Search Results (415)

Cardiovascular disease (CVD) is the leading cause of mortality worldwide. The healthy adult heart depends on flexible energy use, but a diseased or injured heart is associated with a loss of flexibility and metabolic remodeling. Since metabolism plays a central role in cardiac health and disease, there is a growing need to understand how metabolic reprogramming contributes to cardiac dysfunction and impaired CM maturation. Human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) are widely used as a platform to study human cardiac development and disease mechanisms. However, current models are limited by metabolic and structural immaturity. This review provides an overview of the dynamic shifts in cardiac metabolic states from fetal development to senescence, while delineating the metabolic signatures of healthy versus disease states. These metabolic switches are orchestrated by a complex interplay of upstream signals driven by variations in substrate availability, post-translational modifications and key transcriptional regulatory networks, which ultimately regulate downstream cardiac remodeling and pathological cascades. As cardiac metabolic function is affected by a coordinated multicellular network, this review also includes the metabolic crosstalk between CMs and non-CMs, including fibroblasts, endothelial cells and immune cells. In addition, various strategies to further mature hiPSC-CMs are summarized to enhance their metabolic profiles. Investigating cardiac metabolic shifts bridges developmental biology, stem cell biology, and regenerative cardiology by revealing how energy metabolism governs cellular identity, maturation, and regenerative potential. These insights are essential for improving stem-cell-derived CMs for disease modeling, drug discovery, and heart repair. Full article

Background/Objectives: Only substantial quantities of xeno-free human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) (hiPSC-CMs) with stable quality and structural and functional maturity can meet the demand for cardiac cell therapy. The use of xeno-free microcarriers can significantly increase cell yield. Co-culturing with hematopoietic stem cells (HSCs) simulates the environment in vivo and has a necessary impact on the development of CMs. However, no microcarrier-based protocol for xeno-free hiPSC-CM culture has yet been established, and the effects of HSCs on CM development and their underlying mechanisms remain unclear. Therefore, this study aims to investigate these issues. Methods: We used a xeno-free microcarrier (plastic) culture system coated by a defined xeno-free matrix (MatriClone) to expand hiPSCs and hiPSC-CMs with human hematopoietic stem cells (hHSCs). Using RNA sequencing (RNA-seq), cytokine assay, and various cellular molecular techniques, we investigated the role of hHSCs in cardiac differentiation and maturation, and underlying mechanisms. Results: hiPSCs were evenly distributed on the surface of plastic coated with 1 μg/cm² MatriClone (MatriClone-Plastic), increasing and sustaining pluripotency marker levels. Directed differentiation of hiPSCs on 1 μg/cm² MatriClone-Plastic induced a larger number of CMs, and the level of cardiac differentiation was also significantly improved. When hHSCs were co-cultured with cells at the cardiac progenitor cell stage, results from electron microscopy, electrophysiology, and qPCR showed that hiPSC-CMs significantly promoted cardiac structural and functional maturation. The co-cultured hHSCs released multiple cytokines that were changed dynamically at different time points, and that were highly likely to activate the epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway to promote cardiac development and maturation. Conclusions: hHSCs can efficiently promote differentiation and maturation of xeno-free hiPSC-CMs on MatriClone-Plastic via the EGFR/MAPK/ERK signaling pathway. Full article

