A large human stem-cell initiative is underway to help the field explore genetic variants for different forms of dementia. In the April 7 Neuron, Michael Ward, National Institute of Neurological Disorders and Stroke, and Mark Cookson at the National Institute on Aging, both in Bethesda, Maryland, detailed the human induced pluripotent stem cell Neurodegenerative Disease Initiative (iNDI). The researchers will engineer iPSCs from healthy donor cells to carry one of 134 variants associated with either Alzheimer’s disease or a related dementia. After differentiating the cells into neurons and glia, the researchers will characterize their morphology, transcriptomics, and proteomics, then make the data freely available in an open repository. The cells will be cultured and sold by the Jackson Laboratory for Genomic Medicine in Farmington, Connecticut. JAX started in Bar Harbor, Maine, but now operates five locations in the United States and China.
- Each isogenic iPSC line will contain one of 100+ dementia variants.
- Derived neurons and glia will be characterized.
- iNDI will store the open-access data; JAX to distribute cells.
Although many iPSC-based models of dementia are being studied in dozens of labs across the world, there are no standards in the field. “We are trying to break the paradigm of each investigator looking at variants in their own well-characterized system, which is then difficult to compare with others’ models,” Cookson told Alzforum. By creating a series of isogenic cell lines from the same starter cells, scientists will be able to study all the mutations in the same genetic background. “Knocking in a host of different pathogenic mutations that cover AD, FTD, DLB, and ALS against a background of a few standard cell lines originating from healthy donors is a marked improvement in standardization,” Douglas Galasko, University of California, San Diego, wrote to Alzforum (comment below).
John Hardy, University College London, thinks iNDI is a great initiative. “Establishing common cell lines will certainly speed research and reproducibility,” he wrote to Alzforum. Jessica Young, University of Washington, Seattle, said iNDI will open up iPSC research to many investigators. “This resource will be incredibly useful to scientists who want to analyze human data without the burden of setting up a cell culture program,” Young wrote to Alzforum (comment below).
Last year, the NIH created the Center for Alzheimer’s and Related Dementias (CARD), a new research center directed by Andrew Singleton, NIA, to capitalize on cross-disciplinary collaboration between the NIA and NINDS (NIH press release). iNDI is one of four ongoing CARD projects. “This is one of the largest genome engineering initiatives to be done in human stem cells, regardless of disease subtype,” Ward told Alzforum.
iNDI Production. iNDI plans to knock 134 dementia risk variants into iPSCs derived from healthy donor cells. Phase 1 is for creating isogenic homozygous and heterozygous variant cell lines, and distributing them to JAX. Phase 2 is for characterizing the lines. [Courtesy of Ramos et al., Neuron, 2021.]
First author Daniel Ramos and colleagues at CARD aim to build a library of isogenic iPSC lines, each carrying a different dementia risk variant (see image above). They will take a healthy parental line and use CRISPR to introduce one of 134 variants, from 73 genes, that have been associated with one of various types of dementia, including AD, frontotemporal dementia/amyotrophic lateral sclerosis, or dementia with Lewy bodies/Parkinson’s disease dementia.
For now, the researchers have started on a series of cell lines based on donor cells from a healthy white man. Ward said they started with eight lines, which came from either the NIH or the U.K.-based Human iPSC Initiative (HipSci). After weeding out those cells that did not grow robustly, did not properly differentiate into neurons, or had cancerous mutations, they had one cell line with which to move forward. A second line will come from a Caucasian woman; subsequent lines will be from people of diverse backgrounds.
For now, Ramos and colleagues are not disclosing which variants they are introducing into the cells. “We’re still in the engineering process and know some variants will not be easy to incorporate, so we do not want to raise expectations we can’t meet,” Cookson said. However, he did elaborate on the selection process.
The researchers reviewed variants in the Genomics England PanelApp, a public, crowdsourced database of disease genes and variants, and in the Human Gene Mutation Database, a collection of published disease variants in the scientific literature. They chose variants that are well-studied, highly penetrant, and known to be pathogenic or increase risk of dementias. In the end, they selected 134 variants: 65 related to FTD/ALS, 23 to DLB/PDD, 21 to AD, and 25 to other dementias.
For each of these 134, the scientists then used CRISPR to create cell lines from iPSCs from that one male donor. To control for off-target mutations CRISPR may introduce into the variant line, the scientists will correct each of the intended ADRD genetic edits back to wild-type, creating a revertant line. If these revertants behave differently than wild-type control cells, then that will signify that something went awry during the gene editing. “We envision investigators performing experiments in parallel with the wild-type control, the mutant, and the revertant control,” Ward said.
The project is currently in Phase 1, in which the parental line is selected and edited to create six daughter lines. Four of these six are gene-based: heterozygotes, homozygotes, revertants for each of the 134 variants, and knockouts for each of the 73 genes. Two are protein-based: they are cell lines engineered to express each of the 73 genes’ proteins, wild-type and variant forms, with HaloTag, a peptide adjunct used for tracking and protein interaction experiments.
So far, CARD researchers and collaborators at Jackson Lab have created the heterozygous and homozygous lines. The researchers are conducting quality control on those lines and expect them to be available later this year. Within the next 18 months, they expect to complete the revertant, knockout, and Halo-tagged lines. “Now that we have gotten this first series almost done, we anticipate the next rounds to go so much faster,” Ward said.
In Phase 2, all these lines will be characterized. The scientists will differentiate the iPSCs into neurons and glia, then study morphology, cell biology, transcriptomics, proteomics, and gene interactions. The cells that make tagged proteins will show how the mutant proteins interact and where they localize within the cell. The final cell lines will be distributed by Jax.
Researchers interested in obtaining particular cells should email Ward (email@example.com), Cookson (firstname.lastname@example.org), or Singleton (email@example.com). Now is the time to express interest.—Chelsea Weidman Burke
- Ramos DM, Skarnes WC, Singleton AB, Cookson MR, Ward ME. Tackling neurodegenerative diseases with genomic engineering: A new stem cell initiative from the NIH. Neuron. 2021 Apr 7;109(7):1080-1083. PubMed.