An integrative miRNA-mRNA expression analysis identifies miRNA signatures associated with SOD1 and TARDBP patient-derived motor neurons.

MicroRNAs (miRNAs) are a subset of small non-coding single-stranded RNA molecules involved in the regulation of post-transcriptional gene expression of a variety of transcript targets. Therefore altered miRNA expression may result in the dysregulation of key genes and biological pathways that has been reported with the onset and progression of neurodegenerative diseases, such as Amyotrophic lateral sclerosis (ALS). ALS is marked by a progressive degeneration of motor neurons (MNs) present in the spinal cord, brain stem and motor cortex. Although the pathomechanism underlying molecular interactions of ALS remains poorly understood, alterations in RNA metabolism, including dysregulation of miRNA expression in familial as well as sporadic forms are still scarcely studied. In this study, we performed combined transcriptomic data and miRNA profiling in MN samples of the same samples of iPSC-derived MNs from SOD1- and TARDBP (TDP-43 protein)-mutant-ALS patients and healthy controls. We report a global upregulation of mature miRNAs, and suggest that differentially expressed (DE) miRNAs have a significant impact on mRNA-level in SOD1-, but not in TARDBP-linked ALS. Furthermore, in SOD1-ALS we identified dysregulated miRNAs such as miR-124-3p, miR-19b-3p and miR-218 and their potential targets previously implicated in important functional process and pathogenic pathways underlying ALS. These miRNAs may play key roles in the neuronal development and cell survival related functions in SOD1-ALS. Altogether, we provide evidence of miRNA regulated genes expression mainly in SOD1 rather than TDP43-ALS.

RANBP17 Overexpression Restores Nucleocytoplasmic Transport and Ameliorates Neurodevelopment in Induced DYT1 Dystonia Motor Neurons.

DYT1 dystonia is a debilitating neurological movement disorder, and it represents the most frequent and severe form of hereditary primary dystonia. There is currently no cure for this disease due to its unclear pathogenesis. In our previous study utilizing patient-specific motor neurons (MNs), we identified distinct cellular deficits associated with the disease, including a deformed nucleus, disrupted neurodevelopment, and compromised nucleocytoplasmic transport (NCT) functions. However, the precise molecular mechanisms underlying these cellular impairments have remained elusive. In this study, we revealed the genome-wide changes in gene expression in DYT1 MNs through transcriptomic analysis. We found that those dysregulated genes are intricately involved in neurodevelopment and various biological processes. Interestingly, we identified that the expression level of RANBP17, a RAN-binding protein crucial for NCT regulation, exhibited a significant reduction in DYT1 MNs. By manipulating RANBP17 expression, we further demonstrated that RANBP17 plays an important role in facilitating the nuclear transport of both protein and transcript cargos in induced human neurons. Excitingly, the overexpression of RANBP17 emerged as a substantial mitigating factor, effectively restoring impaired NCT activity and rescuing neurodevelopmental deficits observed in DYT1 MNs. These findings shed light on the intricate molecular underpinnings of impaired NCT in DYT1 neurons and provide novel insights into the pathophysiology of DYT1 dystonia, potentially leading to the development of innovative treatment strategies.

Cardiomyocyte differentiation from human induced pluripotent stem cells is delayed following knockout of Bcl-2.

Anti-apoptotic B-cell lymphoma 2 (Bcl-2) regulates a wide array of cellular functions involved in cell death, cell survival and autophagy. Less known is its involvement in the differentiation of cardiomyocytes. As a consequence, mechanisms by which Bcl-2 contributes to cardiac differentiation remain to be elucidated. To address this, we used CRISPR/Cas9 to knockout (KO) BCL2 in human induced pluripotent stem cells (hiPSCs) and investigated the consequence of this KO for differentiation towards cardiomyocytes. Our results indicate that differentiation of hiPSCs to cardiomyocytes was delayed following BCL2 KO. This was not related to the canonical anti-apoptotic function of Bcl-2. This delay led to reduced expression and activity of the cardiomyocyte Ca2+ toolkit. Finally, Bcl-2 KO reduced c-Myc expression and nuclear localization in the early phase of the cardiac differentiation process, which accounts at least in part for the observed delay in the cardiac differentiation. These results suggest that there is a central role for Bcl-2 in cardiomyocyte differentiation and maturation.

