Search Results (2,619)

Developing robust methods to differentiate pluripotent stem cells (PSCs) into specific neuronal subtypes is crucial for advancing neuroscience research, including disease modelling and regenerative medicine. Research in this area has primarily focused on generating and studying excitatory neurons, often in co-culture with primary astrocytes to support maturation. Due to the shared ectodermal lineage of these cell types, any mesoderm derived cells, such as microglia, are absent using traditional methods of culture. To more accurately model the intricate complexity of the brain and its normal neuronal physiology, it is important to incorporate other critical neural subtypes, such as inhibitory interneurons and various glial cells. This review highlights recent progress in using transcription factor-based in vitro differentiation strategies to generate these diverse neural populations. A major advantage of this approach is the ability to rapidly produce highly specific cell types in a controlled manner, allowing for the precise seeding of cells at defined anatomical and physiological ratios. This controlled methodology enables the creation of more accurate and reproducible in vitro models, including two-dimensional (2D) and three-dimensional (3D) cultures and organoids, thereby moving beyond the limitations of random differentiation from neuronal progenitor cells. Despite these advances, key challenges remain, including reproducibility between pluripotent stem cell lines, off-target transcriptional effects of exogenous factors, and incomplete phenotypic maturation of derived cells. Addressing these constraints is essential for translating transcription factor-based approaches into robust and clinically relevant neural models. Full article

Background/Objectives: Approximately 20% of familial ALS (fALS) cases are linked to mutations in Cu/Zn superoxide dismutase (SOD1). Through a gain function, SOD1 misfolding exerts a toxic effect on motor neurons, leading to their degradation and ALS symptomology in both fALS cases and sporadic ALS (sALS) cases with no known genetic cause. To further our understanding of SOD1-ALS etiology, identifying motor neuron-specific SOD1 post-translational modifications (PTMs) and studying their structural influence is necessary. To this end, we have conducted a study on the influence of the deamidation of Asn53, a PTM proximal to key stabilizing motifs in SOD1, which has scarcely been addressed in the literature to date. Methods: Deamidation to N53 was identified by tandem mass spectrometry of SOD1 immunoprecipitated from motor neuron (MN) cultures derived from wild-type (WT) human induced pluripotent stem cells (iPSCs). WT SOD1 and N53D SOD1, a mutant mimicking the deamidation, were expressed in Escherichia coli and purified for in vitro analyses. Differences between species were measured by experiments probing metal cofactors, relative monomer populations, and aggregation propensity. Furthermore, molecular dynamics experiments were conducted to model and determine the influence of the PTM on SOD1 structure. Results: In contrast to WT, N53D SOD1 showed non-native incorporation of metal cofactors, coordinating more Zn²⁺ cofactors than total Zn-binding sites, and more readily adopted monomeric forms, unfolded, and aggregated with heating, possibly while releasing coordinated metals. Conclusions: Deamidation to N53 in SOD1 encourages the adoption of non-native conformers, and its detection in WT MN cultures suggests relevance to sALS pathophysiology. Full article

Cardiac muscle has limited proliferative potential; therefore, loss of cardiomyocytes is irreversible and can cause or exacerbate heart failure. Although both pharmacological and non-pharmacological therapies are available, these interventions act primarily on surviving myocardium to manage symptoms and reduce—rather than reverse—adverse remodeling. The only curative option for end-stage heart failure remains heart transplantation; however, its clinical use is severely constrained by the shortage of donor organs. Consequently, regenerative therapies have gained increasing attention as potential novel treatments. Among these, cardiomyocytes derived from patient-specific pluripotent stem cells (PSCs) represent a particularly promising experimental platform for cardiac regeneration. To evaluate the potential of PSCs for cardiac repair through both in vivo and in vitro approaches, we (1) examined the hallmarks of cardiomyocyte maturation and the regulatory systems that coordinate these processes, (2) reviewed recent advances in maturation protocols and derivation techniques, (3) discussed how the cellular microenvironment enhances maturation and function, and (4) identified current barriers to clinical translation. Importantly, we integrated developmental biology with protocol design to provide a mechanistic foundation for PSC-based regeneration. Specifically, insights from cardiac development—such as signaling pathways governing proliferation, alignment, and excitation-contraction coupling—were explicitly linked to the refinement of PSC differentiation and maturation protocols. This developmental perspective allows us to bridge pathology and stem-cell methodology, explaining how disruptions in native cardiac maturation can inform strategies to produce functionally mature PSC-derived cardiomyocytes. Finally, we assessed the clinical prospects of PSC-derived cardiomyocytes, highlighting both the most recent advances and the persistent translational challenges that must be addressed before widespread therapeutic use. Full article

GNAO1 is an alpha subunit of the G-protein complex involved in signal transduction in neurons. The G203R mutation in the GNAO1 gene arises recurrently de novo and causes epileptic encephalopathy and movement disorder. GNAO1 has two main isoforms, GNAO1-A and GNAO1-B, but their functional or expression differences are poorly understood. Molecular functions of GNAO1 are mainly studied in neurons, yet glial cells also express GNAO1 and participate in the pathogenesis of epilepsy. Here, we used human-induced pluripotent stem cell-based models to investigate the localization and expression of GNAO1 isoforms in astrocytes. We showed that in astrocytes, almost 100% of GNAO1 transcripts encoded GNAO1-B with very low GNAO1-A expression. We showed that there were no differences in localization between GNAO1-A and GNAO1-B, both in WT and G203R states. We also showed that GNAO1 localized in astrocytic retraction fibers and migrasomes, structures not previously described in this cell type. We showed that GNAO1-positive retraction fibers of neighboring cells provided cell-to-cell contacts and also provided calcium waves during astrocytic excitation. Overexpression of both GNAO1-A and GNAO1-B tends to lower calcium activity in astrocytes, with GNAO1-A providing the most severe impairment of activity. Our results demonstrate that astrocytes, in addition to neurons, should be used as a model for studying GNAO1-related disorders and that GNAO1 mutations should be evaluated in the context of both the GNAO1-A and GNAO1-B isoforms. Full article

