Now that they can make induced pluripotent stem cells from anyone, scientists are near-giddy with excitement over the potential to model diseases in cell lines, as well as someday correct genetic mutations in people (see ARF news series). But researchers still need a good way to repair disease-causing mutations. In last week’s Cell, a team led by Rudolf Jaenisch at The Whitehead Institute in Cambridge, Massachusetts, report that they have successfully corrected an α-synuclein mutation in induced pluripotent stem (iPS) cells from a person who had Parkinson’s disease. The technology may open the door to better cellular studies and therapies.

“It is a remarkable demonstration of the power of two new technologies—iPS cells and genome editing—to revolutionize our ability to model human diseases,” wrote George Daley of Children’s Hospital Boston in an e-mail to ARF. Daley was not involved in the work.

Patient-specific iPS cells and their derivatives are “the perfect model,” said first author Frank Soldner. Since they are human cells with the same genetic makeup as an affected person, he thinks they will represent disease more accurately than other animal or cellular models. In addition, scientists can observe developmental processes as they differentiate stem cells into neurons, potentially discovering the earliest roots of pathology, he suggested. Researchers could also use these cells to screen potential treatments.

But there is a caveat. “One of the big questions in the field at the moment is, What is the perfect control?” Soldner said. Researchers could compare patient-derived lines with those from unaffected control donors, but there would be a multitude of genetic differences between the two. Beyond the one key genetic change—such as an α-synuclein mutation in a PD patient, or a superoxide dismutase mutation in an amyotrophic lateral sclerosis patient—other genetic modifiers across the genome could affect pathology. “Even if people have defined monogenetic diseases, the age of onset, the severity of the disease are different,” Soldner noted (for review, see Summers, 1996 and also ARF related news story on Kauwe et al., 2008). Plus, even if researchers follow similar protocols to make stem cells, the cell lines often fail to match each other in key characteristics, such as their ability to differentiate into certain cell types (Bock et al., 2011).

Soldner and Jaenisch set out to make the perfect case/control pair—cell lines that are identical twins with the exception of a single, disease-linked mutation. Collaborating with Philip Gregory and others at Sangamo Biosciences, Inc., of Richmond, California, they used the company’s zinc finger nucleases to either fix the mutation in a patient-derived line, or add it into an embryonic cell line. These enzymes consist of a sequence-specific DNA-binding domain plus a nuclease. They work by making a double-stranded break at a specific site in the DNA, activating the cell’s DNA repair machinery. If a donor DNA is present, with a sequence similar to the broken one, there is a good chance the repair complex will use the donor as a template, resulting in a genome with the desired code.

The researchers customized zinc finger nucleases to recognize sequences flanking ones they wanted to change. To edit the genome, they electroporated plasmid DNA for the nucleases, plus a separate donor DNA plasmid with the new, desired sequence, into the stem cells.

Soldner experimented with several methods to add α-synuclein mutations to wild-type human embryonic stem cells. He found the process was most efficient when he employed two selection markers—one to identify cells that adopted the new sequence, and a second to weed out cells that took up extra, undesired chunks of the donor DNA. He was then able to use Cre recombinase to excise the selection markers from his final product, leaving only a loxP Cre recognition site behind.

Not satisfied having a loxP scar on the genome, Soldner refined the technology even further. He dropped the selection markers altogether, instead, transfecting the cells with green fluorescent protein DNA at the same time as he added the nucleases, to cut the DNA, and the donor plasmid to serve as a repair template. He picked out the glowing green cells as the most likely to have taken up the α-synuclein donor DNA, and then confirmed their genotype with sequencing. In his final attempt, Soldner simplified the process further by eliminating the homemade plasmid and using a synthesized oligonucleotide as the donor DNA, along with the same zinc finger nuclease treatment to cut the DNA. With all versions of the procedure, he was able to engineer the genome to his desired sequence.