The myocardium possesses one of the highest vascular densities in the body. The outermost wall layer of large and medium-sized vessels, the adventitia, forms a critical interface between the vasculature and the myocardium and serves as a reservoir for stem and progenitor cells capable of differentiating into all vascular wall lineages as well as innate immune cells, including macrophages. Current cardiac organoid models intrinsically develop networks of endothelial cords and small capillary-like structures that resemble cardiac microvessels. However, these microvessels mostly lack an adventitial compartment in vivo. Here, we present a potential alternative assembloid strategy that combines vascular segments from mouse and human origin with either cardiomyocytes or cardiac spheroids derived from human induced pluripotent stem cells, thereby incorporating large diameter vessels and the vascular adventitia into a cardiac tissue model. Within the assembloids, the myocardial component remained contractile and connected to the vascular adventitia, which displayed cellular sprouting toward the hiPSC-derived cardiac tissue. Immunostaining for vascular and immune markers revealed that the adventitia gave rise to endothelial sprouts and macrophage-like cells which integrated into the myocardial tissue. In summary, we present proof of concept for complex assembloids composed of vessel segments and human iPSC-derived cardiomyocytes which contain and maintain an in vivo-like adventitial compartment. We suggest this model may serve as a platform for investigating myocardial–stromal interactions, cardiac tissue repair, and functional remodeling under both physiological and pathological conditions. Furthermore, the incorporation of large-lumen vessel segments may enable future experimental perfusion, rendering the model particularly suitable for drug testing via intravascular delivery. Full article

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by progressive memory impairment and cognitive decline. The APOE4 allele represents one of the most prominent genetic risk factors. In this study, we investigated the impact of APOE4 on the cholinergic neuronal development and on the neuronal inflammatory response to TNF-α stimulation. To address this, human induced pluripotent stem cells (hiPSCs) carrying a homozygous APOE4 genotype and an isogenic APOE3 control were differentiated into cholinergic-like induced neurons (iNs) by LHX8 overexpression. APOE4 was associated with accelerated early neuronal differentiation, as reflected by earlier downregulation of the progenitor marker Nestin. However, delayed expression of synaptophysin indicated impaired synaptic maturation. Functionally, APOE3 iNs exhibited a robust but temporally regulated response to TNF-α, whereas APOE4 iNs were characterized by a delayed yet sustained induction of inflammatory signaling. Moreover, APOE4 iNs displayed an enhanced stress-associated transcriptional response at early differentiation stages. Collectively, these findings suggest that APOE4 influences both neuronal development and the timing and persistence of inflammatory responses, potentially predisposing cholinergic neurons to later dysfunction in AD. Full article

Current protocols for generating cortical interneurons from human pluripotent stem cells are hindered by slow differentiation kinetics and poor reproducibility across cell lines. Here, we present a defined small-molecule-based strategy that efficiently directs human-induced neural stem cells (hiNSCs) toward cortical GABAergic interneurons within 14–18 days, which is substantially faster than conventional methods. Short-term dual-SMAD and WNT inhibition rapidly commits hiNSCs to an interneuron progenitor fate, reaching transcriptional states equivalent to those obtained with prolonged protocols. Prolonged activation of Sonic Hedgehog (via SAG) further enhances lineage specification, markedly upregulating NKX2.1, FOXG1, GABA, somatostatin (SST), and parvalbumin (PV) expression, and enriching pathways associated with early functional maturation. Importantly, RNA-sequencing reveals that under identical induction conditions, hiNSCs differentiate more rapidly and homogeneously than human-induced pluripotent stem cells (hiPSCs), which exhibit broader, less lineage-focused transcriptional trajectories. This differentiation strategy is highly reproducible across four genetically distinct hiNSC lines, with minimal off-target populations. Functionally, hiNSC-derived cortical interneurons display robust migratory behavior, produce abundant GABA, and survive transplantation into the adult mouse hippocampus, where they extend processes and form synapse-like structures. These findings establish a rapid, scalable, and robust approach for generating human cortical interneurons, supporting their safety and integration potential as a foundation for future cell replacement strategies in neurological disorders. Full article

