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Cellular disease models are useful tools for Alzheimer's disease (AD) research. Pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), are promising materials for creating cellular models of such diseases. In the present study, we established cellular models of AD in hESCs that overexpressed the mutant Presenilin 1 (PS1) gene with the use of a site-specific gene integration system. The overexpression of PS1 did not affect the undifferentiated status or the neural differentiation ability of the hESCs. We found increases in the ratios of amyloid-β 42 (Aβ42)/Aβ40 and Aβ43/Aβ40. Furthermore, synaptic dysfunction was observed in a cellular model of AD that overexpressed mutant PS1. These results suggest that the AD phenotypes, in particular, the electrophysiological abnormality of the synapses in our AD models might be useful for AD research and drug discovery.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative motor neuron (MN) disease. The gene encoding superoxide dismutase 1 (SOD1) is a causative element of familial ALS. Animal ALS models involving SOD1 gene mutations are widely used to study the underlying mechanisms of disease and facilitate drug discovery. Unfortunately, most drug candidates have failed in clinical trials, potentially due to species differences among rodents and humans. It is unclear, however, whether there are different responses to drugs among the causative genes of ALS or their associated mutations. In this study, to evaluate different SOD1 mutations, we generated SOD1-ALS models derived from human embryonic stem cells with identical genetic backgrounds, except for the overexpression of mutant variants of SOD1. The overexpression of mutant SOD1 did not affect pluripotency or MN differentiation. However, mutation-dependent reductions in neurite length were observed in MNs. Moreover, experiments investigating the effects of specific compounds revealed that each ALS model displayed different responses with respect to MN neurite length. These results suggest that SOD1 mutations could be classified based the response of MNs to drug treatment. This classification could be useful for the development of mutant-specific strategies for drug discovery and clinical trials.

The generation of amyotrophic lateral sclerosis (ALS) disease models is an important subject for investigating disease mechanisms and pharmaceutical applications. In transgenic mice, expression of a mutant form of superoxide dismutase 1 (SOD1) can lead to the development of ALS that closely mimics the familial type of ALS (FALS). Although SOD1 mutant mice show phenotypes similar to FALS, dissimilar drug responses and size differences limit their usefulness to study the disease mechanism(s) and identify potential therapeutic compounds. Development of an in vitro model system for ALS is expected to help in obtaining novel insights into disease mechanisms and discovery of therapeutics. We report the establishment of an in vitro FALS model from human embryonic stem cells overexpressing either a wild-type (WT) or a mutant SOD1 (G93A) gene and the evaluation of the phenotypes and survival of the spinal motor neurons (sMNs), which are the neurons affected in ALS patients. The in vitro FALS model that we developed mimics the in vivo human ALS disease in terms of the following: (a) selective degeneration of sMNs expressing the G93A SOD1 but not those expressing the WT gene; (b) susceptibility of G93A SOD1-derived sMNs to form ubiquitinated inclusions; (c) astrocyte-derived factor(s) in the selective degeneration of G93A SOD1 sMNs; and (d) cell-autonomous, as well as non-cell-autonomous, dependent sMN degeneration. Thus, this model is expected to help unravel the disease mechanisms involved in the development of FALS and also lead to potential drug discoveries based on the prevention of neurodegeneration.

Low efficiencies of gene targeting via homologous recombination (HR) have limited basic research and applications using human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Here, we show highly and equally efficient gene knockout and knock-in at both transcriptionally active (HPRT1, KU80, LIG1, LIG3) and inactive (HB9) loci in these cells using high-capacity helper-dependent adenoviral vectors (HDAdVs). Without the necessity of introducing artificial DNA double-strand breaks, 7–81% of drug-resistant colonies were gene-targeted by accurate HR, which were not accompanied with additional ectopic integrations. Even at the motor neuron-specific HB9 locus, the enhanced green fluorescent protein (EGFP) gene was accurately knocked in in 23–57% of drug-resistant colonies. In these clones, induced differentiation into the HB9-positive motor neuron correlated with EGFP expression. Furthermore, HDAdV infection had no detectable adverse effects on the undifferentiated state and pluripotency of hESCs and hiPSCs. These results suggest that HDAdV is one of the best methods for efficient and accurate gene targeting in hESCs and hiPSCs and might be especially useful for therapeutic applications.