ARTICLE IN BRIEF
Investigators were able to correct the Huntington's disease (HD) phenotype in in vitro and in vivo models derived from induced pluripotent stem cells from HD patients.
Almost three decades after scientists implicated mutant huntingtin in Huntington's disease (HD), investigators are unraveling new insights about the pathophysiology of the disorder.
In a new development, a team of investigators from multiple institutions — comprising the NINDS-supported HD iPSC (induced pluripotent stem cell) consortium — used stem cell technology to isolate and study human cells with the HD mutation — the pure human form with more than 36 polyglutamine (CAG) repeats.
Consistent results from consortium labs working collaboratively using parallel techniques demonstrate conclusively that the HD cells show neurodegenerative phenotypes remarkably similar to the cell degeneration seen in the brains of human HD patients.
Furthermore, in preliminary studies, they have identified small molecules that can block HD iPSC degeneration and have the potential to be developed into drugs for human therapeutics.
The consortium is licensing the cells to pharmaceutical and biotechnology companies for further experiments.
“Having these cells will allow us to screen for therapeutics in a way we haven't been able to do before in Huntington's,” said Christopher A. Ross, MD, PhD, professor of psychiatry and behavioral sciences, neurology, pharmacology and neuroscience at the Johns Hopkins University School of Medicine, who led the study reported in the June 28 online edition of Cell Stem Cell. “For the first time, we will be able to study how drugs work on human HD neurons and hopefully take those findings directly to the clinic.”
Dr. Ross and Hongjun Song, PhD, a professor of neurology at Hopkins, induced stem cells from the fibroblasts of a 7-year-old girl who suffered from a severe, juvenile form of the disorder. She had developed the first signs of HD at around three. They also programmed stem cells from a healthy volunteer.
The Johns Hopkins team collaborated with other members of the iPSC consortium — Clive Svendsen, PhD, director of the Cedars-Sinai Regenerative Medicine Institute in Los Angeles, and Leslie M. Thompson, PhD, director of the interdepartmental neurosciences program at the University of California, Irvine — who induced similar HD iPSC lines from the fibroblasts of 14 patients with the mutation.
The consortium scientists added growth factors that allowed the cells to become medium spiny neurons, the same cells hard hit in HD. In the lab dish, the neurons showed rapid degeneration compared with the cells made from a healthy person.
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The teams of investigators used microarray profiling and quantitative proteomics to show that the CAG repeat expansion-associated gene and protein expression patterns could separate the patient lines from the controls, and the early onset patients from those who developed the disease later in life.
They conducted an exhaustive series of tests on the cells, including genetics, cell imaging, electrophysiology, metabolism, cell adhesion and cell death assays. They also compared cell lines made from medium and longer CAG repeat expansions. The longer repeats, Dr. Ross said, were more vulnerable to cellular stressors and the withdrawal of brain derived neurotrophic factor (BDNF). This may explain why people with longer CAG repeat expansions develop the disease, on average, earlier than those with shorter CAG repeat expansions, Dr. Ross said.
The investigators also used the iPS cells to make other cell types and used RNA-based technology to knock down the HD mutant and thus eliminate the phenotype.
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“The results in the paper represent a truly international effort to utilize iPSC technology to further our understanding of HD, and begin to determine if CAG repeat length or other factors contribute to phenotype severity,” said Margaret Sutherland, PhD, a program director at the NINDS. “The consortium strategy enabled the same iPSC lines to be shared across laboratories where different technologies and assays were applied to identify HD specific phenotypes.
“These experiments suggest that there are clear phenotypes associated with expanded CAG repeats, and that the choice of assay and exact tissue culture conditions will determine the extent of the phenotypic gradation with the length of the CAG expansion that can be detected.”
By and large, scientists have developed cell models of human diseases but such feats have generally failed to represent the appropriate cellular background of disease. iPS-derived cells from patients will advance the field in enormous ways, said Christopher Austin, MD, director of the Division of Preclinical Innovation at the NIH National Center for Advancing Translational Sciences.
“The technology is enabling scientists to make disease-relevant models that can immediately be used to test drugs,” he said. “It is very exciting.”
ANOTHER STEM CELL APPROACH TO UNDERSTAND HUNTINGTON'S
In a June 28 online report in Cell Stem Cell, Lisa Ellerby, PhD, an associate professor at the Buck Institute for Research on Aging in California, and her colleagues used a novel technology to correct the CAG expansion mutation in iPS cells from an HD patient into a normal CAG repeat. The HD iPS-derived cell line had been generated by George Daley, MD, PhD, director of the Stem Cell Transplantation Program and associate director of the Stem Cell Program at Harvard. Again, the HD-related phenotypes disappeared.
Dr. Ellerby's goal was to show that genetic techniques could be used to fix the mutation. The investigators used a targeting vector attached to a normal CAG repeat length and transfected the HD cells. This so-called homologous recombination exchanges the normal length CAG repeat in place of the mutated (expanded CAG) DNA.
The corrected human induced pluripotent stem cells (iPSCs) derived from HD patients persisted in vitro and in vivo. The corrected HD-iPSCs normalized the pathogenic HD signaling pathways — cadherin, transforming growth factor-beta, and caspase activation — and no longer expressed the HD phenotype; they had normal levels of brain-derived neurotrophic factor, which are significantly lower in HD, and mitochondria were also healthy.
Then, they checked the cells to make sure they were still stem cells and proved that there was no alteration to the chromosome itself. “It was a true homologous event,” she said. “The cells were fine and it corrected the HD mutation.”
They are now doing studies to see whether the corrected cells have the ability to rescue the phenotype in a mouse model.
The investigators are also using the HD iPS cells to screen for small molecules. “It is so exciting to see the potential of these technologies,” said Dr. Ellerby. “I was surprised how many cells survived when we put them in the mouse brain and how good the cells looked.”
Commenting on the study, Margaret Sutherland, PhD, program director at the NINDS, said, “It will be of interest to test if there is preferential cell toxicity in neurons expressing striatal markers, as contrasted with other neurons in the culture. This kind of preferential toxicity was seen for dopamine neurons in a 2011 report [in Cell Stem Cell by HN Nguyen, et al.] on iPSCs derived from a patient with a LRRK2 mutation. However, in HD, unlike in Parkinson's disease, there is widespread neuronal dysfunction and death, especially in cases with long repeats, so cell toxicity may not be limited purely to striatal neurons.”
Dr. Sutherland said that an allelic series of HD iPSC lines will be available through the NINDS repository by September.