EGCG Disrupts the LIN28B/Let-7 Interaction and Reduces Neuroblastoma Aggressiveness.

Neuroblastoma (NB) is the most commonly diagnosed extracranial solid tumor in children, accounting for 15% of all childhood cancer deaths. Although the 5-year survival rate of patients with a high-risk disease has increased in recent decades, NB remains a challenge in pediatric oncology, and the identification of novel potential therapeutic targets and agents is an urgent clinical need. The RNA-binding protein LIN28B has been identified as an oncogene in NB and is associated with a poor prognosis. Given that LIN28B acts by negatively regulating the biogenesis of the tumor suppressor let-7 miRNAs, we reasoned that selective interference with the LIN28B/let-7 miRNA interaction would increase let-7 miRNA levels, ultimately leading to reduced NB aggressiveness. Here, we selected (-)-epigallocatechin 3-gallate (EGCG) out of 4959 molecules screened as the molecule with the best inhibitory activity on LIN28B/let-7 miRNA interaction and showed that treatment with PLC/PLGA-PEG nanoparticles containing EGCG (EGCG-NPs) led to an increase in mature let-7 miRNAs and a consequent inhibition of NB cell growth. In addition, EGCG-NP pretreatment reduced the tumorigenic potential of NB cells in vivo. These experiments suggest that the LIN28B/let-7 miRNA axis is a good therapeutic target in NB and that EGCG, which can interfere with this interaction, deserves further preclinical evaluation.

Optimized and Scalable Precoating-Free Reprogramming of Human Peripheral Blood Mononuclear Cells into iPSCs.

Human disease modeling has been profoundly transformed by the introduction of human induced pluripotent stem cells (iPSCs), marking the onset of a new era. This ground-breaking development offers a tailored framework for generating pluripotent cells from any individual, effectively enabling the development of cellular models for the study of human physiology and diseases on an unprecedented scale. Although technologies for iPSCs generation have advanced rapidly over the past two decades, protocols for reprogramming patient-derived somatic cells into stem cells still pose a major challenge for the development of automated pipelines capable of generating iPSCs at scales that are cost-effective, reproducible, and easy to implement. Most methods commonly rely on extracellular matrix protein mixtures or synthetic substrates to promote efficient proliferation of iPSCs. Nonetheless, employing these substances entails a laborious and time-consuming process, as the culture surface requires coating treatments before cell seeding. Here we describe a method for reprogramming blood-derived mononucleated cells that eliminates the need to precoat culture surfaces for the entire experimental flow. This procedure is suitable for fresh or frozen purified peripheral blood mononuclear cells (PBMCs) and allows seeding of reprogrammed cells in a culture medium containing a fragment of laminin-511, regardless of the method of reprogramming employed. Our protocol incorporates a streamlined workflow that optimizes key factors, including cell density, culture medium composition, and iPSC culture propagation techniques. Using a precoating-free approach, we eliminate the time-consuming steps, while our optimized subcloning method improves the scalability of the protocol, making it suitable for large-scale applications. Additionally, the automation-friendly nature of our protocol allows for high-throughput processing, reducing the labor and costs associated with manual handling. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Miniaturized and time efficient precoating-free reprogramming of fresh or frozen PBMCs Alternate Protocol: Erythroid progenitor cells (EPCs) enrichment and reprogramming into iPSCs using Sendai viral vectors Basic Protocol 2: Picking and precoating-free optimized expansion of iPSC clones.

CAG repeat expansion in the Huntington's disease gene shapes linear and circular RNAs biogenesis.