Vascularized Brain Assembloids With Enhanced Cellular Complexity Provide Insights Into the Cellular Deficits of Tauopathy.

Advanced technologies have enabled the engineering of self-organized 3-dimensional (3D) cellular structures from human induced pluripotent stem cells (hiPSCs), namely organoids, which recapitulate some key features of tissue development and functions of the human central nervous system (CNS). While hiPSC-derived 3D CNS organoids hold promise in providing a human-specific platform for studying CNS development and diseases, most of them do not incorporate the full range of implicated cell types, including vascular cell components and microglia, limiting their ability to accurately recreate the CNS environment and their utility in the study of certain aspects of the disease. Here we have developed a novel approach, called vascularized brain assembloids, for constructing hiPSC-derived 3D CNS structures with a higher level of cellular complexity. This is achieved by integrating forebrain organoids with common myeloid progenitors and phenotypically stabilized human umbilical vein endothelial cells (VeraVecs), which can be cultured and expanded in serum-free conditions. Compared with organoids, these assembloids exhibited enhanced neuroepithelial proliferation, advanced astrocytic maturation, and increased synapse numbers. Strikingly, the assembloids derived from hiPSCs harboring the tauP301S mutation exhibited increased levels of total tau and phosphorylated tau, along with a higher proportion of rod-like microglia-like cells and enhanced astrocytic activation, when compared to the assembloids derived from isogenic hiPSCs. Additionally, the tauP301S assembloids showed an altered profile of neuroinflammatory cytokines. This innovative assembloid technology serves as a compelling proof-of-concept model, opening new avenues for unraveling the intricate complexities of the human brain and accelerating progress in the development of effective treatments for neurological disorders.

Utility of human induced pluripotent stem cell-derived small intestinal epithelial cells for pharmacokinetic, toxicological, and immunological studies.

The small intestine, which plays a crucial role in the absorption and metabolism of drugs and foods, serves as a target organ for drug-induced toxicity and immune interactions with functional foods and intestinal bacteria. Current alternative models of the human small intestine, such as Caco-2 cells and experimental animals, have limitations due to variations in the expression levels of metabolic enzymes, transporters, and receptors. This study presents investigations into the utility of human induced pluripotent stem cell-derived small intestinal epithelial cells (hiSIECs) for pharmacokinetic, toxicological, and immunological studies, respectively. While hiSIECs displayed small intestinal epithelial cell characteristics and barrier function, they demonstrated pharmacokinetic properties such as cytochrome P450 3A4/5 activity equivalent to human primary enterocytes and stable P-glycoprotein activity. These cells also demonstrated potential for assessing two forms of intestinal toxicity caused by anticancer drugs and gamma-secretase inhibitors, displaying immune responses mediated by toll-like and fatty acid receptors while serving as an inflammatory gut model through the addition of tumor necrosis factor alpha and interferon gamma. Overall, hiSIECs hold promise as an in vitro model for assessing pharmacokinetics, toxicity, and effects on the intestinal immunity of pharmaceuticals, functional foods, supplements, and intestinal bacteria.

Engineering Stem Cell Fate Controlling Biomaterials to Develop Muscle Connective Tissue Layered Myofibers.

Skeletal muscle connective tissue (MCT) surrounds myofiber bundles to provide structural support, produce force transduction from tendons, and regulate satellite cell differentiation during muscle regeneration. Engineered muscle tissue composed of myofibers layered within MCT has not yet been developed. Herein, a bioengineering strategy to create MCT-layered myofibers through the development of stem cell fate-controlling biomaterials that achieve both myogenesis and fibroblast differentiation in a locally controlled manner at the single construct is introduced. The reciprocal role of transforming growth factor-beta 1 (TGF-ß1) and its inhibitor as well as 3D matrix stiffness to achieve co-differentiation of MCT fibroblasts and myofibers from a human-induced pluripotent stem cell (hiPSC)-derived paraxial mesoderm is studied. To avoid myogenic inhibition, TGF-ß1 is conjugated on the gelatin-based hydrogel to control the fibroblasts' populations locally; the TGF-ß1 degrades after 2 weeks, resulting in increased MCT-specific extracellular matrix (ECM) production. The locations of myofibers and fibroblasts are precisely controlled by using photolithography and co-axial wet spinning techniques, which results in the formation of MCT-layered functional myofibers in 3D constructs. This advanced engineering strategy is envisioned as a possible method for obtaining biomimetic human muscle grafts for various biomedical applications.

