Life Technologies, the company formed from the merger of Invitrogen and Applied Biosystems, last week received a two-year, $870,000 grant from the California Institute for Regenerative Medicine to develop disease models for neurodegenerative disorders, especially amyotrophic lateral sclerosis, that use gene targeting in human embryonic stem cells.
The company said its goal is to create a proof-of-principle model to better understand the pathogenesis of ALS in human tissues by using homologous recombination to establish in vitro disease models in hESCs.
Ying Liu, a research scientist at Life Technologies and the principal investigator on the grant, told CBA News this week that she chose ALS because she had previously done some research on astrocytes and motor neurons.
“In addition, in ALS, only one gene, the Cu-Zn superoxide dismutase gene, is mutated,” said Liu. It is therefore easy for people to use ALS as a starting point to see if hESCs can be a disease model at all because, although hESCs have the potential to be a model, “people have not yet determined if that is really possible,” she said.
“If we determine that hESCs can be a good model for ALS, then maybe we can extend the use of hESCs as a model to other” neural and non-neural lineage diseases of which genetic defects have been identified, including Parkinson’s and Huntington’s diseases.
Because drug developers must verify in human cells the data and conclusions that they get from animal models, Liu explained that in the grant proposal she tried to provide three things. “One is a very efficient protocol for homologous recombination in hESCs. Not many people can currently do this, because human ESCs are difficult to culture,” she said. Only a few labs have been able to do this successfully, but with limited efficiency.
Secondly, Liu said she wanted to provide hESC-derived motor neuron and astrocyte reporters for disease and developmental studies.
Third, she wanted to provide a platform of mutated hESC lines to serve as an in vitro model of ALS, and as an unlimited source of “diseased” motor neurons and astrocytes.
“If we determine that hESCs can be a good model for ALS, then maybe we can extend the use of hESCs as a model to other diseases.”
As a proof of principle, three SOD1 missense mutations, G37R, G85R, and G93A, which have been determined to cause familial ALS, will be inserted into hESC lines using gene targeting technology similar to that used to establish rodent models of ALS.
After obtaining the SOD1 missense mutants in astrocyte and motor neuron reporter cell lines, Liu said that she and her colleagues will characterize these cell lines. “I need to build the vector first,” she said.
That will probably take several months, she said, adding that her team had already designed the vector when they wrote the grant.
After building the vector, “I can do the transfection and isolate the cells, grow them, characterize them, and do the differentiation,” Liu said. Then she can start work using those cell populations.
Liu’s CIRM grant comes two weeks after investigators at Harvard University and the Salk Institute published in Stem Cell separate studies of ALS using hESCs (see CBA News, 12/5/08).
In their study, researchers in Fred Gage’s lab at the Salk Institute developed a novel in vitro model of ALS by co-culturing hESC-derived motor neurons with human primary astrocytes expressing mutated SOD1. Simultaneously, the investigators at Harvard University developed a method for the large-scale production of human embryonic stem cell-derived human motor neurons, which they used in co-culture experiments to determine if they were sensitive to the toxic, non-cell-autonomous effect of SOD1-mutated glial cells.
“The difference between my plan and [Gage’s] plan is that he is using primary astrocytes,” Liu explained. She said Gage also over-expressed the mutated SOD1 gene, which is not what happens in patients who have only one copy of the mutated SOD1 gene. This SOD1 overexpression might “sort of exaggerate the function” of the mutated SOD1 gene, Liu said.
“What I am trying to do is put one copy of the mutated SOD1 gene in my astrocytes, which truly reflects what is happening in ALS patients.”
Liu mentioned that the commercialization of this assay has not yet been discussed “in much detail,” although “if we can come up with a highly efficient protocol or a disease model that we can provide to our customers, I think that Life Technologies will be very happy to do that.”