S3‐02‐03: Modelling Microglia with Human Pluripotent Stem Cells

Background:Our aging society is confronted with a dramatic increase in patients suffering from dementias, such as Alzheimer’s disease (AD), for which no mechanism-based cures are available. Animal models, although useful for understanding aspects of AD pathology, do not capture key aspects of the disease and have limited use in developing treatments. A human AD model that reproducibly develops disease-relevant molecular pathology in cortical neurons would enhance functional studies and improve screening for drug development. Recent advances in stem cell research have allowed reprogramming of mutant patient-derived cells to induced pluripotent stem cells (iPSCs) and subsequent differentiation into cortical neurons. Although first studies using this approach have shown promising results for AD, the field still lacks optimized technology for generating isogenic lines to confirm phenotype specificity. CRISPR/Cas9 has recently been developed into a versatile gene editing tool holding promise for generating models of human diseases, e.g. in iPSCs. Although CRISPR/Cas9 is used extensively to engineer gene knock-outs, editing cells by homology-directed repair (HDR) to introduce or gene-correct disease-associated mutations remains inefficient and inaccurate. Furthermore, targeted mutation introduction at single alleles, to model diseases caused by heterozygous mutations, such as early-onset Alzheimer’s disease (EOAD), has not been reported. Methods:We developed a CRISPR/Cas9-based genome-editing framework that allows selective introduction of monoand bi-allelic sequence changes with high efficiency, accuracy and predictable control of zygosity. Homozygous introduction requires using a guide RNA targeting close to the intended mutation, whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by using mixed repair templates. Results: Using this approach, we generated the first human induced pluripotent stem cells (iPSCs) with heterozygous and homozygous dominant EOAD mutations in amyloid precursor protein (APPSwe) and presenilin 1 (PSEN1M146V). We then differentiated mutant iPSCs into disease-relevant cortical neurons to study phenotypic changes in the neurons affected in the patients. In doing so we found distinct genotype-dependent diseaseassociated phenotypes, particularly in the pathological amyloidogenic processing of APP and related molecular downstream changes. Conclusions:Taken together, our findings not only enable efficient introduction of disease-associated mutations with CRISPR/Cas9, but shed light onto the molecular mechanisms underlying human dementia.