BackgroundAlzheimer’s disease (AD) is the most common type of dementia, affecting one in eight adults over 65 years of age. The majority of AD cases are sporadic, with unknown etiology, and only 5% of all patients with AD present the familial monogenic form of the disease. In the present study, our aim was to establish an in vitro cell model based on patient-specific human neurons to study the pathomechanism of sporadic AD.MethodsWe compared neurons derived from induced pluripotent stem cell (iPSC) lines of patients with early-onset familial Alzheimer’s disease (fAD), all caused by mutations in the PSEN1 gene; patients with late-onset sporadic Alzheimer’s disease (sAD); and three control individuals without dementia. The iPSC lines were differentiated toward mature cortical neurons, and AD pathological hallmarks were analyzed by RT-qPCR, enzyme-linked immunosorbent assay, and Western blotting methods.ResultsNeurons from patients with fAD and patients with sAD showed increased phosphorylation of TAU protein at all investigated phosphorylation sites. Relative to the control neurons, neurons derived from patients with fAD and patients with sAD exhibited higher levels of extracellular amyloid-β 1–40 (Aβ1–40) and amyloid-β 1–42 (Aβ1–42). However, significantly increased Aβ1–42/Aβ1–40 ratios, which is one of the pathological markers of fAD, were observed only in samples of patients with fAD. Additionally, we detected increased levels of active glycogen synthase kinase 3 β, a physiological kinase of TAU, in neurons derived from AD iPSCs, as well as significant upregulation of amyloid precursor protein (APP) synthesis and APP carboxy-terminal fragment cleavage. Moreover, elevated sensitivity to oxidative stress, as induced by amyloid oligomers or peroxide, was detected in both fAD- and sAD-derived neurons.ConclusionsOn the basis of the experiments we performed, we can conclude there is no evident difference except secreted Aβ1–40 levels in phenotype between fAD and sAD samples. To our knowledge, this is the first study in which the hyperphosphorylation of TAU protein has been compared in fAD and sAD iPSC-derived neurons. Our findings demonstrate that iPSC technology is suitable to model both fAD and sAD and may provide a platform for developing new treatment strategies for these conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13195-017-0317-z) contains supplementary material, which is available to authorized users.

Neural progenitor cells (NPCs) from human induced pluripotent stem cells (hiPSCs) are frequently induced using 3D culture methodologies however, it is unknown whether spheroid-based (3D) neural induction is actually superior to monolayer (2D) neural induction. Our aim was to compare the efficiency of 2D induction with 3D induction method in their ability to generate NPCs, and subsequently neurons and astrocytes. Neural differentiation was analysed at the protein level qualitatively by immunocytochemistry and quantitatively by flow cytometry for NPC (SOX1, PAX6, NESTIN), neuronal (MAP2, TUBB3), cortical layer (TBR1, CUX1) and glial markers (SOX9, GFAP, AQP4). Electron microscopy demonstrated that both methods resulted in morphologically similar neural rosettes. However, quantification of NPCs derived from 3D neural induction exhibited an increase in the number of PAX6/NESTIN double positive cells and the derived neurons exhibited longer neurites. In contrast, 2D neural induction resulted in more SOX1 positive cells. While 2D monolayer induction resulted in slightly less mature neurons, at an early stage of differentiation, the patch clamp analysis failed to reveal any significant differences between the electrophysiological properties between the two induction methods. In conclusion, 3D neural induction increases the yield of PAX6/NESTIN cells and gives rise to neurons with longer neurites, which might be an advantage for the production of forebrain cortical neurons, highlighting the potential of 3D neural induction, independent of iPSCs' genetic background.

No abstract

The aim of the present study was to establish an in vitro Kleefstra syndrome (KS) disease model using the human induced pluripotent stem cell (hiPSC) technology. Previously, an autism spectrum disorder (ASD) patient with Kleefstra syndrome (KS-ASD) carrying a deleterious premature termination codon mutation in the EHMT1 gene was identified. Patient specific hiPSCs generated from peripheral blood mononuclear cells of the KS-ASD patient were differentiated into post-mitotic cortical neurons. Lower levels of EHMT1 mRNA as well as protein expression were confirmed in these cells. Morphological analysis on neuronal cells differentiated from the KS-ASD patient-derived hiPSC clones showed significantly shorter neurites and reduced arborization compared to cells generated from healthy controls. Moreover, density of dendritic protrusions of neuronal cells derived from KS-ASD hiPSCs was lower than that of control cells. Synaptic connections and spontaneous neuronal activity measured by live cell calcium imaging could be detected after 5 weeks of differentiation, when KS-ASD cells exhibited higher sensitivity of calcium responses to acetylcholine stimulation indicating a lower nicotinic cholinergic tone at baseline condition in KS-ASD cells. In addition, gene expression profiling of differentiated neuronal cells from the KS-ASD patient revealed higher expression of proliferation-related genes and lower mRNA levels of genes involved in neuronal maturation and migration. Our data demonstrate anomalous neuronal morphology, functional activity and gene expression in KS-ASD patient-specific hiPSC-derived neuronal cultures, which offers an in vitro system that contributes to a better understanding of KS and potentially other neurodevelopmental disorders including ASD.

We present a hiPSC-based 3D in vitro system suitable to test neurotoxicity (NT). Human iPSCs-derived 3D neurospheres grown in 96-well plate format were characterized timewise for 6-weeks. Changes in complexity and homogeneity were followed by immunocytochemistry and transmission electron microscopy. Transcriptional activity of major developmental, structural, and cell-type-specific markers was investigated at weekly intervals to present the differentiation of neurons, astrocytes, and oligodendrocytes. Neurospheres were exposed to different well-known toxicants with or without neurotoxic effect (e.g., paraquat, acrylamide, or ibuprofen) and examined at various stages of the differentiation with an ATP-based cell viability assay optimized for 3D-tissues. Concentration responses were investigated after acute (72 h) exposure. Moreover, the compound-specific effect of rotenone was investigated by a panel of ER-stress assay, TUNEL assay, immunocytochemistry, electron microscopy, and in 3D-spheroid based neurite outgrowth assay. The acute exposure to different classes of toxicants revealed distinct susceptibility profiles in a differentiation stage-dependent manner, indicating that hiPSC-based 3D in vitro neurosphere models could be used effectively to evaluate NT, and can be developed further to detect developmental neurotoxicity (DNT) and thus replace or complement the use of animal models in various basic research and pharmaceutical applications.