Modeling neurological disorders using induced pluripotent stem cells (iPSCs) and tissue bioengineering
Induced pluripotent stem cell (iPSC) technologies provide powerful tools to study human disease in relevant cell types derived from human sources. Our lab has generated numerous iPSC lines by reprogramming fibroblasts from healthy individuals, as well as from late onset sporadic Alzheimer’s disease (LOAD), early onset familial AD (fAD), and Down syndrome patients. To assess the phenotypic consequences of disease-associated genetic variants, we additionally apply the CRISPR/Cas9 genome-editing technique to create isogenic cell lines, as well as genome-engineering approaches such as the dCAS9 system to examine the impact of non-coding genetic variants of AD on gene expression. Cell-type specific interrogations can be carried out by differentiating iPSCs into the major brain cell types – including excitatory neurons, astrocytes, oligodendrocytes, and microglia – that can be used for a number of basic and applied research purposes. For example, we recently reported the possibility of modeling LOAD caused by the APOE4 allele. Using CRISPR/Cas9-editing to generate two isogenic pairs of iPSC lines – with conversion of APOE3 to APOE4 and conversely, correction of APOE4 to E3 – we demonstrated that APOE4 neurons are hyperexcitable and secrete elevated levels of Aβ42 peptide. In addition, we found that APOE4 astrocytes show increased cholesterol biosynthesis and reduced Aβ uptake, and APOE4 microglia are more prone to inflammation and impaired in Aβ uptake. These results provide strong evidence that APOE4 impacts the function of all major brain cell types to facilitate the development of AD pathology. Furthermore, in illuminating how specific gene perturbations effect AD-like pathology directly in human neurons and glia, these phenotypes can be applied in screening libraries of drug-like chemicals in a high-throughput fashion to determine new potential therapeutic candidates.
In addition to conventional 2D cultures, the Tsai Lab is also developing and utilizing complex culture systems in 3D and with multiple cell types in co-culture. Using techniques of bioengineering combined with multiphoton deep imaging, optogenetics, and electrophysiology, we can recapitulate and study complex in vitro models of human brain tissue. In “mini-brain” or organoid cultures, we can examine neuronal and glial activity and relevant disease phenotypes such as protein aggregation, neuronal connectivity, and synapse loss. For instance, we showed that the organoids derived from fAD iPSC lines – including those harboring APP gene duplication or presenilin mutations – develop extracellular amyloid aggregates and tau hyperphosphorylation following 60 days in culture. These pathological phenotypes could be ameliorated by treating these cultures with beta- and gamma-secretase inhibitors. Current efforts aim to better recapitulate the in vivo brain environment by adding engineered vasculature to our 3D organoids to mimic the blood brain barrier. Ultimately, we hope that these techniques will facilitate and expedite drug screening and discovery by allowing us to use engineered human brain organoids to directly screen for compounds and therapies likely to work in the in vivo human brain.