Having succeeded with embryonic stem cells, Soldner tried the technique in iPS cells from a person with Parkinson’s. Using a donor plasmid and GFP sorting, he was able to correct the α-synuclein mutation. This, indeed, is a “perfect control…a feat of molecular engineering” said Ole Isacson of McLean Hospital in Belmont, Massachusetts, who was not involved in the study.

Isacson and Jaenisch have been collaborating on studies of iPS-based models for Parkinson’s. Isacson said they would soon publish the phenotypes of these lines. In some cases, the pathology is so strong, and so different from even non-twinned control cells, that he thinks minor variations in the genome are unlikely to alter the results. Thus, making the perfect control might not always be necessary, especially for preliminary experiments, Isacson suggested. Of course, this kind of editing will be crucial in repairing iPS cells for therapeutic, autologous transplants.

Zinc finger nucleases may not be the final word on stem cell editing, Isacson added. Jaenisch’s group recently published a method using transcription activator-like effector nucleases (TALENs) (Hockemeyer et al., 2011). Exchanging fingers for TALENs “may make this technology more applicable, and easier and faster,” Isacson said.

As far as fixing and replacing a sick person’s own cells, “It is a long-term goal, but people are starting to think about it,” Soldner said (Daley and Scadden, 2008). Researchers have long hoped to replace dying dopaminergic neurons in people with Parkinson’s (reviewed in Sayles et al., 2004), but Soldner noted that neurodegenerative diseases are unlikely to be the first targets for this kind of therapy; since most cases are idiopathic, doctors would not even know which gene to fix.—Amber Dance


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News Citations

  1. Variations in Tau Gene Linked to Age of Onset for AD

Paper Citations

  1. . Relationship between genotype and phenotype in monogenic diseases: relevance to polygenic diseases. Hum Mutat. 1996;7(4):283-93. PubMed.
  2. . Variation in MAPT is associated with cerebrospinal fluid tau levels in the presence of amyloid-beta deposition. Proc Natl Acad Sci U S A. 2008 Jun 10;105(23):8050-4. PubMed.
  3. . Reference Maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines. Cell. 2011 Feb 4;144(3):439-52. PubMed.
  4. . Genetic engineering of human pluripotent cells using TALE nucleases. Nat Biotechnol. 2011 Aug;29(8):731-4. PubMed.
  5. . Prospects for stem cell-based therapy. Cell. 2008 Feb 22;132(4):544-8. PubMed.
  6. . The cellular repair of the brain in Parkinson's disease--past, present and future. Transpl Immunol. 2004 Apr;12(3-4):321-42. PubMed.

Other Citations

  1. ARF news series

Further Reading


  1. . Induced pluripotent stem cells as a model for accelerated patient- and disease-specific drug discovery. Curr Med Chem. 2010;17(8):759-66. PubMed.
  2. . Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science. 2008 Aug 29;321(5893):1218-21. PubMed.
  3. . Induced pluripotent stem cells and neurodegenerative diseases. Neurosci Bull. 2011 Apr;27(2):107-14. PubMed.
  4. . Characterization of Human Huntington's Disease Cell Model from Induced Pluripotent Stem Cells. PLoS Curr. 2010;2:RRN1193. PubMed.
  5. . Transplantation of human neural stem cells exerts neuroprotection in a rat model of Parkinson's disease. J Neurosci. 2006 Nov 29;26(48):12497-511. PubMed.
  6. . Simultaneous intrastriatal and intranigral fetal dopaminergic grafts in patients with Parkinson disease: a pilot study. Report of three cases. J Neurosurg. 2002 Mar;96(3):589-96. PubMed.
  7. . Cellular replacement therapy for Parkinson's disease--where we are today?. Neuroscientist. 2002 Oct;8(5):457-88. PubMed.
  8. . Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease. Proc Natl Acad Sci U S A. 2008 Apr 15;105(15):5856-61. PubMed.

Primary Papers

  1. . Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations. Cell. 2011 Jul 22;146(2):318-31. PubMed.