Comparative transcriptomic and phenotypic analysis of induced pluripotent stem cell hepatocyte-like cells and primary human hepatocytes.

Primary human hepatocytes (PHHs) are used extensively for in vitro liver cultures to study hepatic functions. However, limited availability and invasive retrieval prevent their widespread use. Induced pluripotent stem cells exhibit significant potential since they can be obtained non-invasively and differentiated into hepatic lineages, such as hepatocyte-like cells (iHLCs). However, there are concerns about their fetal phenotypic characteristics and their hepatic functions compared to PHHs in culture. Therefore, we performed an RNA-sequencing (RNA-seq) analysis to understand pathways that are either up- or downregulated in each cell type. Analysis of the RNA-seq data showed an upregulation in the bile secretion pathway where genes such as AQP9 and UGT1A1 were higher expressed in PHHs compared to iHLCs by 455- and 15-fold, respectively. Upon immunostaining, bile canaliculi were shown to be present in PHHs. The TCA cycle in PHHs was upregulated compared to iHLCs. Cellular analysis showed a 2-2.5-fold increase in normalized urea production in PHHs compared to iHLCs. In addition, drug metabolism pathways, including cytochrome P450 (CYP450) and UDP-glucuronosyltransferase enzymes, were upregulated in PHHs compared to iHLCs. Of note, CYP2E1 gene expression was significantly higher (21,810-fold) in PHHs. Acetaminophen and ethanol were administered to PHH and iHLC cultures to investigate differences in biotransformation. CYP450 activity of baseline and toxicant-treated samples was significantly higher in PHHs compared to iHLCs. Our analysis revealed that iHLCs have substantial differences from PHHs in critical hepatic functions. These results have highlighted the differences in gene expression and hepatic functions between PHHs and iHLCs to motivate future investigation.

RNA-Seq time-course analysis of neural precursor cell transcriptome in response to herpes simplex Virus-1 infection.

The neurogenic niches within the central nervous system serve as essential reservoirs for neural precursor cells (NPCs), playing a crucial role in neurogenesis. However, these NPCs are particularly vulnerable to infection by the herpes simplex virus 1 (HSV-1). In the present study, we investigated the changes in the transcriptome of NPCs in response to HSV-1 infection using bulk RNA-Seq, compared to those of uninfected samples, at different time points post infection and in the presence or absence of antivirals. The results showed that NPCs upon HSV-1 infection undergo a significant dysregulation of genes playing a crucial role in aspects of neurogenesis, including genes affecting NPC proliferation, migration, and differentiation. Our analysis revealed that the CREB signaling, which plays a crucial role in the regulation of neurogenesis and memory consolidation, was the most consistantly downregulated pathway, even in the presence of antivirals. Additionally, cholesterol biosynthesis was significantly downregulated in HSV-1-infected NPCs. The findings from this study, for the first time, offer insights into the intricate molecular mechanisms that underlie the neurogenesis impairment associated with HSV-1 infection.

iPSC-Derived Airway Epithelial Cells: Progress, Promise, and Challenges.

Induced pluripotent stem cells (iPSCs) generated from somatic cell sources are pluripotent and capable of indefinite expansion in vitro. They provide an unlimited source of cells that can be differentiated into lung progenitor cells for potential clinical use in pulmonary regenerative medicine. This review gives a comprehensive overview of recent progress toward the use of iPSCs to generate proximal and distal airway epithelial cells and mix lung organoids. Furthermore, their potential applications and future challenges for the field are discussed, with a focus on the technological hurdles that must be cleared before stem cell therapeutics can be used for clinical treatment.

ETV2 Upregulation Marks the Specification of Early Cardiomyocytes and Endothelial Cells During Co-differentiation.

