Tatiana Shnaider

Introduction: Human cerebral organoids (COs) are sophisticated three-dimensional (3D) models that successfully replicate the 3D cytoarchitecture of human brain development in its early stages. Co-cultivation of COs obtained from human-induced pluripotent stem cells with 3D U-87 MG glioblastoma cell spheroids (glioblastoma–CO assembloid [GCOA]) represents a robust strategy for modeling brain cancer behavior. Objectives: This study aims to evaluate the feasibility of using GCOAs and 3D U-87 MG glioblastoma cell spheroids as in vitro cell models to evaluate the cytotoxic effect of the oncolytic recombinant vaccinia virus-granulocyte–macrophage colony-stimulating factor (VV-GM-CSF)-Lact compared to the VV-GMCSF-del virus. Methods: To assess the cytotoxicity of two virus strains, we used fluorescent and confocal microscopy, spectrophotometry, the MTT assay, and real-time polymerase chain reaction Results: Glioblastoma cells died faster in the presence of VV-GMCSF-Lact virus, and 3D spheroids infected with this virus contained more caspase-3-positive cells. On the 10th day in GCOA, glioblastoma cells began to invade the interior of the CO. Under conditions of 3D spheroid and GCOA infection with recombinant viruses, three differentially expressed genes – FSCN1, SCUBE2, and TNFRSF9 – associated with invasion were analyzed. Conclusion: These 3D models of glioblastoma can be used in the development of antitumor drugs to study their cytotoxic effects.

Human accelerated regions (HARs) are short genomic sequences that, despite being highly conserved across vertebrates, have undergone an unusually high rate of nucleotide substitutions in the human lineage. Although most HARs are located in non-coding regions, accumulating evidence suggests that many function as developmental regulatory elements, fine-tuning gene expression during fundamental neurodevelopmental processes such as radial glial cell proliferation, neurogenesis, cortical lamination, and others. Consequently, deciphering the functional consequences of human-specific sequence changes in HARs represents a major challenge in evolutionary genomics, as it holds the key to understanding the genetic basis of human uniqueness. In this review, we summarize recent advances in experimental approaches for dissecting HAR functions. We discuss how epigenomic profiling and chromatin conformation techniques pinpoint HARs with regulatory potential and connect them to target genes. We then detail functional validation strategies, ranging from classic in vitro and in vivo reporter systems to high-throughput screens like massively parallel reporter assays (MPRAs). Furthermore, we describe genome editing approaches, particularly CRISPR–Cas9, CRISPRi/a, and base editing, that enable causal testing of HAR functions in human cellular and animal models. The integration of these complementary methods provides a comprehensive framework for elucidating how specific sequence changes in HARs have reshaped gene regulatory networks during human evolution. We conclude by outlining current methodological challenges and future directions toward a systems-level understanding of HAR functions across cellular, developmental, and organismal contexts.

It is thought that changes in mechanisms of gene regulation have played the critical role in human evolution, rather than changes in protein-coding sequences. Recent studies have discovered a special class of genome elements, human accelerated regions (HARs). These elements are conserved non-coding DNA sequences of mammals that have been accumulating human-specific mutations throughout the evolution. Starting from their discovery, the actual role of HARs in human evolution has remained unclear, since they are almost exclusively represented by non-coding sequences with no annotations. It is now known that HAR elements are enriched with binding motifs of transcription factors and histone markers of active chromatin. Recent investigations using functional genomics, computational methods, and genetic analysis have demonstrated that many HARs participate in the genetic regulation of development and have made a major contribution to the evolution of the human brain—in particular, the enlargement of the cerebral cortex. Furthermore, there is much evidence that there is relationship between the polymorphisms of HAR sequences and development of various neurological diseases, such as autism spectrum disorders, schizophrenia, and Huntington’s disease. Such functional methods of analysis as massively parallel reporter assay and screenings using the CRISPR system significantly increase the amount of described regulatory elements specific for human beings. Further investigation of HARs and other evolutionary dynamic genome regions might clarify sophisticated evolutionary changes underlying the unique cytoarchitecture and cognitive abilities of the human brain. Here, we have elucidated the approaches to HAR identification in the genome and their role in regulation of gene activity and effect on the evolution of the human brain and reviewed certain pathological effects of mutations in HAR sequences.

The development of the human brain is a complex multi-stage process including the formation of various types of neural cells and their interactions. Many fundamental mechanisms of neurogenesis have been established due to the studying of model animals. However, significant differences in the brain structure compared to other animals do not allow considering all aspects of the human brain formation, which could play the main role in the development of unique cognitive abilities for human. Four years ago, Lancaster's group elaborated human pluripotent stem cell-derived three-dimensional cerebral organoid technology, which opened a unique opportunity for researchers to model early stages of human neurogenesis in vitro. Cerebral organoids closely remodel many endogenous brain regions with specific cell composition like ventricular zone with radial glia, choroid plexus, and cortical plate with upper and deeper-layer neurons. Moreover, human brain development includes interactions between different brain regions. Generation of hybrid three-dimensional cerebral organoids with different brain region identity allows remodeling some of them, including long-distance neuronal migration or formation of major axonal tracts. In this review, we consider the technology of obtaining human pluripotent stem cell-derived three-dimensional cerebral organoids with different modifications and with different brain region identity. In addition, we discuss successful implementation of this technology in-fundamental and applied research like modeling of different neuro-developmental disorders and drug screening. Finally, we regard existing problems and prospects for development of human pluripotent stem cell-derived three-dimensional cerebral organoid technology.