Thyroid hormones (THs) are essential regulators of human brain development, and disrupted TH availability during pregnancy or early life is linked to adverse neurodevelopmental outcomes. Concerns that environmental chemicals interfere with TH signalling have increased the need for human-relevant in vitro systems to identify thyroid hormone system-disrupting chemicals (THSDCs) for risk assessment. Here, we compared two human-induced pluripotent stem cell (hiPSC)-derived brain organoid models for THSDC assessment: (i) human cortical organoids (COs) generated by unguided differentiation, offering higher architectural complexity but lower throughput; and (ii) neural stem cell-derived organoids (NSCOs), designed for scalability with reduced cellular diversity. Both models expressed key TH handling components, including the transporter SLC16A2 (MCT8) and the inactivating enzyme DIO3. Using LC–MS/MS, we show that exogenous T3 is depleted from culture media and metabolized to 3,3′-T2 and 3′-T1 in both models, alongside upregulation of T3-responsive genes (HR, KLF9, DIO3, SEMA3C). Pulse and chronic co-exposures to reference disruptors iopanoic acid (IA, deiodinase inhibitor) and silychristin (SC, MCT8 inhibitor) altered T3 metabolism and modulated T3-responsive transcriptional endpoints. In NSCOs, high-content imaging revealed treatment-associated changes in cell composition, with chronic T3 reducing the SOX2-positive progenitor pool and THSDCs blocking this effect. Together, these findings provide a framework for organoid qualification—linking TH handling, transcriptomic responsiveness, and scalable phenotypic readouts—as a necessary step toward model validation and implementation of brain organoids in THSDC risk assessment pipelines. Full article

Background/Objectives: The blood–brain barrier (BBB) presents a major challenge for delivering therapeutics to the central nervous system (CNS) due to its highly selective permeability. Human brain microvascular endothelial cells (hBMECs), the principal cellular component of the BBB, tightly regulate molecular transport and restrict the entry of many CNS-targeted therapies. Lipid-based nanoparticles have emerged as promising carriers for BBB transport because of their biocompatibility, tunable surface properties, and cargo encapsulation capabilities. One strategy to enhance nanoparticle transport involves surface functionalization with ligands that exploit endogenous transcytosis pathways. Mannose-6-phosphate (M6P), a glycan implicated in the brain entry of certain proteins and viruses, represents a potential targeting ligand for this purpose. Methods: In this study, we established a physiologically relevant in vitro BBB model using human-induced pluripotent stem cell-derived brain microvascular endothelial cells (hiPSC-BMECs) to evaluate M6P-functionalized liposomes for BBB transport. Fluorophore-labeled liposomes were used to monitor nanoparticle uptake and transcytosis. Results: M6P-functionalized liposomes exhibited significantly enhanced uptake in hiPSC-BMECs compared with non-functionalized control liposomes. Pharmacological inhibition studies supported the involvement of a clathrin-sensitive endocytic pathway. Transcytosis assays demonstrated enhanced BBB crossing of M6P-functionalized liposomes, with transport increasing according to ligand density and reaching approximately 55% of the transport observed for transferrin under the same experimental conditions. Following transcytosis, intact M6P-functionalized liposomes showed significantly higher uptake by downstream hiPSC-derived neurons and astrocytoma cells compared with control formulations. Conclusions: Together, these findings support M6P-functionalization as a promising strategy to enhance liposome uptake and transcytosis across a human-relevant in vitro BBB model. This work provides a proof-of-concept framework for the development and optimization of glycan-functionalized nanocarriers for CNS-directed delivery. Full article

Paraspeckles are subnuclear ribonucleoprotein condensates that regulate host stress responses, including those triggered by viral infection. In vitro studies using non-neuronal cells have shown the involvement of specific paraspeckle components in facilitating the replication of certain viruses, including Herpes Simplex Virus 1 (HSV-1), but these processes have not been investigated in human neuronal cells, which represent a relevant target of the virus. We employed human neural precursor cells (NPCs), neurons, and brain organoids derived from hiPSCs to investigate the previously unexplored dynamics of paraspeckle components in HSV-1-infected human neuronal cells. Our results reveal cell-type-specific differences in the expression of paraspeckle genes in response to HSV-1 infection. Unlike other viruses, HSV-1 orchestrates a previously unreported redistribution of paraspeckle proteins, leading to their accumulation in viral replication compartments (VRCs). Importantly, the expression of the paraspeckle proteins NONO and SFPQ correlates with HSV-1 permissiveness in human neuronal cells and may be required to establish a nuclear environment favoring viral transcription/replication. This enhances our understanding of how stress-response pathways in cells can be exploited by viruses in a cell-type-specific manner. Full article

