Directed Differentiation Of Human Induced Pluripotent Stem Cells Through Neurogenin 2 as a Platform for Investigating Alzheimer's Disease Mechanisms

Abstract

Studies involving human neurological diseases are often limited by the availability of patient-derived neurons. While direct differentiation of neurons from fibroblasts or other somatic cells is viable, the directed differentiation of human induced pluripotent stem cells (iPSCs) offers a renewable and scalable source of patient-specific neurons. However, current growth factor-based protocols for iPSC neuronal differentiation are cumbersome and require weeks to yield mature neurons and typically result in mixed cell populations. Thus, the manual isolation of desired neurons not only reduces their yield but also compromises specificity. Moreover, growth factor-induced neuronal differentiation tends to be highly variable, further compromising reproducibility. To overcome these shortcomings, we utilized a rapid single-step induced neuron (iN) methodology from iPSCs. Using a lentiviral delivery system, we induced constitutive tetracycline expression to overexpress exogeneous neurogenin-2 (NGN2) driven by the tetO promoter. The forced NGN2 expression aided in the direct lineage conversion of iPSCs into neuronal cells. The lentiviral construct also encoded eGFP and a puromycin resistance gene to enable both visualization and selection of successfully transduced cells. Within one-week post-transduction, surviving cells exhibited characteristic neuronal morphologies. To better model Alzheimer’s disease (AD), we generated neurons from patient-derived iPSCs with varying polygenic risk scores (PRS) based on ADNI samples. We confirmed the neuronal identity of these induced cells via immunostaining of key neuronal markers, demonstrating the protocol’s robustness and reproducibility. We confirmed that the NGN2-derived neurons were functionally active using multi-electrode array recordings, which showed differences in activity patterns across patient-derived lines. In parallel, molecular testing was used to examine AD-related features, specifically Aβ and pTau, with elevated levels observed so far in lines with high polygenic risk scores. Early findings suggest this model may aid in capturing patient-specific aspects of AD pathology and how genetic changes contribute to disease-related changes in neurons. Standardization of iPSCs-derived induced-neurons protocol will contribute to increasing the yield and specificity of isolated neurons with low cell-to-cell variability, which is necessary for determining disease pathogenesis and drug targets.