect people would take the hurdle in the next half-year or year -- probably half-year," he said. Then he added, "I think maybe people have taken the hurdle already. It's possible."

SOURCES: Matthias Stadtfeld, Ph.D., postdoctoral fellow, Massachusetts General Hospital Cancer Center and Harvard Stem Cell Institute, Boston; Kevin Eggan Ph.D., assistant professor, Stem Cell and Regenerative Biology, and assistant investigator, Stowers Medical Institute, Harvard University; Rudolf Jaenisch, M.D., professor, biology, Whitehead Institute, and Department of Biology, Massachusetts Institute of Technology; Sept. 25, 2008,Science

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In the "second generation" embryonic stem cell technology, they take an adult skin cell, and introduce a small number of genes which direct the "committed" adult skin cell to revert all the way back to an embryonic stem cell.

All cells from a single individual have the same DNA. It's only a matter of controlling which part of the total DNA is active. There is no reason a cell cannot be reprogrammed to return to precisely the state it was in which it was a primitive embryonic stem cell or the original stem cell (the fertilized egg itself).

This technology would have the same genetic material and the same capabilities as the "first generation" embryonic stem cell technology. It would be the same cell as it was at the time it was a newly fertilized egg. It would genetically be an identical twin, a clone of the original fertilized egg, in every sense of the word.

In terms of them being 100% identical, save for four extraneous genes introduced to turn the non-pluripotent skin (somatic) cell back into a true embryonic stem cell, these genes would either silence themselves spontaneously or could be silenced using already available technology (e.g. RNA interference).

RNA interference uses small molecules that are an important regulatory component in the machinery of living cells. It allows scientists to "silence" certain genes. In RNA interference, certain molecules trigger the destruction of RNA from a particular gene, so that no protein is produced. Thus, the gene is effectively silenced.

While adult stem cells can be reprogrammed into embryonic stem cells by the introduction of four specific genes, direct reprogramming carries a theoretical risk of cancer for the recipients of tissue from these cells.

Normally, the four genes are delivered using retroviruses, which integrate their viral DNA into the cells' chromosomes. It seems like they've circumvented this problem by delivering the genes using adenoviruses instead, which do not insert their viral DNA into a cell's chromosomes. The cells then become pluripotent stem cells (iPS cells).

Making induced pluripotent stem cells (iPS cells) without using retroviruses (changing the DNA of their host cells, which can trigger cancer) is a safer way to make iPS cells. Pluripotent means they can become any type of cell.

Scientists used adenoviruses to insert the four genes needed to cause an adult cell to transform into an iPS cell. Adenoviruses do not change the DNA of their host. They go into the nucleus of the host and work directly on the proteins and leave the chromosomes alone (you don't need integration of the virus into the genome to produce iPS cells).

Researchers have also been looking at using chemicals instead of viruses as ways to introduce the four genes needed to make normal cells into iPS cells.

Human iPS cells have many potential uses. In research they could be used to find out how genes trigger diseases like cancer and Parkinson's, and one day there may be drugs tailored to individual patients that cause their own bodies to prevent genetic diseases from being triggered, or even to regenerate damaged tissue (e.g. brain cells damaged by neurodegenerative diseases).

"Induced Pluripotent Stem Cells Generated Without Viral Integration." Matthias Stadtfeld, Masaki Nagaya, Jochen Utikal, Gordon Weir, and Konrad Hochedlinger Science, Published Online September 25, 2008 DOI: 10.1126/science.1162494

http://www.sciencemag.org/cgi/content/abstract/1162494

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Researchers find easier way to make stem cells

Sun Oct 12, 2008
By Maggie Fox, Health and Science Editor

WASHINGTON (Reuters) - Researchers trying to find ways to transform ordinary skin cells into powerful stem cells said on Sunday they found a shortcut by "sprinkling" a chemical onto the cells.

Adding the chemical allowed the team at the Harvard Stem Cell Institute in Massachusetts to use just two genes to transform ordinary human skin cells into more powerful induced pluripotent stem cells or iPS cells.

"This study demonstrates there's a possibility that instead of using genes and viruses to reprogram cells, one can use chemicals," said Dr. Doug Melton, who directed the study published in the journal Nature Biotechnology.

To get these genes into the cells, they have had to use retroviruses, which integrate their own genetic material into the cells they infect. This can be dangerous and can cause tumors and perhaps other effects.

Last month U.S. researchers did the same thing using a harmless virus called an adenovirus, but the method was not efficient. And last week, Shinya Yamanaka of Kyoto University in Japan, who discovered iPS cells in mice, used a loop of genetic material called a plasmid to reformat the cells.

http://www.reuters.com/articlePrint?articleId=USTRE49B2M320081012

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In two papers published in the journal Nature, researchers described how they reprogrammed stem cells using an even safer technique called electroporation. This allowed the scientists to do away with viruses and ferry genes into the cells through pores. Once the genes do their job, they are removed, leaving the cells healthy and intact.

Scientists could make stem cells from adult cells but the cells could never be used in patients because the procedure involved injecting viruses that could cause cancer. Electroporation allows the scientists to achieve the same feat without using viruses, making induced pluripotent stem (iPS) cell therapies a realistic prospect for the first time. The development of iPS cells was heavily dependent on the knowledge previously gained from embryonic stem cell research, which told scientists what the properties of a pluripotent stem cell are.

Electroporation (electropermeabilization) is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. It is a way of introducing a piece of coding DNA. Because the cells can be made from a patient's own skin, they carry the same DNA and could be used without the risk of being rejected by the immune system.

http://www.nature.com/nature/index.html