Nanogrooves provide instructive cues to cells in culture. Several nanofabrication techniques have been developed to create biomimetic substrates, advancing our understanding of cell adhesion. Their integration into nervous system models highlights the critical role of the extracellular matrix (ECM) in developing functional tissue constructs for in vitro platforms such as Brain-on-Chip (BoC) and Nervous System-on-Chip (NoC). This study presents a nanofabrication approach that integrates photolithography and microtransfer molding (μTM) to pattern nanogrooves using photocurable polymer NOA81 onto microelectrode array (MEA) plates. The resulting nanogrooves exhibited a pattern periodicity of 976 nm and a ridge width of 232 nm, as confirmed by scanning electron microscopy and atomic force microscopy. We assessed the biocompatibility and functional impact of these modified substrates using human induced pluripotent stem cell (hiPSC)-derived neuronal cultures. Neurons cultured on nanogroove-modified MEAs exhibited aligned neural processes due to the anisotropic surface features and expressed vivid spiking behavior and higher burst frequency compared to randomly cultured neuronal networks. In conclusion, the proposed fabrication technique integrates nanogrooves with commercial MEAs using a combination of microtransfer molding and photolithography, resulting in modified culture substrates that enhance spike activity and network organization, aiding in the development of more in vivo-like neural models. Full article

Activating PIK3CA mutations occur in approximately 40% of hormone receptor-positive (HR+)/HER2-negative breast cancers and represent a major driver of endocrine resistance. The PI3Kα-selective inhibitor alpelisib, in combination with fulvestrant, significantly improves progression-free survival in patients with PIK3CA-mutant disease, as demonstrated in the SOLAR-1 trial. However, this therapeutic strategy is frequently complicated by treatment-induced hyperglycemia, a metabolic disturbance that promotes oxidative stress, mitochondrial dysfunction, and inflammatory signaling, thereby increasing cardiovascular vulnerability. Sodium–glucose cotransporter-2 (SGLT2) inhibitors have emerged as cardiometabolic modulators with benefits extending beyond glucose lowering. In this study, we used a human cardiomyocyte in vitro model designed to recapitulate the hyperglycemic metabolic milieu observed in breast cancer patients receiving PI3Kα-targeted therapy, to investigate whether the SGLT2 inhibitor dapagliflozin directly protects cardiomyocytes from alpelisib- and fulvestrant-induced injury. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were cultured under hyperglycemic conditions (25 mM glucose) to mimic the metabolic environment associated with PI3Kα inhibitor-induced dysglycemia. Cells were exposed to alpelisib (100 nM) and fulvestrant (100 nM), alone or in combination, in the absence or presence of dapagliflozin (1 μM). Cardiomyocyte viability was assessed using the MTS assay, mitochondrial function by TMRM-based mitochondrial membrane potential (ΔΨm) measurements, and apoptosis by caspase-3 quantification. Cardiomyocyte injury was evaluated by release of cardiac troponin I and heart-type fatty acid binding protein (H-FABP). Lipid peroxidation markers (MDA and 4-HNE) were measured to assess oxidative membrane damage. Intracellular inflammasome-related signaling (NLRP3 and MyD88) and secreted inflammatory mediators (IL-1β, IL-18, IL-6, TNF-α, and CCL2) were quantified by ELISA. Exposure to alpelisib, particularly in combination with fulvestrant, significantly reduced cardiomyocyte viability, induced mitochondrial depolarization, and increased caspase-3-mediated apoptotic signaling. These alterations were accompanied by elevated lipid peroxidation (MDA and 4-HNE) and increased release of cardiac injury biomarkers (troponin I and H-FABP). Alpelisib-based treatments also activated inflammasome-related signaling, as indicated by increased intracellular NLRP3 and MyD88 levels and enhanced secretion of pro-inflammatory mediators (IL-1β, IL-18, IL-6, TNF-α, and CCL2). Co-treatment with dapagliflozin significantly attenuated these alterations, preserving mitochondrial membrane potential, reducing apoptotic signaling, limiting oxidative membrane damage, and suppressing inflammatory cytokine release. This study provides evidence that alpelisib-based therapy under hyperglycemic conditions is associated with oxidative, mitochondrial, and inflammatory stress responses in human cardiomyocytes, recapitulating key features of cardiometabolic stress relevant to PI3Kα-targeted therapy. Importantly, dapagliflozin markedly attenuated these alterations, supporting a potential cardioprotective role that may extend beyond glycemic control. These findings provide a mechanistic rationale for further investigation of SGLT2 inhibition as a cardiometabolic protective strategy in patients receiving PI3Kα inhibitor-based cancer therapy. Full article

