The ability to target a specific locus in the mouse genome and to alter it in a specific fashion has fundamentally changed experimental design and made mice the preeminent model for studying human diseases . However, pathogenesis in humans have unique pathways that may not be revealed by only using mouse or other animal models. An approach that combines the advantages of mouse models with parallel experiments in human embryonic stem cells (hESCs) offers significant advantages over current methodologies. With the large number of hESC lines available, the ability to grow cells in defined media, the development of drug resistant feeders and the reports of strategies to insert DNA with increasing efficiency into hESC, it would only be a matter of time to obtain homologous recombinants in hESCs.
In order to provide direct clues to pathogenesis in human tissues, we propose to use homologous recombination to establish in vitro human disease models in hESCs. As a proof of principle, we have chosen Lou Gehrig's disease (or amyotrophic lateral sclerosis, ALS). ALS is a disease that progressively and selectively attacks motoneurons in the brain and the spinal cord. It becomes fatal when motoneurons controlling breathing are affected. Approximately 2% of ALS cases have been identified to be caused by mutations of the the Cu-Zn superoxide dismutase (SOD1) gene in an autosomal dominant trait. Animal models have been established and researchers have been able to propose disease mechanisms which led to potential treatments, although no cure has been offered yet. This in part might be due to lack of human cell based models and varied mutant copy numbers in transgenic animals as well as the random nature of their integration into the genome.
Here, we propose to generate hESC lines by gene targeting to harbor point mutations in the SOD1 gene, which recapitulates the genetic defects in SOD1 mutated ALS patients. We will further target these mutations in hESC reporter lines of the two important cell types in ALS: motoneurons and astrocytes. The availability of these SOD1 mutated hESC and hESC reporter lines will allow researchers to obtain purified “diseased” motoneurons and astrocytes, which will facilitate the dissection of ALS pathogenesis. The completion of this proposal will provide (1) a highly efficient protocol for performing homologous recombination in hESCs, (2) a package of motoneuron and astrocyte reporters which are useful for both disease and developmental studies along the neural lineages, and (3) a set of ALS disease platforms of hESC lines to serve as an hESC ALS disease in vitro model, as well as a virtually unlimited source of “diseased” motoneurons and astrocytes. This work not only will provide tools to move pathogenesis research for ALS, but also can be reliably extended into other neural and non-neural lineage diseases, of which genetic defects have been identified, including Huntington's disease (HD) and Parkinson’s disease (PD).
The overall objectives for this proposal are to create in vitro human neurodegenerative disease models using human embryonic stem cells (hESCs), and as a proof of principle, three point mutations of the SOD1 gene which cause familial amyotrophic lateral sclerosis (FALS) will be tested first. These SOD1 missense mutations, G37R, G85R and G93A, have been identified in FALS patients and widely used in rodent models of FALS. We propose to create SOD1 mutations in hESC lines by gene targeting technology which has been proven to be revolutionary in establishing disease models in animals. In addition, we will use similar protocol to generate motoneuron and astrocyte reporter lines in hESCs, since these two cell types and the interaction between them play the most critical roles in the pathogenesis of ALS. After obtaining the three SOD1 missense mutants in motoneuron and astrocyte reporter lines, we will extend our efforts to characterization of these lines, by examining their growth, survival, cell death and other biochemical properties. We will also perform large scale comparisons for genomic and proteomic profiles of the diseased hESC lines with wild type hESCs, as well as comparing the “diseased” and wild type hESC-derived populations of motoneurons and astrocytes.
These experiments will not only provide direct clues for ALS pathogenesis research but also serve as a proof of principle for general disease research using hESCs as a model system. The protocols and reagents developed in this work will be available for Californian researchers and physicians to use for similar neurodegenerative diseases or diseases of other systems. This work will eventually facilitate the scale-up in establishment of human diseases models using human tissues or human cell culture systems for our colleagues in California and around the world.
This proposal focuses on the development of methods to model human neurodegenerative diseases in human embryonic stem cells (hESCs). Specifically, the Principal Investigator (PI) proposes to use homologous recombination (HR) to target and modify the hESC genome to express disease-causing genes. As proof-of-principle, the research team will focus on amyotrophic lateral sclerosis (ALS). In Aim1, they will introduce three common ALS point mutations by HR in hESCs. In Aim 2 they plan to further target these mutations in hESC reporter lines of the two important cell types in ALS: motoneurons and astrocytes. Finally, in Aim 3, they will analyze cell survival, apoptosis and SOD1 protein expression in the SOD1 mutant hESC lines they create. The availability of these SOD1 mutant hESCs and hESC reporter lines will allow researchers to obtain purified “diseased” motoneurons and astrocytes, which offer direct clues to ALS pathogenesis.
The reviewers agreed that this proposal could have a significant impact on the field of stem cell biology. The disease models that exist for neurodegenerative diseases show poor correlation to the human disease state(s). If successful, this proposal would provide an unlimited source of purified “diseased” motoneurons and astrocytes: novel and valuable tools for the study of ALS. Importantly, because the cells would be generated from a normal starting population, the appropriate, genetically identical, controls will be available for comparative experiments. In addition, the HR technology developed in this proposal could easily be applied to other neurodegenerative diseases for which genetic defects have been identified, including Huntington’s and Parkinson’s disease. Finally, a reviewer noted that this approach is complementary to those generating disease models using induced pluripotent stem cells (iPSCs) from ALS patients.
Overall, reviewers found the proposal very well written, carefully designed and ultimately feasible. They commented that the strategies outlined are sensible and achievable and the preliminary data strongly suggests that the team is capable of carrying out gene targeting in hESC cells. However, a few comments were raised concerning the ambitious nature of the proposal and the lack of detail provided in some cases. Reviewers expressed doubts about the timelines presented, which they thought were based on best-case scenarios. Reviewers expect most aspects of the proposal will take considerably longer than stated, particularly in cases where serial gene targeting is required. One reviewer appreciated that the applicants intend to generate independent clones derived from independent parental cell lines, originating from independent laboratories, even while noting that it makes the proposal more ambitious. A reviewer also commented that because the applicants intend to introduce these SOD mutations into the motoneuron and astrocyte reporter lines, the benefit of generating similar SOD mutant lines without reporter genes is unclear and this aspect of the first aim could be eliminated. Another reviewer was frustrated by a lack of experimental detail provided for the third aim. This reviewer questioned how the applicants will measure survival of mutant and wild-type hESCs. This reviewer also commented that the genomic and proteomic analyses are not well defined and no preliminary data for this specific project was presented. This reviewer also noted that the targeting efficiency in the preliminary data from a different knockin line (Olig2-GFP) was low (4-5%) and wondered if it was typical. While the reviewers generally praised the discussion of pitfalls and alternative approaches in the proposal, one felt that the section proposing to use a transgene strategy to generate the desired reporter lines should the HR strategy fail was poorly justified.
Reviewers praised the research team’s molecular and cell biology expertise but noted a lack of experience on neurodegenerative disease. One reviewer strongly recommended including a consultant from the ALS field in this proposal. Reviewers generally found the budget appropriate, although one commented that not all of the proposed rounds of gene targeting on all of the proposed lines are absolutely necessary to demonstrate proof-of-principle (as detailed above) and if some were eliminated the budget would be smaller.
Overall, reviewers found this to be a strong, well-organized proposal that clearly addresses a roadblock in stem cell biology and provides new tools for therapeutic research. They raised a few concerns about it feasibility but were ultimately convinced by the quality of the preliminary data and research team.