Search Results (195)

Organoids allow to model healthy and diseased human tissues. and have applications in developmental biology, drug discovery, and cell therapy. Traditionally cultured in immersion/suspension, organoids face issues like lack of standardization, fusion, hypoxia-induced necrosis, continuous agitation, and high media volume requirements. To address these issues, we developed an air–liquid interface (ALi) technology for culturing organoids, termed AirLiwell. It uses non-adhesive microwells for generating and maintaining individualized organoids on an air–liquid interface. This method ensures high standardization, prevents organoid fusion, eliminates the need for agitation, simplifies media changes, reduces media volume, and is compatible with Good Manufacturing Practices. We compared the ALi method to standard immersion culture for midbrain organoids, detailing the process from human pluripotent stem cell (hPSC) culture to organoid maturation and analysis. Air–liquid interface organoids (3D-ALi) showed optimized size and shape standardization. RNA sequencing and immunostaining confirmed neural/dopaminergic specification. Single-cell RNA sequencing revealed that immersion organoids (3D-i) contained 16% fibroblast-like, 23% myeloid-like, and 61% neural cells (49% neurons), whereas 3D-ALi organoids comprised 99% neural cells (86% neurons). Functionally, 3D-ALi organoids showed a striking electrophysiological synchronization, unlike the heterogeneous activity of 3D-i organoids. This standardized organoid platform improves reproducibility and scalability, demonstrated here with midbrain organoids. The use of midbrain organoids is particularly relevant for neuroscience and neurodegenerative diseases, such as Parkinson’s disease, due to their high incidence, opening new perspectives in disease modeling and cell therapy. In addition to hPSC-derived organoids, the method’s versatility extends to cancer organoids and 3D cultures from primary human cells. Full article

Diabetes mellitus, both type 1 (T1D) and type 2 (T2D), has become the epidemic of the century and a major public health concern given its rising prevalence and the increasing adoption of a sedentary lifestyle globally. This multifaceted disease is characterized by impaired pancreatic beta cell function and insulin resistance (IR) in peripheral organs, namely the liver, skeletal muscle, and adipose tissue. Additional insulin target tissues, including cardiomyocytes and neuronal cells, are also affected. The advent of stem cell research has opened new avenues for tackling this disease, particularly through the regeneration of insulin target cells and the establishment of disease models for further investigation. Human-induced pluripotent stem cells (iPSCs) have emerged as a valuable resource for generating specialized cell types, such as hepatocytes, myocytes, adipocytes, cardiomyocytes, and neuronal cells, with diverse applications ranging from drug screening to disease modeling and, importantly, treating IR in T2D. This review aims to elucidate the significant applications of iPSC-derived insulin target cells in studying the pathogenesis of insulin resistance and T2D. Furthermore, recent differentiation strategies, protocols, signaling pathways, growth factors, and advancements in this field of therapeutic research for each specific iPSC-derived cell type are discussed. Full article

As the global population continues to age, the incidence of neurodegenerative diseases and neural injuries is increasing, presenting major challenges for healthcare systems. Due to the brain’s limited regenerative capacity, there is an urgent need for strategies that promote neuronal repair and functional integration. Brain-derived neurotrophic factor (BDNF) is a key regulator of synaptic plasticity and neuronal development. In this study, we investigated whether constitutive BDNF expression in human induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) enhances their neurogenic and integrative potential in vitro. We found that NPCs engineered to overexpress BDNF produced neuronal cultures with increased numbers of mature and spontaneously active neurons, without altering the overall structure or organization of functional networks. Furthermore, BDNF-expressing neurons exhibited significantly greater axonal outgrowth, including directed axon extension in a compartmentalized microfluidic system, suggesting a chemoattractive effect of localized BDNF secretion. These effects were comparable to those observed with the early supplementation of recombinant BDNF. Our results demonstrate that sustained BDNF expression enhances neuronal maturation and axonal projection without disrupting network integrity. These findings support the use of BDNF not only as a therapeutic agent to improve cell therapy outcomes but also as a tool to accelerate the development of functional neural networks in vitro. Full article