The ability to differentiate human-induced pluripotent stem cells (hiPSCs) efficiently into defined cardiac lineages, such as cardiomyocytes and cardiac endothelial cells, is crucial to study human heart development and model cardiovascular diseases in vitro. The mechanisms underlying the specification of these cell types during human development are not well understood which limits fine-tuning and broader application of cardiac model systems. Here, we used the expression of ETV2, a master regulator of hematoendothelial specification in mice, to identify functionally distinct subpopulations during the co-differentiation of endothelial cells and cardiomyocytes from hiPSCs. Targeted analysis of single-cell RNA-sequencing data revealed differential ETV2 dynamics in the 2 lineages. A newly created fluorescent reporter line allowed us to identify early lineage-predisposed states and show that a transient ETV2-high-state initiates the specification of endothelial cells. We further demonstrated, unexpectedly, that functional cardiomyocytes can originate from progenitors expressing ETV2 at a low level. Our study thus sheds light on the in vitro differentiation dynamics of 2 important cardiac lineages.

In vitro platform to model the function of ionocytes in the human airway epithelium.

BACKGROUND: Pulmonary ionocytes have been identified in the airway epithelium as a small population of ion transporting cells expressing high levels of CFTR (cystic fibrosis transmembrane conductance regulator), the gene mutated in cystic fibrosis. By providing an infinite source of airway epithelial cells (AECs), the use of human induced pluripotent stem cells (hiPSCs) could overcome some challenges of studying ionocytes. However, the production of AEC epithelia containing ionocytes from hiPSCs has proven difficult. Here, we present a platform to produce hiPSC-derived AECs (hiPSC-AECs) including ionocytes and investigate their role in the airway epithelium. METHODS: hiPSCs were differentiated into lung progenitors, which were expanded as 3D organoids and matured by air-liquid interface culture as polarised hiPSC-AEC epithelia. Using CRISPR/Cas9 technology, we generated a hiPSCs knockout (KO) for FOXI1, a transcription factor that is essential for ionocyte specification. Differences between FOXI1 KO hiPSC-AECs and their wild-type (WT) isogenic controls were investigated by assessing gene and protein expression, epithelial composition, cilia coverage and motility, pH and transepithelial barrier properties. RESULTS: Mature hiPSC-AEC epithelia contained basal cells, secretory cells, ciliated cells with motile cilia, pulmonary neuroendocrine cells (PNECs) and ionocytes. There was no difference between FOXI1 WT and KO hiPSCs in terms of their capacity to differentiate into airway progenitors. However, FOXI1 KO led to mature hiPSC-AEC epithelia without ionocytes with reduced capacity to produce ciliated cells. CONCLUSION: Our results suggest that ionocytes could have role beyond transepithelial ion transport by regulating epithelial properties and homeostasis in the airway epithelium.

Good Manufacturing Practice-compliant human induced pluripotent stem cells: from bench to putative clinical products.

BACKGROUND AIMS: Few human induced pluripotent stem cell (hiPSC) lines are Good Manufacturing Practice (GMP)-compliant, limiting the clinical use of hiPSC-derived products. Here, we addressed this by establishing and validating an in-house platform to produce GMP-compliant hiPSCs that would be appropriate for producing both allogeneic and autologous hiPSC-derived products. METHODS: Our standard research protocol for hiPSCs production was adapted and translated into a GMP-compliant platform. In addition to the generation of GMP-compliant hiPSC, the platform entails the methodology for donor recruitment, consent and screening, donor material procurement, hiPSCs manufacture, in-process control, specific QC test validation, QC testing, product release, hiPSCs storage and stability testing. For platform validation, one test run and three production runs were performed. Highest-quality lines were selected to establish master cell banks (MCBs). RESULTS: Two MCBs were successfully released under GMP conditions. They demonstrated safety (sterility, negative mycoplasma, endotoxins <5.0 EU/mL and negative adventitious agents), cell identity (>75% of cells expressing markers of undifferentiated state, identical STR profile, normal karyotype in >20 metaphases), purity (negative residual vectors and no plasmid integration in the genome) and potency (expression of at least two of the three markers for each of the three germ layers). In addition, directed differentiation to somitoids (skeletal muscle precursors) and six potential clinical products from all three germ layers was achieved: pancreatic islets (endoderm), kidney organoids and cardiomyocytes (mesoderm), and keratinocytes, GABAergic interneurons and inner-ear organoids (ectoderm). CONCLUSIONS: We successfully developed and validated a platform for generating GMP-compliant hiPSC lines. The two MCBs released were shown to differentiate into clinical products relevant for our own and other regenerative medicine interests.

