Amyloid Precursor Protein (APP) Regulates Gliogenesis and Neurogenesis of Human Neural Stem Cells by Several Signaling Pathways.

Numerous studies have focused on the pathophysiological role of amyloid precursor protein (APP) because the proteolytic processing of APP to ß-amyloid (Aß) peptide is a central event in Alzheimer's disease (AD). However, many authors consider that alterations in the physiological functions of APP are likely to play a key role in AD. Previous studies in our laboratory revealed that APP plays an important role in the differentiation of human neural stem cells (hNSCs), favoring glial differentiation (gliogenesis) and preventing their differentiation toward a neuronal phenotype (neurogenesis). In the present study, we have evaluated the effects of APP overexpression in hNSCs at a global gene level by a transcriptomic analysis using the massive RNA sequencing (RNA-seq) technology. Specifically, we have focused on differentially expressed genes that are related to neuronal and glial differentiation processes, as well as on groups of differentially expressed genes associated with different signaling pathways, in order to find a possible interaction between them and APP. Our data indicate a differential expression in genes related to Notch, Wnt, PI3K-AKT, and JAK-STAT signaling, among others. Knowledge of APP biological functions, as well as the possible signaling pathways that could be related to this protein, are essential to advance our understanding of AD.

Human cerebral organoids: cellular composition and subcellular morphological features.

Introduction: Human cerebral organoids (hCOs) derived from pluripotent stem cells are very promising for the study of neurodevelopment and the investigation of the healthy or diseased brain. To help establish hCOs as a powerful research model, it is essential to perform the morphological characterization of their cellular components in depth. Methods: In this study, we analyzed the cell types consisting of hCOs after culturing for 45 days using immunofluorescence and reverse transcriptase qualitative polymerase chain reaction (RT-qPCR) assays. We also analyzed their subcellular morphological characteristics by transmission electron microscopy (TEM). Results: Our results show the development of proliferative zones to be remarkably similar to those found in human brain development with cells having a polarized structure surrounding a central cavity with tight junctions and cilia. In addition, we describe the presence of immature and mature migrating neurons, astrocytes, oligodendrocyte precursor cells, and microglia-like cells. Discussion: The ultrastructural characterization presented in this study provides valuable information on the structural development and morphology of the hCO, and this information is of general interest for future research on the mechanisms that alter the cell structure or function of hCOs.

Efficient generation of human cerebral organoids directly from adherent cultures of pluripotent stem cells.

Human cerebral organoids (hCOs) offer the possibility of deepening the knowledge of human brain development, as well as the pathologies that affect it. The method developed here describes the efficient generation of hCOs by going directly from two-dimensional (2D) pluripotent stem cell (PSC) cultures to three-dimensional (3D) neuroepithelial tissue, avoiding dissociation and aggregation steps. This has been achieved by subjecting 2D cultures, from the beginning of the neural induction step, to dual-SMAD inhibition in combination with CHIR99021. This is a simple and reproducible protocol in which the hCOs generated develop properly presenting proliferative ventricular zones (VZs) formed by neural precursor and radial glia (RG) that differentiate to give rise to mature neurons and glial cells. The hCOs present additional cell types such as oligodendrocyte precursors, astrocytes, microglia-like cells, and endothelial-like cells. This new approach could help to overcome some of the existing limitations in the field of organoid biotechnology, facilitating its execution in any laboratory setting.