The nervous system maintains homeostasis and coordinated behavior through complex neuronal and glial cells. Traditional models, such as primary rodent neurons and human-induced pluripotent stem cell (hIPSC)-derived neurons, have advanced our understanding of neuronal function and neurotoxic damage; however, they are costly and labor-intensive. SH-SY5Y cells, an immortalized human neuroblastoma cell line, provide a more accessible alternative for studying neuronal processes and neurotoxicity. However, their limited capacity to differentiate into specific neuronal phenotypes remains a challenge. To address this limitation, differentiation protocols using neuronal factors and vitamins have been developed, primarily in two-dimensional (2D) cultures, which reduces physiological relevance. Here, we present a novel three-dimensional (3D) SH-SY5Y model incorporating 2D differentiation protocols to generate cholinergic neurons (ChAT+). This model enhances neurotoxicity studies related to pesticides and mycotoxins. Our protocol produces homogeneous spheroids differentiated into cholinergic neurons using serum restriction and specific factors, maintaining viability and circularity for up to 22 days. Differentiation was validated by immunofluorescence and Western blot by Choline acetyltransferase (ChAT) expression. This scalable and reproducible 3D model provides a valuable in vitro tool for neurotoxicological research, improving physiological relevance and enabling the study of cholinergic neuron differentiation and function. Full article

Alzheimer’s disease (AD), the leading cause of dementia, remains poorly understood despite decades of intensive research, which continues to hinder the development of effective treatments. As a complex multifactorial disorder, AD lacks a cure to halt the progressive neurodegeneration, and the precise mechanisms underlying its onset and progression remain elusive, limiting therapeutic options. Due to the challenges of studying neuronal cells in vivo, technologies such as clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) and human-induced pluripotent stem cells (hiPSCs) are key for identifying therapeutic targets, although they face technical and ethical hurdles in their early stages. CRISPR/Cas9 and hiPSCs are promising for disease modeling and therapy, but off-target effects and the complexity of gene editing in the brain limit their use. CRISPR technology enables specific genetic modifications in key AD-related genes, such as APP, PSEN1, PSEN2, and APOE, providing valuable insights into disease mechanisms. iPSC-derived neurons, astrocytes, microglia, and 3D organoids can recapitulate key aspects of human AD pathology, but they do not fully replicate the complexity of the human brain, limiting clinical applicability. These technologies advance studies of amyloid processing, tau aggregation, neuroinflammation, and oxidative stress, yet translating them into clinical therapies remains challenging. Despite the promise of CRISPR/Cas9 and iPSCs for precision medicine, gaps in knowledge about their long-term safety and efficacy must be addressed before clinical implementation. Full article

Cerebral organoids (cORGs) obtained from induced pluripotent stem cells (iPSCs) have become significant instruments for investigating human neurophysiology, with the possibility of simulating diseases and enhancing drug discovery. The current approaches require a strict process of manual inclusion in animal-derived matrix Matrigel^® and are challenged by unpredictability, operators’ skill and expertise, elevated costs, and restricted scalability, impeding their extensive applicability and translational potential. In this study, we present a novel method to generate brain organoids that address these limitations. Our approach does not require a manual, operator-dependent embedding. Instead, it employs a chemically defined hydrogel in which the Matrigel^® is diluted in a solution enriched with sodium alginate (SA) and sodium carboxymethylcellulose (CMC) and used as a bioink to print neural embryoid bodies (nEBs). Immunohistochemical, immunofluorescence, and gene expression analyses confirmed that SA-CMC-Matrigel^® hydrogel can sustain the generation of iPSC-derived cortical cORGs as the conventional Matrigel^®-based approach does. By day 40 of differentiation, hydrogel-based 3D-bioprinted cORGs showed heterogeneous and consistent masses, with a cytoarchitecture resembling an early-stage developmental fetal brain composed of neural progenitor cells PAX6⁺/Ki67⁺ organized into tubular structures, and densely packed cell somas with extensive neurites SYP⁺, suggestive of cortical tissue-like neuronal layer formation. Full article

