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.

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.

Neurogenesis Is Increased in Human Neural Stem Cells by Aß40 Peptide.

Amyloid-ß 40 peptides [Aß1-40 (Aß40)] are present within amyloid plaques in the brains of patients with Alzheimer's disease (AD). Even though Aß peptides are considered neurotoxic, they can mediate many biological processes, both in adult brains and throughout brain development. However, the physiological function of these Aß peptides remains poorly understood, and the existing data are sometimes controversial. Here, we analyze and compare the effects of monomeric Aß40 on the biology of differentiating human neural stem cells (human NSCs). For that purpose, we used a model of human NSCs called hNS1. Our data demonstrated that Aß40 at high concentrations provokes apoptotic cellular death and the damage of DNA in human NSCs while also increasing the proliferation and favors neurogenesis by raising the percentage of proliferating neuronal precursors. These effects can be mediated, at least in part, by ß-catenin. These results provide evidence of how Aß modulate/regulate human NSC proliferation and differentiation, suggesting Aß40 may be a pro-neurogenic factor. Our data could contribute to a better understanding of the molecular mechanisms involved in AD pathology and to the development of human NSC-based therapies for AD treatment, since these results could then be used in diagnosing the disease at early stages and be applied to the development of new treatment options.

Oligomeric and Fibrillar Species of Aß42 Diversely Affect Human Neural Stem Cells.

Amyloid-ß 42 peptide (Aß1-42 (Aß42)) is well-known for its involvement in the development of Alzheimer's disease (AD). Aß42 accumulates and aggregates in fibers that precipitate in the form of plaques in the brain causing toxicity; however, like other forms of Aß peptide, the role of these peptides remains unclear. Here we analyze and compare the effects of oligomeric and fibrillary Aß42 peptide on the biology (cell death, proliferative rate, and cell fate specification) of differentiating human neural stem cells (hNS1 cell line). By using the hNS1 cells we found that, at high concentrations, oligomeric and fibrillary Aß42 peptides provoke apoptotic cellular death and damage of DNA in these cells, but Aß42 fibrils have the strongest effect. The data also show that both oligomeric and fibrillar Aß42 peptides decrease cellular proliferation but Aß42 oligomers have the greatest effect. Finally, both, oligomers and fibrils favor gliogenesis and neurogenesis in hNS1 cells, although, in this case, the effect is more prominent in oligomers. All together the findings of this study may contribute to a better understanding of the molecular mechanisms involved in the pathology of AD and to the development of human neural stem cell-based therapies for AD treatment.

Evaluation and Optimization of Poly-d-Lysine as a Non-Natural Cationic Polypeptide for Gene Transfer in Neuroblastoma Cells.

Cationic polypeptides and cationic polymers have cell-penetrating capacities and have been used in gene transfer studies. In this study, we investigate the capability of a polymer of d-lysine (PDL), a chiral form of α-Poly-lysine, as a possible nonviral vector for releasing genetic materials to neuroblastoma cells and evaluate its stability against proteases. We tested and compared its transfection effectiveness in vitro as a vehicle for the EGFP plasmid DNA (pDNA) reporter in the SH-SY5Y human neuroblastoma, HeLa, and 3T3 cell lines. Using fluorescent microscopy and flow cytometry, we demonstrated high transfection efficiencies based on EGFP fluorescence in SH-SY5Y cells, compared with HeLa and 3T3. Our results reveal PDL as an efficient vector for gene delivery specifically in the SH-SY5Y cell line and suggest that PDL can be used as a synthetic cell-penetrating polypeptide for gene therapy in neuroblastoma cells.

Physiological and pathological effects of amyloid-ß species in neural stem cell biology.

Although amyloid-ß peptide is considered neurotoxic, it may mediate several physiological processes during embryonic development and in the adult brain. The pathological function of amyloid-ß peptide has been extensively studied due to its implication in Alzheimer's disease, but its physiological function remains poorly understood. Amyloid-ß peptide can be detected in non-aggregated (monomeric) and aggregated (oligomeric and fibrillary) forms. Each form has different cytotoxic and/or physiological properties, so amyloid-ß peptide and its role in Alzheimer's disease need to be studied further. Neural stem cells and neural precursor cells are good tools for the study on neurodegenerative diseases and can provide future therapeutic applications in diseases such as Alzheimer's disease. In this review, we provide an outline of the effects of amyloid-ß peptide, in monomeric and aggregated forms, on the biology of neural stem cells/neural precursor cells, and discuss the controversies. We also describe the possible molecular targets that could be implicated in these effects, especially GSK3ß. A better understanding of amyloid-ß peptide (both physiological and pathological), and the signaling pathways involved are essential to advance the field of Alzheimer's disease.

Physiological effects of amyloid precursor protein and its derivatives on neural stem cell biology and signaling pathways involved.