High and low permeability of human pluripotent stem cell-derived blood-brain barrier models depend on epithelial or endothelial features.

The search for reliable human blood-brain barrier (BBB) models represents a challenge for the development/testing of strategies aiming to enhance brain delivery of drugs. Human-induced pluripotent stem cells (hiPSCs) have raised hopes in the development of predictive BBB models. Differentiating strategies are thus required to generate endothelial cells (ECs), a major component of the BBB. Several hiPSC-based protocols have reported the generation of in vitro models with significant differences in barrier properties. We studied in depth the properties of iPSCs byproducts from two protocols that have been established to yield these in vitro barrier models. Our analysis/study reveals that iPSCs derivatives endowed with EC features yield high permeability models while the cells that exhibit outstanding barrier properties show principally epithelial cell-like (EpC) features. We found that models containing EpC-like cells express tight junction proteins, transporters/efflux pumps and display a high functional tightness with very low permeability, which are features commonly shared between BBB and epithelial barriers. Our study demonstrates that hiPSC-based BBB models need extensive characterization beforehand and that a reliable human BBB model containing EC-like cells and displaying low permeability is still needed.

A highly efficient cell culture method using oxygen-permeable PDMS-based honeycomb microwells produces functional liver organoids from human induced pluripotent stem cell-derived carboxypeptidase M liver progenitor cells.

Recent advancements in bioengineering have introduced potential alternatives to liver transplantation via the development of self-assembled liver organoids, derived from human-induced pluripotent stem cells (hiPSCs). However, the limited maturity of the tissue makes it challenging to implement this technology on a large scale in clinical settings. In this study, we developed a highly efficient method for generating functional liver organoids from hiPSC-derived carboxypeptidase M liver progenitor cells (CPM+ LPCs), using a microwell structure, and enhanced maturation through direct oxygenation in oxygen-permeable culture plates. We compared the morphology, gene expression profile, and function of the liver organoid with those of cells cultured under conventional conditions using either monolayer or spheroid culture systems. Our results revealed that liver organoids generated using polydimethylsiloxane-based honeycomb microwells significantly exhibited enhanced albumin secretion, hepatic marker expression, and cytochrome P450-mediated metabolism. Additionally, the oxygenated organoids consisted of both hepatocytes and cholangiocytes, which showed increased expression of bile transporter-related genes as well as enhanced bile transport function. Oxygen-permeable polydimethylsiloxane membranes may offer an efficient approach to generating highly mature liver organoids consisting of diverse cell populations.

Brain organoids: A revolutionary tool for modeling neurological disorders and development of therapeutics.

Brain organoids are self-organized, three-dimensional (3D) aggregates derived from pluripotent stem cells that have cell types and cellular architectures resembling those of the developing human brain. The current understanding of human brain developmental processes and neurological disorders has advanced significantly with the introduction of this in vitro model. Brain organoids serve as a translational link between two-dimensional (2D) cultures and in vivo models which imitate the neural tube formation at the early and late stages and the differentiation of neuroepithelium with whole-brain regionalization. In addition, the generation of region-specific brain organoids made it possible to investigate the pathogenic and etiological aspects of acquired and inherited brain disease along with drug discovery and drug toxicity testing. In this review article, we first summarize an overview of the existing methods and platforms used for generating brain organoids and their limitations and then discuss the recent advancement in brain organoid technology. In addition, we discuss how brain organoids have been used to model aspects of neurodevelopmental and neurodegenerative diseases, including autism spectrum disorder (ASD), Rett syndrome, Zika virus-related microcephaly, Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD).

RBPMS2 Is a Myocardial-Enriched Splicing Regulator Required for Cardiac Function.