Background: Amyotrophic lateral sclerosis (ALS) is a multifactorial neurodegenerative disease characterized by the involvement of multiple pathways and mechanisms. The complexity of its pathophysiology is reflected in the diverse hypotheses relating to its underlying causes. Given this intricate interplay of processes, a combination therapy approach offers a promising strategy. Combination therapies have demonstrated significant success in treating complex diseases, where they aim to achieve synergistic therapeutic effects and reduce drug dosage. PrimeC is an oral combination treatment composed of a patented novel formulation consisting of specific and unique doses of two well-characterized drugs (ciprofloxacin and celecoxib). It aims to synergistically inhibit the progression of ALS by addressing key elements of its pathophysiology. Objectives: Demonstrating the synergistic effect of the PrimeC combination compared to each of its individual components, celecoxib and ciprofloxacin, and assessing its ability to improve the drug concentration profile and efficacy. Methods: The efficacy of the PrimeC combination was assessed in a survival assay using human induced pluripotent stem cell (iPSC)-derived motor neurons. Additionally, a drug profiling study was conducted, measuring drug levels in the brain and serum of C57BL mice treated with a single compound versus the combination. Results: Motor neurons modeling ALS treated with the PrimeC combination exhibited better survival rates compared to treatment with either individual compound alone. The enhanced efficacy of the combination was further supported by a drug concentration profiling study in rodents, demonstrating that the PrimeC combination resulted in increased ciprofloxacin concentrations in both brain tissue and serum—highlighting the optimized interaction and synergistic potential of its two comprising agents. Conclusions: Our findings support the potential of combination therapy as an effective strategy for ALS treatment. Specifically, the PrimeC combination demonstrated promising therapeutic effects, providing a strong rationale for its ongoing development as a targeted treatment for ALS. Full article

Background: All the processes leading to neurodegeneration cannot be addressed with just one medication. Combinations of drugs affecting various disease mechanisms concurrently could demonstrate improved effect in slowing the course of Parkinson’s disease (PD). Objective: This was a drug-repurposing experiment designed to assess several combinations of nine drugs for possible added or synergistic efficacy using in vitro models of PD. Methods: We evaluated 44 combinations of the nine medications (sodium phenylbutyrate, terazosin, exenatide, ambroxol, deferiprone, coenzyme-Q10, creatine, dasatinib and tauroursodeoxycholic acid) selected for their previously demonstrated evidence of their impact on different targets, showing neuroprotective properties in preclinical models of PD. We utilized wild-type induced pluripotent stem-cell-derived human dopaminergic neurons treated with 1-methyl-4-phenylpyridinium for initial screening. We retested some combinations using an idiopathic PD patient-derived induced pluripotent stem cell line and alpha-synuclein triplication line. We assessed anti-neuroinflammatory effects using human microglia cells. As metrics, we evaluated neurite length, number of branch points per mm², the number of live neurons, neurofilament heavy chain and pro-inflammatory cytokines. Results: We have identified four combinations of two to three drugs that showed an additive protective effect in some endpoints. Only the combination of sodium phenylbutyrate, exenatide and tauroursodeoxycholic acid showed improvement in four endpoints studied. Conclusions: We demonstrated that some of the medications, used in combination, can exert an additive neuroprotective effect in preclinical models of PD that is superior to that of each of the compounds individually. This project can lead to the development of the first treatment for PD that can slow or prevent its progression. Full article

Background/Objectives: TDP-43 mutation-driven Amyotrophic Lateral Sclerosis (ALS) motor neuron disease is one of the most prominent forms (approximately 97%) in cases of sporadic ALS. Dysfunctional autophagy and lysosomal function are the prime mechanisms behind ALS. Mitoxantrone (Mito), a synthetic doxorubicin analog, is an inhibitor of DNA and RNA synthesis/repair via intercalating with nitrogenous bases and inhibiting topoisomerase II. The therapeutic potential of miRNAs associated with disease conditions has also been reported. This study explores the therapeutic potential of Mito along with miRNAs against mutated TDP-43 protein-induced proteinopathy in human-induced pluripotent stem cell (hiPSC)-derived human neural progenitor cells (hNPCs). Methods: HiPSCs mutated for TDP-43 were differentiated into hNPCs and used to explore the therapeutic potential of Mito at a concentration of 1 μM for 24 h (the identified non-cytotoxic dose). The therapeutic effects of Mito on miRNA expression and various cellular parameters such as mitochondrial dynamics, autophagy, and stress granules were assessed using the high-throughput Open Array technique, immunocytochemistry, flow cytometry, immunoblotting, and mitochondrial bioenergetic assay. Results: Mutated TDP-43 protein accumulation causes stress granule formation (G3BP1), mitochondrial bioenergetic dysfunction, SOD1 accumulation, hyperactivated autophagy, and ER stress in hNPCs. The mutated hNPCs also show dysregulation in six miRNAs (miR-543, miR-34a, miR-200c, miR-22, miR-29b, and miR-29c) in mutated hNPCs. A significant restoration of TDP-43 mutation-induced alterations could be witnessed upon the exposure of mutated hNPCs to Mito. Conclusions: Our study indicates that miR-543, miR-29b, miR-22, miR-200c, and miR-34a have antisense therapeutic potential alone and in combination with Mitoxantrone. Full article

