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 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.

Keywords: 3D cell culture; Alzheimer’s disease; disease modeling; iPSC-derived neurons; personalized therapy.

Figure 1

Generation of induced pluripotent stem cells (iPSCs). (A) Day 0. Human fibroblast culture. Scale 50 μm, 20× amplification. (B) Representative derived iPSCs using lentiviral vectors containing Yamanaka’s factors in feeder-free conditions after 10 passages. iPSCs have flat, rounded, and compact morphology, a high ratio of nucleus to the cytoplasm and prominent nucleoli embryonic stem cell-like (ESC-like). From left to right, iPSCs 4× amplification, 100 μm scale; 10× amplification, 100 μm scale; and 20× amplification, scale 50 μm.

Figure 2

iPSC pluripotency expression. iPSCs were characterized using standard pluripotency assays. Representative images are shown. (A) iPSCs showed alkaline phosphatase activity. (B) Immunofluorescence staining showed nuclear marker (red) and cell surface (green) pluripotency marker expressions in iPSCs, including NANOG, OCT4, SOX2, and SSEA4, and TRA-1-60. Hoechst nuclear staining in blue. Scale bars 50 μm. (C) Expression of pluripotency-related endogenous genes, OCT4 and Nanog, was detected in derived iPSCs by RT-PCR analysis. hESCs were used as a positive control (Ctrl+), and human fibroblasts (HFs) were used as a negative control (Ctrl−) for endogenous pluripotency gene expression. (D) Some iPSC clones expressed the transgenes (data not shown), whereas fully reprogrammed iPSCs consistently exhibited lentiviral silencing or attenuation (iFLAG and iFLN). HFs at day 5 p.t. were used as a positive control (Ctrl+ HFs D5 p.t.) for transgene expression, and HFs were used as a negative control (Ctrl− HFs). GAPDH was used as an internal control.

Figure 3

Immunofluorescence staining of neural precursor cell (NPC) marker Nestin. Representative images indicate Nestin expression in NPCs on day 5 post neural induction. Nuclear staining in blue with Hoechst. Scale bars 50 μm.

Figure 4

Neuronal marker expression in the three-dimensional (3D) cultures. Representative images are shown. At day 14, we performed immunofluorescence and iPSC-derived neurons in 3D culture expressed TUJ-1 and MAP2 neuronal markers in red. Nuclear staining in blue with Hoechst. Scale bars 100 μm.

Figure 5

3D iPSC neural differentiation scheme. iPSCs were plated onto hESC-qualified Matrigel-coated 24-well plates in an E8 medium (D0) until 80% of confluence. Then the E8 medium was replaced by neural induction medium (NIM) for neural induction with daily medium replacement for 5 days. On the fifth day (D5) of neural induction, 3D Matrigel basement matrices were prepared in accordance with Kim et al. (2015) with modifications. A Matrigel basement matrix stock solution was diluted (1:1 dilution ratio) with ice-cold neural differentiation medium (NDM) and then vortexed for 5 s. Immediately, the prepared dilution was transferred into 48-well plates (200 μl in each well); then the plates were incubated for 20 min at 37°C to form a 3D gel layer at the bottom of the well. Subsequently, NPCs were dissociated into clumps using 0.1% collagenase IV and replated (Split 1:2) onto a Matrigel basement matrix in prewarmed NDM for neural maturation. The next day, the 3D cell culture was exposed to 5 μM DAPT for 24 h. The 3D cultures were maintained for 2 weeks, with media replacement every 2 days. At D14, the 3D neural cell cultures were observed and analyzed.

Figure 6

Amyloid-β (Aβ) marker expression and Aβ aggregation in the Alzheimer’s disease (AD) 3D model. Representative images are shown. (A) Immunofluorescence staining indicates Aβ marker expression in the AD 3D model. TUJ-1 (green), Aβ (red), and MAP2 (red) and Aβ (green). Co-3D model, Aβ marker expression was not detected. TUJ-1 (red) and Aβ (green). Nuclei were stained in blue with Hoechst. Scale bars 25 μm. (B) Aβ oligomers were detected by western blot (WB) analysis in the AD 3D model as a protein band with a molecular mass of ~50 kDa and were absent in the non-mutated 3D model. β-Actin was expressed by mutated and non-mutated cells. These assays were performed at 14 days post neural induction.