BACKGROUND: RBPs (RNA-binding proteins) perform indispensable functions in the post-transcriptional regulation of gene expression. Numerous RBPs have been implicated in cardiac development or physiology based on gene knockout studies and the identification of pathogenic RBP gene mutations in monogenic heart disorders. The discovery and characterization of additional RBPs performing indispensable functions in the heart will advance basic and translational cardiovascular research. METHODS: We performed a differential expression screen in zebrafish embryos to identify genes enriched in nkx2.5-positive cardiomyocytes or cardiopharyngeal progenitors compared to nkx2.5-negative cells from the same embryos. We investigated the myocardial-enriched gene RNA-binding protein with multiple splicing (variants) 2 [RBPMS2)] by generating and characterizing rbpms2 knockout zebrafish and human cardiomyocytes derived from RBPMS2-deficient induced pluripotent stem cells. RESULTS: We identified 1848 genes enriched in the nkx2.5-positive population. Among the most highly enriched genes, most with well-established functions in the heart, we discovered the ohnologs rbpms2a and rbpms2b, which encode an evolutionarily conserved RBP. Rbpms2 localizes selectively to cardiomyocytes during zebrafish heart development and strong cardiomyocyte expression persists into adulthood. Rbpms2-deficient embryos suffer from early cardiac dysfunction characterized by reduced ejection fraction. The functional deficit is accompanied by myofibril disarray, altered calcium handling, and differential alternative splicing events in mutant cardiomyocytes. These phenotypes are also observed in RBPMS2-deficient human cardiomyocytes, indicative of conserved molecular and cellular function. RNA-sequencing and comparative analysis of genes mis-spliced in RBPMS2-deficient zebrafish and human cardiomyocytes uncovered a conserved network of 29 ortholog pairs that require RBPMS2 for alternative splicing regulation, including RBFOX2, SLC8A1, and MYBPC3. CONCLUSIONS: Our study identifies RBPMS2 as a conserved regulator of alternative splicing, myofibrillar organization, and calcium handling in zebrafish and human cardiomyocytes.

Aberrant early hematopoietic progenitor formation marks the onset of hematopoietic defects in Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure disorder that often presents at infancy. Progress has been made in revealing causal mutated genes (SBDS and others), ribosome defects, and hematopoietic aberrations in SDS. However, the mechanism underlying the hematopoietic failure remained unknown, and treatment options are limited. Herein, we investigated the onset of SDS embryonic hematopoietic impairments. We generated SDS and control human-derived induced pluripotent stem cells (iPSCs). SDS iPSCs recapitulated the SDS hematological phenotype. Detailed stepwise evaluation of definitive hematopoiesis revealed defects that started at the early emerging hematopoietic progenitor (EHP) stage after mesoderm and hemogenic endothelium were normally induced. Hematopoietic potential of EHPs was markedly reduced, and the introduction of SBDS in SDS iPSCs improved colony formation. Transcriptome analysis revealed reduced expression of ribosome and oxidative phosphorylation-related genes in undifferentiated and differentiated iPSCs. However, certain pathways (e.g., DNA replication) and genes (e.g., CHCHD2) were exclusively or more severely dysregulated in EHPs compared with earlier and later stages. To our knowledge, this study offers for the first time an insight into the embryonic onset of human hematopoietic defects in an inherited bone marrow failure syndrome and reveals cellular and molecular aberrations at critical stages of hematopoietic development toward EHPs.

A review of local anesthetic-induced heart toxicity using human induced pluripotent stem cell-derived cardiomyocytes.