Cerebral organoids derived from human induced pluripotent stem cells (iPSCs) have emerged as powerful in vitro models for studying human brain development and neurological disorders. Understanding the electrophysiological properties of these organoids is crucial for evaluating their functional maturity and potential applications. However, the differentiation and maturation of stem cells into cerebral organoids is a long, slow, and error-prone process. Hence, it is vitally crucial to establish a non-invasive method of monitoring the process over a long period of time. In this study, a planar microelectrode array (MEA) with platinum (Pt) black electroplating is designed to monitor the electrophysiological activities and pharmacological responses of cerebral organoids using an external neural signal acquisition system interfaced with the MEA. The planar MEA with Pt black electroplating has a significantly reduced electrode impedance and exhibits a robust capability for the real-time detection of spontaneous neural activities, including extracellular spikes and local field potentials. Distinct electrophysiological signal strengths in cerebral organoids were observed at early and late developmental stages. Further pharmacological stimulations showed that 30 mM KCl would induce a marked increase in spike rate, indicating an enhancement of neuronal depolarization and an elevation of network excitability. This robust response to KCl stimulation in mature networks serves as a reliable indicator of neural maturity in cerebral organoids and underscores the platform’s potential for drug screening applications. This work highlights the integration of MEA technology with cerebral organoids, offering a powerful platform for real-time electrophysiological monitoring. It provides new insights into the functional maturation of neural networks and establishes a reliable system for drug screening and disease modeling, facilitating future research into human brain physiology and pathology. Full article

The transplantation of human neurons into the central nervous system (CNS) offers transformative opportunities for modeling neurodegenerative diseases in vivo. This study evaluated the survival, integration, and functional properties of cryopreserved forebrain GABAergic neurons (iGABAs) derived from human induced pluripotent stem cells (iPSCs) across three species used in translational research. iGABAs were stereotactically injected into the striatum of Sprague–Dawley rats, immunodeficient RNU rats, R6/2 Huntington’s disease (HD) mice, wild-type controls, and Cynomolgus monkeys. Post-transplantation, long-term assessments revealed robust neuronal survival, extensive neurite outgrowth, and integration with host CNS environments. In immunodeficient rats, iGABAs innervated the neuraxis, extending from the prefrontal cortex to the midbrain, while maintaining mature neuronal phenotypes without uncontrolled proliferation. Similarly, grafts in nonhuman primates showed localized survival and stable phenotype at one month. In the neurodegenerative milieu of HD mice, iGABAs survived up to six months, projecting into the host striatum and white matter, with evidence of mutant huntingtin aggregates localized within the graft, indicating pathological protein transfer. These findings underscore the utility of cryopreserved iGABAs as a reproducible, scalable model for disease-specific CNS investigations and mechanistic studies of proteinopathic propagation. This work establishes a critical platform for studying neurodegenerative diseases and developing therapeutic interventions. Full article

Amyotrophic lateral sclerosis (ALS) is a terminal complex neurodegenerative disease, with 10–15% of cases being familial and the majority being sporadic with no known cause. There are no animal models for the 85–90% of sporadic ALS cases. More creative, sophisticated models of ALS disease are required to unravel the mysteries of this complicated disease. While ALS patients urgently require new medications and treatments, suitable preclinical in vitro models for drug screening are lacking. Therefore, human-derived induced pluripotent stem cell (hiPSC) technology offers the opportunity to model diverse and unreachable cell types in a culture dish. In this review, we focus on recent hiPSC-derived ALS neuronal and non-neuronal models to examine the research progress of current ALS 2D monocultures, co-cultures, and more complex 3D-model organoids. Despite the challenges inherent to hiPSC-based models, their application to preclinical drug studies is enormous. Full article