Alternative splicing (AS) appears to be altered in Huntington's disease (HD), but its significance for early, pre-symptomatic disease stages has not been inspected. Here, taking advantage of Htt CAG knock-in mouse in vitro and in vivo models, we demonstrate a correlation between Htt CAG repeat length and increased aberrant linear AS, specifically affecting neural progenitors and, in vivo, the striatum prior to overt behavioral phenotypes stages. Remarkably, a significant proportion (36%) of the aberrantly spliced isoforms are not-functional and meant to non-sense mediated decay (NMD). The expanded Htt CAG repeats further reflect on a previously neglected, global impairment of back-splicing, leading to decreased circular RNAs production in neural progenitors. Integrative transcriptomic analyses unveil a network of transcriptionally altered micro-RNAs and RNA-binding proteins (Celf, hnRNPs, Ptbp, Srsf, Upf1, Ythd2) which might influence the AS machinery, primarily in neural cells. We suggest that this unbalanced expression of linear and circular RNAs might alter neural fitness, contributing to HD pathogenesis.

Cortical Astrocyte Progenitors and Astrocytes from Human Pluripotent Stem Cells.

Astrocytes coordinate several homeostatic processes of the central nervous system and play essential roles for normal brain development and response to disease conditions. Protocols for the conversion of human induced pluripotent stem cells (hiPSCs) into mature astrocytes have opened to the generation of in vitro systems to explore astrocytes' functions in living human cell contexts and patient-specific settings. In this study, we present an optimized monolayer procedure to commit hiPSC-derived cortical progenitors into enriched populations of cortical astrocyte progenitor cells (CX APCs) that can be further amplified and efficiently differentiated into mature astrocytes. Our optimized system provides a valid tool to explore the role of these cells in neurodevelopmental and neuropsychiatric diseases, opening it up to applications in drug development and biomarkers discovery/validation.

One-shot analysis of translated mammalian lncRNAs with AHARIBO.

A vast portion of the mammalian genome is transcribed as long non-coding RNAs (lncRNAs) acting in the cytoplasm with largely unknown functions. Surprisingly, lncRNAs have been shown to interact with ribosomes, encode peptides, or act as ribosome sponges. These functions still remain mostly undetected and understudied owing to the lack of efficient tools for genome-wide simultaneous identification of ribosome-associated and peptide-producing lncRNAs. Here, we present AHA-mediated RIBOsome isolation (AHARIBO), a method for the detection of lncRNAs either untranslated, but associated with ribosomes, or encoding small peptides. Using AHARIBO in mouse embryonic stem cells during neuronal differentiation, we isolated ribosome-protected RNA fragments, translated RNAs, and corresponding de novo synthesized peptides. Besides identifying mRNAs under active translation and associated ribosomes, we found and distinguished lncRNAs acting as ribosome sponges or encoding micropeptides, laying the ground for a better functional understanding of hundreds of lncRNAs.

Artificial cells drive neural differentiation.

We report the construction of artificial cells that chemically communicate with mammalian cells under physiological conditions. The artificial cells respond to the presence of a small molecule in the environment by synthesizing and releasing a potent protein signal, brain-derived neurotrophic factor. Genetically controlled artificial cells communicate with engineered human embryonic kidney cells and murine neural stem cells. The data suggest that artificial cells are a versatile chassis for the in situ synthesis and on-demand release of chemical signals that elicit desired phenotypic changes of eukaryotic cells, including neuronal differentiation. In the future, artificial cells could be engineered to go beyond the capabilities of typical smart drug delivery vehicles by synthesizing and delivering specific therapeutic molecules tailored to distinct physiological conditions.

Epsins Regulate Mouse Embryonic Stem Cell Exit from Pluripotency and Neural Commitment by Controlling Notch Activation.

Epsins are part of the internalization machinery pivotal to control clathrin-mediated endocytosis. Here, we report that epsin family members are expressed in mouse embryonic stem cells (mESCs) and that epsin1/2 knockdown alters both mESC exits from pluripotency and their differentiation. Furthermore, we show that epsin1/2 knockdown compromises the correct polarization and division of mESC-derived neural progenitors and their conversion into expandable radial glia-like neural stem cells. Finally, we provide evidence that Notch signaling is impaired following epsin1/2 knockdown and that experimental restoration of Notch signaling rescues the epsin-mediated phenotypes. We conclude that epsins contribute to control mESC exit from pluripotency and allow their neural differentiation by appropriate modulation of Notch signaling.

