Abstract
Alzheimer’s disease (AD) is a chronic brain disorder characterized by progressive intellectual decline and memory and neuronal loss, caused mainly by extracellular deposition of amyloid-β (Aβ) and intracellular accumulation of hyperphosphorylated tau protein, primarily in areas implicated in memory and learning as prefrontal cortex and hippocampus. There are two forms of AD, a late-onset form that affects people over 65 years old, and the early-onset form, which is hereditable and affect people at early ages ~45 years. To date, there is no cure for the disease; consequently, it is essential to develop new tools for the study of processes implicated in the disease. Currently, in vitro AD three-dimensional (3D) models using induced pluripotent stem cells (iPSC)-derived neurons have broadened the horizon for in vitro disease modeling and gained interest for mechanistic studies and preclinical drug discovery due to their potential advantages in providing a better physiologically relevant information and more predictive data for in vivo tests. Therefore, this study aimed to establish a 3D cell culture model of AD in vitro using iPSCs carrying the A246E mutation. We generated human iPSCs from fibroblasts from a patient with AD harboring the A246E mutation in the PSEN1 gene. Cell reprogramming was performed using lentiviral vectors with Yamanaka’s factors (OSKM: Oct4, Sox2, Klf4, and c-Myc). The resulting iPSCs expressed pluripotency genes (such as Nanog and Oct4), alkaline phosphatase activity, and pluripotency stem cell marker expression, such as OCT4, SOX2, TRA-1-60, and SSEA4. iPSCs exhibited the ability to differentiate into neuronal lineage in a 3D environment through dual SMAD inhibition as confirmed by Nestin, MAP2, and Tuj1 neural marker expression. These iPSC-derived neurons harbored Aβ oligomers confirmed by Western Blot (WB) and immunostaining. With human iPSC-derived neurons able to produce Aβ oligomers, we established a novel human hydrogel-based 3D cell culture model that recapitulates Aβ aggregation without the need for mutation induction or synthetic Aβ exposure. This model will allow the study of processes implicated in disease spread throughout the brain, the screening of molecules or compounds with therapeutic potential, and the development of personalized therapeutic strategies.
Highlights
Alzheimer’s disease (AD) is a major degenerative disorder of the central nervous system characterized by continuous neuronal loss mostly in the cerebral cortex and the hippocampus, mainly caused by the accumulations of amyloid plaques and neurofibrillary tangles, which leads to pathological changes in the functional organization and the internal structure of the brain and to progressive loss of memory and cognitive impairment (Sanabria-Castro et al, 2017; Reiss et al, 2018)
Evidence from cellular biology, animal models, and genetics suggests that Aβ protein oligomerization and aggregation play a central role in the initiation and progression of AD
We established two iPSC cultures derived from fibroblasts of healthy and diseased individuals, the latter carrying the PSEN1 mutation A246E, which showed defined morphology, phosphatase alkaline activity, ESC markers, and gene expression and were further expanded for 10 passages, demonstrating that mutated and nonmutated Human fibroblasts (HFs) were successfully reprogrammed and established into iPSC as previously demonstrated (Yagi et al, 2011; Muñoz et al, 2018)
Summary
Alzheimer’s disease (AD) is a major degenerative disorder of the central nervous system characterized by continuous neuronal loss mostly in the cerebral cortex and the hippocampus, mainly caused by the accumulations of amyloid plaques and neurofibrillary tangles, which leads to pathological changes in the functional organization and the internal structure of the brain and to progressive loss of memory and cognitive impairment (Sanabria-Castro et al, 2017; Reiss et al, 2018). Most cases of AD (>95%) are commonly diagnosed in people over 65 years of age, known as late-onset form (LOAD), and is the consequence of the failure of the homeostatic networks in the brain tissue to clear the amyloid-β (Aβ) peptide; there is a less frequent early-onset hereditary form (EOAD; 2–5% of cases), usually caused by an autosomal dominant genetic mutation in three known genes. These genes encode for amyloid precursors protein (APP), presenilin 1 (PS1), and presenilin 2 (PS2), which affect the normal processing of Aβ and cause the development of the disease at early stages (∼45 years; Masters et al, 2015; Cacace et al, 2016). AD represents a significant public health problem and represents an increasing clinical challenge in terms of diagnosis and treatment
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