Optical Genome Mapping Reveals Genomic Alterations upon Gene Editing in hiPSCs: Implications for Neural Tissue Differentiation and Brain Organoid Research.

Genome editing, notably CRISPR (cluster regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9), has revolutionized genetic engineering allowing for precise targeted modifications. This technique's combination with human induced pluripotent stem cells (hiPSCs) is a particularly valuable tool in cerebral organoid (CO) research. In this study, CRISPR/Cas9-generated fluorescently labeled hiPSCs exhibited no significant morphological or growth rate differences compared with unedited controls. However, genomic aberrations during gene editing necessitate efficient genome integrity assessment methods. Optical genome mapping, a high-resolution genome-wide technique, revealed genomic alterations, including chromosomal copy number gain and losses affecting numerous genes. Despite these genomic alterations, hiPSCs retain their pluripotency and capacity to generate COs without major phenotypic changes but one edited cell line showed potential neuroectodermal differentiation impairment. Thus, this study highlights optical genome mapping in assessing genome integrity in CRISPR/Cas9-edited hiPSCs emphasizing the need for comprehensive integration of genomic and morphological analysis to ensure the robustness of hiPSC-based models in cerebral organoid research.

Big dynorphin is a neuroprotector scaffold against amyloid ß-peptide aggregation and cell toxicity.

Amyloid ß-peptide (Aß) misfolding into ß-sheet structures triggers neurotoxicity inducing Alzheimer's disease (AD). Molecules able to reduce or to impair Aß aggregation are highly relevant as possible AD treatments since they should protect against Aß neurotoxicity. We have studied the effects of the interaction of dynorphins, a family of opioid neuropeptides, with Aß40 the most abundant species of Aß. Biophysical measurements indicate that Aß40 interacts with Big Dynorphin (BigDyn), lowering the amount of hydrophobic aggregates, and slowing down the aggregation kinetics. As expected, we found that BigDyn protects against Aß40 aggregates when studied in human neuroblastoma cells by cell survival assays. The cross-interaction between BigDyn and Aß40 provides insight into the mechanism of amyloid pathophysiology and may open up new therapy possibilities.

Role of Intracellular Amyloid ß as Pathway Modulator, Biomarker, and Therapy Target.

The ß- and γ-secretase-driven cleavage of the amyloid precursor protein (APP) gives rise to the amyloid ß peptide, which is believed to be the main driver of neurodegeneration in Alzheimer's disease (AD). As it is prominently detectable in extracellular plaques in post-mortem AD brain samples, research in recent decades focused on the pathological role of extracellular amyloid ß aggregation, widely neglecting the potential meaning of very early generation of amyloid ß inside the cell. In the last few years, the importance of intracellular amyloid ß (iAß) as a strong player in neurodegeneration has been indicated by a rising number of studies. In this review, iAß is highlighted as a crucial APP cleavage fragment, able to manipulate intracellular pathways and foster neurodegeneration. We demonstrate its relevance as a pathological marker and shed light on initial studies aiming to modulate iAß through pharmacological treatment, which has been shown to have beneficial effects on cognitive properties in animal models. Finally, we display the relevance of viral infections on iAß generation and point out future directions urgently needed to manifest the potential relevance of iAß in Alzheimer's disease.

Gene-Edited Fluorescent and Mixed Cerebral Organoids.

Cerebral organoids are a promising model to study human brain function and disease, although the high inter-organoid variability is still challenging. To overcome this limitation, we introduce the method of labeled mixed organoids generated from two different human induced pluripotent stem cell (hiPSC) lines, which enables the identification of cells from different origin within a single organoid. The method combining gene editing and organoid differentiation offers a unique tool to study gene function in a complex human three-dimensional model. Using a CRISPR-Cas9 gene-editing approach, different fluorescent proteins were fused to ß-actin or lamin B1 in hiPSCs, and mixtures of differently edited cells were seeded to induce cerebral organoid differentiation. Consequently, the development of the organoids was detectable by live confocal fluorescence microscopy of whole organoids and immunofluorescence staining in fixed samples. We demonstrate that a direct comparison of the individual cells is possible by having the edited and the control (or the two differentially labeled) cells within the same organoid, thus overcoming the inter-organoid inhomogeneity limitations. Furthermore, the approach enables mosaic analysis of mutant clones in a wild-type three-dimensional cellular environment. It paves the way for the reliable analysis of human genetic disorders using organoids and the gain of fundamental understanding of the molecular mechanisms underlying pathological conditions.

Sera from Patients with NMOSD Reduce the Differentiation Capacity of Precursor Cells in the Central Nervous System.

INTRODUCTION: AQP4 (aquaporin-4)-immunoglobulin G (IgG)-mediated neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory demyelinating disease that affects the central nervous system, particularly the spinal cord and optic nerve; remyelination capacity in neuromyelitis optica is yet to be determined, as is the role of AQP4-IgG in cell differentiation. MATERIAL AND METHODS: We included three groups-a group of patients with AQP4-IgG-positive neuromyelitis optica, a healthy group, and a sham group. We analyzed differentiation capacity in cultures of neurospheres from the subventricular zone of mice by adding serum at two different times: early and advanced stages of differentiation. We also analyzed differentiation into different cell lines. RESULTS AND CONCLUSIONS: The effect of sera from patients with NMOSD on precursor cells differs according to the degree of differentiation, and probably affects oligodendrocyte progenitor cells from NG2 cells to a lesser extent than cells from the subventricular zone; however, the resulting oligodendrocytes may be compromised in terms of maturation and possibly limited in their ability to generate myelin. Furthermore, these cells decrease in number with age. It is very unlikely that the use of drugs favoring the migration and differentiation of oligodendrocyte progenitor cells in multiple sclerosis would be effective in the context of neuromyelitis optica, but cell therapy with oligodendrocyte progenitor cells seems to be a potential alternative.