Vertically-Aligned Functionalized Silicon Micropillars for 3D Culture of Human Pluripotent Stem Cell-Derived Cortical Progenitors.

Silicon is a promising material for tissue engineering since it allows to produce micropatterned scaffolding structures resembling biological tissues. Using specific fabrication methods, it is possible to build aligned 3D network-like structures. In the present study, we exploited vertically-aligned silicon micropillar arrays as culture systems for human iPSC-derived cortical progenitors. In particular, our aim was to mimic the radially-oriented cortical radial glia fibres that during embryonic development play key roles in controlling the expansion, radial migration and differentiation of cortical progenitors, which are, in turn, pivotal to the establishment of the correct multilayered cerebral cortex structure. Here we show that silicon vertical micropillar arrays efficiently promote expansion and stemness preservation of human cortical progenitors when compared to standard monolayer growth conditions. Furthermore, the vertically-oriented micropillars allow the radial migration distinctive of cortical progenitors in vivo. These results indicate that vertical silicon micropillar arrays can offer an optimal system for human cortical progenitors' growth and migration. Furthermore, similar structures present an attractive platform for cortical tissue engineering.

Inducible Alpha-Synuclein Expression Affects Human Neural Stem Cells' Behavior.

Converging evidence suggest that levels of alpha-synuclein (aSyn) expression play a critical role in Parkinson's disease (PD). Several mutations of the SNCA gene, encoding for aSyn have been associated to either the familial or the sporadic forms of PD. Nonetheless, the mechanism underlying wild-type aSyn-mediated neurotoxicity in neuronal cells as well as its specific driving role in PD pathogenesis has yet to be fully clarified. In this view, the development of proper in vitro cellular systems is a crucial step. In this study, we present a novel human Tet-on human neural stem cell (hNSC) line, in which aSyn timing and level of expression can be tightly experimentally tuned. Induction of aSyn in self-renewing hNSCs leads to progressive formation of aSyn aggregates and impairs their proliferation and cell survival. Furthermore, aSyn induction during the neuronal differentiation process results in reduced neuronal differentiation and increased number of astrocytes and undifferentiated cells in culture. Finally, acute aSyn induction in hNSC-derived dopaminergic neuronal cultures results in cell toxicity. This novel conditional in vitro cell model system may be a valuable tool for dissecting of aSyn pathogenic effects in hNSCs and neurons and in developing new potential therapeutic strategies.

Hit and go CAS9 delivered through a lentiviral based self-limiting circuit.

In vivo application of the CRISPR-Cas9 technology is still limited by unwanted Cas9 genomic cleavages. Long-term expression of Cas9 increases the number of genomic loci non-specifically cleaved by the nuclease. Here we develop a Self-Limiting Cas9 circuit for Enhanced Safety and specificity (SLiCES) which consists of an expression unit for Streptococcus pyogenes Cas9 (SpCas9), a self-targeting sgRNA and a second sgRNA targeting a chosen genomic locus. The self-limiting circuit results in increased genome editing specificity by controlling Cas9 levels. For its in vivo utilization, we next integrate SLiCES into a lentiviral delivery system (lentiSLiCES) via circuit inhibition to achieve viral particle production. Upon delivery into target cells, the lentiSLiCES circuit switches on to edit the intended genomic locus while simultaneously stepping up its own neutralization through SpCas9 inactivation. By preserving target cells from residual nuclease activity, our hit and go system increases safety margins for genome editing.

Establishment of induced pluripotent stem cell (iPSC) line from 55-year old male patient with hemorrhagic Moyamoya disease.

Peripheral blood mononuclear cells (PBMCs) were collected from 55-year old male patient with a confirmed diagnosis of hemorrhagic Moyamoya disease (MMD). PBMCs were reprogrammed using Sendai virus particles delivering the four Yamanaka factors. A footprint-free hiPSC line was characterized by the expression of pluripotency markers and a normal karyotype. These cells were able to give rise to Embryoid Bodies and to a progeny of differentiated cells belonging to the 3 germ layers. This hiPSC line represents a suitable tool for modelling in vitro MMD disease to investigate the cellular mechanisms underlying the occurrence of this pathology.