Local anesthetic (LA) cardiotoxicity is one of the main health problems in anesthesiology and pain management. This study reviewed the reported LA-induced cardiac toxicity types, risk factors, management, and mechanisms, with attention to the use of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in heart toxicity research. Important scientific databases were searched to find relevant articles. We briefly assessed the reported cardiotoxic effects of different types of LA drugs, including ester- and amide-linked LA agents. Furthermore, cardiotoxic effects and clinical manifestations, strategies for preventing and managing LA-induced cardiotoxic effects, pharmacokinetics, pharmacodynamics, and sodium channel dynamics regarding individual variability and genetic influences were discussed in this review. The applications and importance of hiPSC-CMs cellular model for evaluating the cardiotoxic effects of LA drugs were discussed in detail. This review also explored hiPSC-CMs' potential in risk assessment, drug screening, and developing targeted therapies. The main mechanisms underlying LA-induced cardiotoxicity included perturbation in sodium channels, ROS production, and disorders in the immune system response due to the presence of LA drugs. Furthermore, drug-specific characteristics including pharmacokinetics and pharmacodynamics are important determinants after LA drug injection. In addition, individual patient factors such as age, comorbidities, and genetic variability emphasize the need for a personalized approach to mitigate risks and enhance patient safety. The strategies outlined for the prevention and management of LA cardiotoxicity underscore the importance of careful dosing, continuous monitoring, and the immediate availability of resuscitation equipment. This comprehensive review can be used to guide future investigations into better understanding LA cardiac toxicities and improving patient safety.

Neurofilament accumulations in amyotrophic lateral sclerosis patients' motor neurons impair axonal initial segment integrity.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron (MN) disease in adults with no curative treatment. Neurofilament (NF) level in patient' fluids have recently emerged as the prime biomarker of ALS disease progression, while NF accumulation in MNs of patients is the oldest and one of the best pathological hallmarks. However, the way NF accumulations could lead to MN degeneration remains unknown. To assess NF accumulations and study the impact on MNs, we compared MNs derived from induced pluripotent stem cells (iPSC) of patients carrying mutations in C9orf72, SOD1 and TARDBP genes, the three main ALS genetic causes. We show that in all mutant MNs, light NF (NF-L) chains rapidly accumulate in MN soma, while the phosphorylated heavy/medium NF (pNF-M/H) chains pile up in axonal proximal regions of only C9orf72 and SOD1 MNs. Excitability abnormalities were also only observed in these latter MNs. We demonstrate that the integrity of the MN axonal initial segment (AIS), the region of action potential initiation and responsible for maintaining axonal integrity, is impaired in the presence of pNF-M/H accumulations in C9orf72 and SOD1 MNs. We establish a strong correlation between these pNF-M/H accumulations, an AIS distal shift, increased axonal calibers and modified repartition of sodium channels. The results expand our understanding of how NF accumulation could dysregulate components of the axonal cytoskeleton and disrupt MN homeostasis. With recent cumulative evidence that AIS alterations are implicated in different brain diseases, preserving AIS integrity could have important therapeutic implications for ALS.

The efficient induction of human retinal ganglion-like cells provides a platform for studying optic neuropathies.

Retinal ganglion cells (RGCs) are essential for vision perception. In glaucoma and other optic neuropathies, RGCs and their optic axons undergo degenerative change and cell death; this can result in irreversible vision loss. Here we developed a rapid protocol for directly inducing RGC differentiation from human induced pluripotent stem cells (hiPSCs) by the overexpression of ATOH7, BRN3B, and SOX4. The hiPSC-derived RGC-like cells (iRGCs) show robust expression of various RGC-specific markers by whole transcriptome profiling. A functional assessment was also carried out and this demonstrated that these iRGCs display stimulus-induced neuronal activity, as well as spontaneous neuronal activity. Ethambutol (EMB), an effective first-line anti-tuberculosis agent, is known to cause serious visual impairment and irreversible vision loss due to the RGC degeneration in a significant number of treated patients. Using our iRGCs, EMB was found to induce significant dose-dependent and time-dependent increases in cell death and neurite degeneration. Western blot analysis revealed that the expression levels of p62 and LC3-II were upregulated, and further investigations revealed that EMB caused a blockade of lysosome-autophagosome fusion; this indicates that impairment of autophagic flux is one of the adverse effects of that EMB has on iRGCs. In addition, EMB was found to elevate intracellular reactive oxygen species (ROS) levels increasing apoptotic cell death. This could be partially rescued by the co-treatment with the ROS scavenger NAC. Taken together, our findings suggest that this iRGC model, which achieves both high yield and high purity, is suitable for investigating optic neuropathies, as well as being useful when searching for potential drugs for therapeutic treatment and/or disease prevention.