A Three-Dimensional Alzheimer’s Disease Cell Culture Model Using iPSC-Derived Neurons Carrying A246E Mutation in PSEN1

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

Human Dental Pulp Stem Cells Display a Potential for Modeling Alzheimer Disease-Related Tau Modifications

“…It is possible to reconstruct the initial steps of pathogenesis via a metaanalysis of independently obtained experimental data, but such reconstructions may not be reliable. Neuronal precursors have been obtained from human induced pluripotent stem cells (51,52), including cells with mutations that cause familial AD (73) or cells that have been genetically manipulated toward AD pathogenesis (74). The potential differentiation of these cells into neurons is the advanced strategy for recapitulating amyloid β and tau pathology in advanced human neural cell culture models of AD (71).…”

“…The potential differentiation of these cells into neurons is the advanced strategy for recapitulating amyloid β and tau pathology in advanced human neural cell culture models of AD (71). This strategy has recently been improved through the use of 3-D AD models, which have emerged as an advanced alternative to 2-D models (73,75). The drawbacks of the currently used 3-D models of AD include insufficient maturation and their inaccessibility for routine employment.…”

3D microenvironments for in vitro modeling of alzheimer’s disease

“…More recently, another group cultured patient iPSC-derived neurons harboring a mutation in the PSEN1 gene inside Matrigel to study the initial stages of disease progression. They reported the production of Aβ oligomers by neurons after 2 weeks of differentiation in the absence of both mutation induction and synthetic Aβ exposure [83].…”

“…Electrospinning is another straightforward for fabricating soft 3D fibrillar matrices, as it can produce uniform and continuous fibers which are structurally similar to native ECM proteins [109]. Electrospinning involves the creation of a strong electric field between a positively charged capillary filled with polymer solution and a grounded collector [83]. Once the electrostatic charge built up inside the solution at the tip of the needle exceeds its surface tension, a Taylor cone is formed, and the polymer solution shoots towards the collector in the form of a fine jet (Figure 2-7).…”

“…The type of collector used can be varied to create fibers having different structures as well as orientation depending on the application. A flat plate can be used to obtain random fibers in the form of a 2D mat, whereas a rotating cylindrical shaped collector can be used to fabricate aligned fibers [83]. A liquid-based collector consisting of a low surface tension liquid such as ethanol can be used to fabricate non-woven, 3D macroporous scaffolds [85].…”