Due to a continuous increase in life expectancy and the progress made in specialized healthcare, the incidence of Alzheimer’s disease (AD) has dramatically increased to the point that it has become one of the main challenges of contemporary medicine. Despite a huge scientific and clinical effort, current treatments manage just a temporary alleviation of symptomatology but offer no cure. Modern trials involving cell transplantation in experimental animals require the involvement of neurosurgeons in the treatment protocol. CSF shunting, intraventricular infusions, or DBS for symptoms relief have been an integral part of the therapeutic arsenal from the very beginning. The development of stereotactic surgery has facilitated the experimental potential of cell transplantation in the hippocampus for Alzheimer’s disease. We conducted a narrative review of the literature in the top three medical databases (PubMed, Science Direct, and Google Scholar) using the keywords “Alzheimer’s disease”, “hippocampus”, and “transplant”. After eliminating duplicates, 241 papers were selected and screened by title and abstract. Two reviewers independently analyzed the 88 papers and chose 32 experiments that involved stereotactic hippocampal transplantation of cells in experimental animals with AD. The stereotactic transplantation of cells such as mesenchymal stem cells (MSCs), neuronal stem cells (NSCs), induced pluripotent cells (iPSCs), astrocytes, and derivates from stem cells was analyzed. The experiments used either a chemically induced or transgenic AD model and observed the impact of the stereotactic transplantation with behavioral testing, MRS spectroscopy, and biochemical analysis. The stereotaxic method delivers minimal invasive treatment option by cell transplantation at the hippocampus. The results showed that amyloid deposits were lower after transplantation, showing a positive impact. Other impactful results involve proliferation of neurogenesis, downregulation of anti-inflammatory response, and increased neuronal plasticity. The increased precision with which the stereotaxic method manages to target deep structures of the brain and the results of the reviewed papers could represent an argument for future human trials. More studies are needed to confirm the viability of the transplanted cells and the long-term effects. Full article

Sleep bruxism (SB) involves involuntary jaw movements during sleep and is potentially caused by motor neuronal hyperexcitability and GABAergic system dysfunction. However, the molecular basis remains unclear. In this study, we aimed to investigate changes in the expression of several genes associated with the pathophysiology of SB. Bulk RNA sequencing (bulk RNA-seq) and single-nucleus RNA sequencing (snRNA-seq) of neurons derived from patient and control human induced pluripotent stem cells (hiPSCs) were performed to comprehensively assess gene expression and cell type-specific alterations, respectively. Bulk RNA-seq revealed significant upregulation of calcium signaling-related genes in SB neurons, including those encoding G protein-coupled receptors and receptor-operated calcium channels. snRNA-seq confirmed the increased expression of GRIN2B (an N-methyl-D-aspartate receptor subunit) and CHRM3 (an M3 muscarinic acetylcholine receptor), particularly in glutamatergic and GABAergic neurons. These alterations were linked to hyperexcitability, with GRIN2B contributing to glutamatergic signaling and CHRM3 contributing to cholinergic signaling. These findings suggest that disrupted calcium signaling and overexpression of GRIN2B and CHRM3 drive neuronal hyperexcitability, providing insight into the pathophysiology of SB. Targeting these pathways may inform therapeutic strategies for SB treatment. Full article

The accumulation of damaged mitochondria has long been considered a hallmark of the aging process. Among various factors, age-related mitochondrial alterations comprise bioenergetic impairments and disturbances in reactive oxygen species (ROS) control, thereby negatively affecting mitochondrial performance and ultimately accelerating aging. Previous studies have revealed that polyamine spermidine appears to exert health-protective and lifespan-promoting effects. Notably, recent findings have also described a spermidine-induced improvement in age-associated mitochondrial dysfunction, but the beneficial effects of spermidine on aged mitochondria have not been entirely examined yet. Here, we show that spermidine positively regulates several parameters related to mitochondrial bioenergetics and mitochondrial redox homeostasis in young and aged human-induced pluripotent stem cell-derived neurons. We report that spermidine treatment increases adenosine triphosphate production and mitochondrial membrane potential, which is accompanied by an attenuation in mitochondrial ROS levels in both age groups. Furthermore, we demonstrate a spermidine-mediated amelioration in mitochondrial respiration in both young and aged neurons. Overall, our findings suggest that nutritional spermidine supplementation might represent an attractive therapeutic approach to enhance mitochondrial function, consequently decelerating aging. Full article