Podocyte injury is a characteristic feature of focal segmental glomerulosclerosis (FSGS) that leads to the development of nephrosis as its loss causes proteinuria and progressive glomerulosclerosis. The physiological function of podocytes is critically dependent on proper intracellular calcium levels; an excess or shortage of calcium influx in these cells may result in foot process effacement, apoptosis, and nephron degeneration. A key protein responsible for the regulation of calcium flux is the canonical transient receptor potential 6 (TRPC6) expressed in podocytes. Several mutations in the TRPC6 gene have been associated with FSGS. Here we present a systematically optimized inducible FSGS model system in human induced pluripotent stem cells (hiPSCs). We generated and phenotypically characterized three transgenic hiPSC lines with regulatable overexpression of TRPC6 wild-type and FSGS-associated gain-of-function (GoF, P112Q) and loss-of-function (LoF, G757D) mutations. Moreover, these cell lines were differentiated into induced podocytes (ipodocytes). We assessed the impact of TRPC6 GoF and LoF mutants on calcium influx in combination with TRPC6 agonists and antagonists. Our data showed relative calcium responses consistent with the GoF and LoF phenotypes. Transgenic iPSC-based models, like the one presented here, are instrumental to studying disease mechanisms in vitro and investigating the outcomes of, and possible therapeutic interventions for, this complex disease. Full article

Human-induced pluripotent stem cells (hiPSCs) have an innate ability to differentiate into the three germ layers: the ectoderm, endoderm, and mesoderm. By using targeted differentiation methods and carefully controlling growth factors, morphogens, and signaling modulators, hiPSCs can be guided to develop into specific lineage cell types. For clinical applications of hiPSCs and their derivatives, it is crucial to use xenogen-free, chemically defined culture media, reagents, recombinant growth factors, morphogens, and extracellular matrix (ECM) scaffolds. One major obstacle is the widespread use of Matrigel as an hiPSC culture matrix. Matrigel, derived from Engelbreth–Holm–Swarm (EHS) mouse sarcoma, is an extract of basement membrane material with a complex, poorly defined, and variable composition. It also exhibits batch-to-batch variability in mechanical and biochemical properties and is difficult to modify, which limits its rational use in the production of therapeutic cells and organoids. Synthetic ECM matrices and scaffolds offer a promising alternative because they can have a fully defined composition, highly tunable physical properties, surface modifications, and functionalization with recombinant signaling peptides and growth factors. This provides a suitable microenvironment for hiPSC culture and the directed differentiation towards lineage-specific cells and organoid development, and can be used in clinical-grade tissue transplantation and regenerative medicine. Full article

