Neural stem cells (NSCs) provide promising approaches for investigating embryonic neurogenesis, modeling of the pathogenesis of diseases of the central nervous system, and for designing drug-screening systems. Such cells also have an application in regenerative medicine. The most convenient and acceptable source of NSCs is pluripotent stem cells (embryonic stem cells or induced pluripotent stem cells). However, there are many different protocols for the induction and differentiation of NSCs, and these result in a wide range of neural cell types. This review is intended to summarize the knowledge accumulated, to date, by workers in this field. It should be particularly useful for researchers who are beginning investigations in this area of cell biology.

The study of human neurons and their interaction with neurochemicals is difficult due to the inability to collect primary biomaterial. However, recent advances in the cultivation of human stem cells, methods for their neuronal differentiation and chimeric fluorescent calcium indicators have allowed the creation of model systems in vitro. In this paper we report on the development of a method to obtain human neurons with the GCaMP6s calcium indicator, based on a human iPSC line with the TetON–NGN2 transgene complex. The protocol we developed allows us quickly, conveniently and efficiently obtain significant amounts of human neurons suitable for the study of various neurochemicals and their effects on specific neurophysiological activity, which can be easily registered using fluorescence microscopy. In the neurons we obtained, glutamate (Glu) induces rises in [Ca²⁺]_i which are caused by ionotropic receptors for Glu, predominantly of the NMDA-type. Taken together, these facts allow us to consider the model we have created to be a useful and successful development of this technology.

Epigenetic modifications (methylation, acetylation, etc.) of core histones play a key role in regulation of gene expression. Thus, the epigenome changes strongly during various biological processes such as cell differentiation and dedifferentiation. Classical methods of analysis of epigenetic modifications such as mass-spectrometry and chromatin immuno-precipitation, work with fixed cells only. Here we present a genetically encoded fluorescent probe, MPP8-Green, for detecting H3K9me3, a histone modification associated with inactive chromatin. This probe, based on the chromodomain of MPP8, allows for visualization of H3K9me3 epigenetic landscapes in single living cells. We used this probe to track changes in H3K9me3 landscapes during the differentiation of induced pluripotent stem cells (iPSCs) into induced neurons. Our findings revealed two major waves of global H3K9me3 reorganization during 4-day differentiation, namely on the first and third days, whereas nearly no changes occurred on the second and fourth days. The proposed method LiveMIEL (Live-cell Microscopic Imaging of Epigenetic Landscapes), which combines genetically encoded epigenetic probes and machine learning approaches, enables classification of multiparametric epigenetic signatures of single cells during stem cell differentiation and potentially in other biological models.

The differentiation of pluripotent stem cells into neurons is an essential area of biomedical research, with significant implications for understanding neural development and treating neurological diseases. This study compares neural cultures derived from a common induced pluripotent stem cell line (KYOU-DXR0109B) generated by two widely adopted methods: DUAL SMAD inhibition and exogenous NGN2 overexpression. The DUAL SMAD inhibition method, which differentiates through the neural stem cell stage, produces heterogeneous cultures containing a mix of neurons, neural precursors, and glial cells. Conversely, NGN2 overexpression generates more homogeneous cultures composed predominantly of mature neurons. Transcriptomic analysis revealed significant differences in neural gene markers expression profiles, with cultures from the DUAL SMAD inhibition method enriched in neural stem cell and glial markers, while NGN2 overexpression cultures showed elevated markers for cholinergic and peripheral sensory neurons. This study underscores the importance of choosing appropriate differentiation protocols based on the desired cell types, as each method yields neural cultures with distinct cellular compositions. Understanding these differences can help optimize protocols for specific research and therapeutic applications.

Cellular plasticity, a critical feature of embryonic development, is often reactivated in cancer, enabling tumor cells to acquire stem-like properties and facilitate uncontrolled growth. This study compares cellular plasticity in cancer and early human development by integrating single-cell data from three cancers and embryoid bodies. The analysis identifies a shared, highly proliferative progenitor-like state, driven by an E2F-TFDP transcriptional axis and enriched in key cell-cycle regulators UBE2C , TOP2A , BIRC5 , and NUSAP1 . Functionally linked to cell-cycle progression and DNA repair, this state acts as a lineage hub within tumors. Spatial transcriptomics revealed that this state is enriched in lung adenocarcinoma and in lung tissue with basal cell hyperplasia, a potential precursor to squamous cell carcinoma. Validation across eight cancer types demonstrates a prevalence of this program in tumors versus normal cells. Collectively, these findings define a conserved mechanism of tumor plasticity with potential for early detection and targeted therapy. Abstract Figure:

The adeno-associated virus (AAV) has become the vector of choice for gene therapy and experimental gene delivery, owing to its non-pathogenic nature and ability to achieve persistent gene expression across diverse tissues. AAV has emerged as a key platform in cellular reprogramming and regenerative medicine, with applications spanning transcription factor delivery for in vivo lineage conversion and tissue repair across the CNS, heart, and musculoskeletal systems. However, significant limitations remain, particularly in the context of induced pluripotent stem cell (iPSC) engineering. We assess barriers to efficient iPSC transduction including receptor-dependent entry deficits and activation of p53-dependent DNA damage responses. Although AAV is widely described as non-integrating, evidence indicates that integration events occur in rapidly proliferating and actively reprogramming cells. Critically, we synthesize evidence that cell-type-specific promoters lose fidelity when paired with neurogenic transgene payloads, a cross-tissue problem not addressed in existing AAV reviews, and that published in vivo reprogramming efficiencies may be substantially confounded by promoter leakage in the absence of formal lineage tracing. These aspects, underrepresented in recent platform-level reviews, are specifically emphasized here as a resource for researchers designing rigorous AAV-based reprogramming and gene therapy strategies.