Establishment of induced pluripotent stem cell (iPSC) line from an 8-year old female patient with ischemic Moyamoya disease.

Peripheral blood mononuclear cells (PBMCs) were collected from an 8-year old female patient affected by ischemic Moyamoya disease (MMD). Patient's PBMCs were reprogrammed using Sendai virus particles delivering the four Yamanaka factors. The footprint free hiPSC line expressed the major pluripotency markers and exhibited a normal karyotype. Cells were competent to give rise to progeny of differentiated cells belonging to the 3 germ layers. This hiPSC line represents a good tool to in vitro model MMD in order to shed light on the cellular and molecular mechanisms responsible for the occurrence of this syndrome.

The activation of M2 muscarinic receptor inhibits cell growth and survival in human glioblastoma cancer stem cells.

The involvement of muscarinic receptors in cancer has been reported. Recently we have demonstrated that the activation of M2 muscarinic receptors, through arecaidine propargyl ester, arrests cell proliferation and induces apoptosis in primary and established glioblastoma cell lines. Considering the inability of conventional drugs to completely counteract the growth of glioblastoma cancer stem cells (GSCs), we have investigated the effect produced by arecaidine on GSC growth and survival. The expression of M2 receptors has been analyzed in GSC cell lines derived from human biopsies. Based on the M2 receptor expression levels, we have selected two gliolastoma cell lines (GB7 and GB8). In both cell lines the treatment with arecaidine decreased GCS cell growth. GB7 cells exhibited a time- and dose-dependent decrease of cell proliferation. Moreover arecaidine caused a reduced cell survival in particular in GB8 cell line. These effects appear to be mediated by M2 receptor activation as suggested by pharmacological experiments performed in the presence of M1 and M3 preferring antagonists (pirenzepine and 4-DAMP respectively) and M2/M4 antagonist methoctramine. M2 receptor silencing by siRNA has further confirmed that the inhibition of cell growth arecaidine-induced was mediated by the M2 receptor activation. These results suggest that the M2 receptors may represent a new interesting therapeutic tool to counteract glioblastoma cancer stem cell growth and survival.

Establishment of a radial glia-like mouse fetal hypothalamic neural stem cell line (AC1) able to differentiate into neuroendocrine cells.

The present study describes the generation and the characterization of a stable cell line of neural stem cells derived from embryonic mouse hypothalamus. These cells (AC1) grow as an adherent culture in defined serum-free medium and express typical markers of neurogenic radial glia and of hypothalamic precursors. After prolonged expansion, AC1 cells may be efficiently induced to differentiate into neurons and astroglial cells in vitro and start to express some hormonal neuropeptides, like TRH, CRH, and POMC. Based on the capabilities of AC1 cells to be stably expanded and to develop neuroendocrine lineages in vitro, these cells might represent a novel tool to elucidate the mechanisms involved in the development of the hypothalamus and in the specific differentiation of neuroendocrine neurons.

Neurodevelopmental origin and adult neurogenesis of the neuroendocrine hypothalamus.

The adult hypothalamus regulates many physiological functions and homeostatic loops, including growth, feeding and reproduction. In mammals, the hypothalamus derives from the ventral diencephalon where two distinct ventricular proliferative zones have been described. Although a set of transcription factors regulating the hypothalamic development has been identified, the exact molecular mechanisms that drive the differentiation of hypothalamic neural precursor cells (NPCs) toward specific neuroendocrine neuronal subtypes is yet not fully disclosed. Neurogenesis has been also reported in the adult hypothalamus at the level of specific niches located in the ventrolateral region of ventricle wall, where NPCs have been identified as radial glia-like tanycytes. Here we review the molecular and cellular systems proposed to support the neurogenic potential of developing and adult hypothalamic NPCs. We also report new insights on the mechanisms by which adult hypothalamic neurogenesis modulates key functions of this brain region. Finally, we discuss how environmental factors may modulate the adult hypothalamic neurogenic cascade.