The pathological implication of amyloid precursor protein (APP) in Alzheimer's disease has been widely documented due to its involvement in the generation of amyloid-ß peptide. However, the physiological functions of APP are still poorly understood. APP is considered a multimodal protein due to its role in a wide variety of processes, both in the embryo and in the adult brain. Specifically, APP seems to play a key role in the proliferation, differentiation and maturation of neural stem cells. In addition, APP can be processed through two canonical processing pathways, generating different functionally active fragments: soluble APP-α, soluble APP-ß, amyloid-ß peptide and the APP intracellular C-terminal domain. These fragments also appear to modulate various functions in neural stem cells, including the processes of proliferation, neurogenesis, gliogenesis or cell death. However, the molecular mechanisms involved in these effects are still unclear. In this review, we summarize the physiological functions of APP and its main proteolytic derivatives in neural stem cells, as well as the possible signaling pathways that could be implicated in these effects. The knowledge of these functions and signaling pathways involved in the onset or during the development of Alzheimer's disease is essential to advance the understanding of the pathogenesis of Alzheimer's disease, and in the search for potential therapeutic targets.

Neuronal and Glial Differentiation of Human Neural Stem Cells Is Regulated by Amyloid Precursor Protein (APP) Levels.

Amyloid precursor protein (APP) is implicated in neural development as well as in the pathology of Alzheimer's disease (AD); however, its biological function still remains unclear. It has been reported that APP stimulates the proliferation and neuronal differentiation of neural stem cells (NSCs), while other studies suggest an important effect enhancing gliogenesis in NSCs. As expected, APP protein/mRNA is detected in hNS1 cells, a model cell line of human NSCs, both under proliferation and throughout the differentiation period. To investigate the potential function that APP plays in cell fate specification and differentiation of hNS1 cells, we transiently increased human APP levels in these cells and analyzed its cell intrinsic effects. Our data indicate that increased levels of APP induce early cell cycle exit and instructively direct hNS1 cell fate towards a glial phenotype, while decreasing neuronal differentiation. Since elevated APP levels also enhanced APP intracellular domain (AICD)-immunoreactivity, these effects could be, in part, mediated by the APP/AICD system. The AICD domain can play a potential role in signal transduction by its molecular interaction with different target genes such as GSK3B, whose expression was also increased in APP-overexpressing cells that, in turn, may contribute to promoting gliogenesis and inhibiting neurogenesis in NSCs. These data suggest an important action of APP in modulating hNSCs differentiation (probably in an AICD-GSK-3ß-dependent manner) and may thus be important for the future development of stem cell therapy strategies for the diseased mammalian brain.

Effects of Amyloid-ß Peptide on the Biology of Human Neural Stem Cells.

The amyloid -ß peptide (Aß) is the main component of the amyloid plaques in Alzheimer's disease (AD). It has been widely demonstrated that Aß is toxic to neurons and is associated with AD pathology. However, Aß also appears to have an important biological function both in the adult brain and throughout embryonic development of the nervous system, acting as a trophic factor at low concentrations.It is known that Neural Stem Cells (NSCs) are capable of self-renewal and differentiate into functional glial and neuronal cells. Therefore, human NSCs may be a hope for future therapeutic application in neurodegenerative diseases such as AD. The effects of Aß peptides on NSCs are still not well understood and remain controversial.In this chapter we outline the materials and methods used for the culture and differentiation of hNS1 cells, a cell line of human NSCs. We describe the preparation of different forms (monomeric, oligomeric and fibrillary) of Aß peptide and subsequent cell treatment, followed by the analysis of the effects on toxicity, cell proliferation and cell fate specification of hNS1 cells.

Role of Amyloid Precursor Protein (APP) and Its Derivatives in the Biology and Cell Fate Specification of Neural Stem Cells.

Amyloid precursor protein (APP) is a member of the APP family of proteins, and different enzymatic processing leads to the production of several derivatives that are shown to have distinct biological functions. APP is involved in the pathology of Alzheimer's disease (AD), the most common neurodegenerative disorder causing dementia. Furthermore, it is believed that individuals with Down syndrome (DS) have increased APP expression, due to an extra copy of chromosome 21 (Hsa21), that contains the gene for APP. Nevertheless, the physiological function of APP remains unclear. It is known that APP plays an important role in neural growth and maturation during brain development, possibly by influencing proliferation, cell fate specification and neurogenesis of neural stem cells (NSCs). Proteolytic cleavage of APP occurs mainly via two mutually exclusive pathways, the non-amyloidogenic pathway or the amyloidogenic pathway. Other alternative pathways (η-secretase, δ-secretase and meprin pathways) have also been described for the physiological processing of APP. The different metabolites generated from these pathways, including soluble APPα (sAPPα), soluble APPß (sAPPß), ß-amyloid (Aß) peptides and the APP intracellular domain (AICD), have different functions determined by their structural differences, equilibrium and concentration with respect to other fragments derived from APP. This review discusses recent observations regarding possible functions of APP and its proteolytic derivatives in the biology and phenotypic specification of NSCs. This can be important for a better understanding of the pathogenesis and the development of future therapeutic applications for AD and/or DS, diseases in which alterations in neurogenesis have been described.