The increasing production and accumulation of plastic waste, coupled with insufficient recycling practices, contribute to the growing presence of plastic in the environment. Nanoplastic particles are of particular concern, as they pose greater (health and environmental) risks and exhibit wider dispersion compared to macroplastics. The blood–brain barrier may be exposed to nanoplastics present in the blood, which could affect its functionality or even pass through and damage the central nervous system. This study examined the effects of polystyrene (PS) nanoparticles with different chemical surface modifications (pristine, carboxylated, aminated) and sizes (50 nm and 100 nm) on cells of the neurovascular unit (NVU): human brain endothelial cells, astrocytes, and pericytes. Results indicated that only high concentrations of nanoparticles (100 μg/mL and 300 μg/mL) applied for 48 h decreased cell viability and barrier integrity significantly. Specifically, 50 nm carboxylated PS particles reduced barrier integrity and altered tight junction gene expression substantially. Fluorescent labelling of the investigated particles enabled to confirm their uptake by all tested cell types of the NVU, but also highlighted that the labelling changes the particles’ properties. Furthermore, cell culture medium-dependent particle agglomeration and increase of size were inversely correlated with cellular internalisation, which has to be considered for future risk assessments. Full article

Background/Objectives: Human induced pluripotent stem cell (hiPSC) models provide a unique platform for testing the effect of genomic variants identified in patients with inherited diseases. In Alström syndrome, a rare multisystem disorder mainly caused by nonsense mutations in the ALMS1 gene, patients often present with infantile cardiomyopathy, retinal dystrophy, type 2 diabetes, and hearing loss in addition to obesity. These diverse clinical manifestations highlight the pleiotropic functions of ALMS1 in cellular processes such as ciliary signalling, cell cycle regulation, and tissue homeostasis. In cats, the ALMS1:c.7384G>C missense variant has been associated with cardiomyopathy in the absence of other symptoms of Alström syndrome, raising questions regarding the impact of this variant on cardiac pathology. Methods: To answer these questions, we generated an hiPSC line carrying the human ALMS1:c.10004G>C missense variant, homologous to the ALMS1:c.7384G>C feline variant, as well as an isogenic control, to investigate the impact of this variant on cardiomyocyte differentiation and function. Results: The introduction of the ALMS1:c.10004G>C variant in the homozygous state in hiPSCs resulted in a significant reduction in cardiomyocyte differentiation efficiency. However, the variant did not affect contractile frequency, sarcomere organisation, sarcomere length, or cardiomyocyte cell size. Together, these results suggest that while the ALMS1:c.10004G>C variant impairs cardiomyocyte differentiation, it does not disrupt the structural or functional properties of the hiPSC-derived cardiomyocytes that do form. Conclusions: We have generated and initiated the characterisation of the third ALMS1 mutant hiPSC line and the first line based on a missense variant, but further research is needed on its relevance in modelling ALMS1-related changes. Our results also support the previous recommendation not to use ALMS1:c.7384G>C for the selection of breeding cats until further data confirm its intrinsic pathogenicity. Full article

Pathological USH2A mutations cause Usher syndrome type II, characterized by progressive retinitis pigmentosa and hearing and balance impairment. This study aims to investigate the cellular mechanisms underlying USH2A-related retinal degeneration using human induced pluripotent stem cell (hiPSC)-derived retinal organoids. The introduction of a homozygous nonsense mutation in the USH2A hotspot exon-13 resulted in normal photoreceptor development but loss of ciliary localization of usherin long form B and its interacting proteins, ADGRV1 and whirlin. Notably, single-cell RNA sequencing revealed unexpected significant transcriptional changes in Müller glial cells (MGCs), suggestive of disruptions in the translation, innate immune response, and endolysosomal system. These findings suggest that, while photoreceptor cells are mildly affected by the exon-13 USH2A mutation, MGCs exhibit major transcriptional changes, potentially contributing to the disease progression and therefore shedding light on potential alternative therapeutic targets. Full article