Neurodevelopmental disorders (NDDs), including autism spectrum disorder, intellectual disability, and attention deficit hyperactivity disorder, are increasingly recognized as disorders of early brain construction arising from defects in neural stem and progenitor cell (NSPC) proliferation. NSPCs are responsible for generating the diverse neuronal and glial lineages that establish cortical architecture and neural circuitry; thus, their expansion must be tightly coordinated by intrinsic cell cycle regulators and extrinsic niche-derived cues. Disruption of these mechanisms—through genetic mutations, epigenetic dysregulation, or environmental insults—can perturb the balance between NSPC self-renewal and differentiation, resulting in aberrant brain size and connectivity. Recent advances using animal models and human pluripotent stem cell-derived brain organoids have identified key signaling pathways, including Notch, Wnt, SHH, and PI3K–mTOR, as central hubs integrating proliferative cues, while transcriptional and chromatin regulators such as PAX6, CHD8, SETD5, and ANKRD11 govern gene expression essential for proper NSPC cycling. Furthermore, prenatal exposure to teratogens such as Zika virus infection, valproic acid, or metabolic stress in phenylketonuria can recapitulate proliferation defects and microcephaly, underscoring the vulnerability of NSPCs to environmental perturbation. This review summarizes emerging insights into the molecular and cellular mechanisms by which defective NSPC proliferation contributes to NDD pathogenesis, highlighting convergence among genetic and environmental factors on cell cycle control. A deeper understanding of these pathways may uncover shared therapeutic targets to restore neurodevelopmental trajectories and mitigate disease burden. Full article

Streptozotocin (STZ) is an alkylating agent that has neurotoxic effects when injected into the cerebral ventricles (ICV) and also models many other features of Alzheimer’s disease. However, the mechanisms of STZ neurotoxicity are not well understood. In this study, we hypothesized that some of the neurotoxic effects of STZ could be due to direct activities on brain endothelial cells and astrocytes, which are key in forming and supporting the functions of the blood–brain barrier (BBB), respectively. To test this hypothesis, we characterized the changes induced by STZ either in cultures of human-induced pluripotent stem cell (iPSC)-derived brain endothelial-like cells (iBECs), which form an in vitro BBB model, or in primary human astrocytes. We found that STZ at a dosage of 5 mM caused a delayed reduction in the transendothelial electrical resistance (TEER) of iBECs at 7–11 days post-treatment, indicating induction of BBB leakage. Additionally, we observed significant increases in albumin leakage across the monolayer, altered iBEC morphology, and reductions in tight junction proteins, suggesting that STZ causes BBB disruption. We further found that the BBB glucose transporter GLUT-1 was reduced in iBECs, as was the total number of iBECs. In astrocytes, the 5 mM dose of STZ reduced the GFAP signal and total number of cells, suggesting that STZ has anti-proliferative and/or toxic effects on astrocytes. Together, these data support that STZ’s neurotoxic effects could be due, in part, to its direct toxic activities on brain endothelial cells and astrocytes. Full article

Cancer stem cells (CSCs) represent a small but critical subpopulation of tumor cells that drive therapy resistance, relapse and metastasis. Gastric cancer stem cells (GCSCs) have been identified through surface markers and transcriptional signatures, revealing their central role in tumor progression. Recently, N⁶-methyladenosine (m⁶A) RNA modification has emerged as a crucial epitranscriptomic regulator of CSC biology. The m⁶A machinery, including “writers” (METTL3, METTL14, WTAP, VIRMA), “erasers” (FTO, ALKBH5) and “readers” (YTHDFs/ YTHDCs, IGF2BPs, hnRNPA2B1), orchestrates RNA stability, splicing, translation and decay, thereby influencing self-renewal and oncogenic signaling. In GCSCs, m⁶A controls pluripotency factors, oncogenic transcripts and non-coding RNAs, collectively reinforcing stemness and malignant potential. Mounting evidence implicates dysregulated m⁶A effectors as not only key drivers of GCSC biology but also as promising biomarkers for patient stratification and therapeutic targets capable of selectively eliminating CSCs. Harnessing this knowledge could enable earlier diagnosis, more accurate patient stratification and more precise treatments. However, challenges remain regarding the resolution of m⁶A profiling, therapeutic selectivity to avoid unwanted toxicity and biomarker validation for clinical use. This review summarizes the discovery and features of CSCs, highlights the functional role of m⁶A in GCSCs, and explores diagnostic and therapeutic opportunities while outlining key difficulties for clinical translation. Full article

Human retinal organoids derived from pluripotent stem cells represent a robust in vitro model for investigating retinal development and disease mechanisms of retinal disorders. However, achieving structural maturation that faithfully recapitulates the intricate architecture of photoreceptors within a feasible and cost-efficient culture timeframe remains a significant challenge. Here, we present an optimized differentiation protocol that enables the generation of retinal organoids exhibiting advanced photoreceptor maturation within 140 days. Photoreceptors in the retinal organoids displayed compartmentalized architecture, including distinct inner and outer segments and connecting cilia. Notably, we observed the emergence of budding calyceal process-like structures—a feature not previously emphasized in photoreceptors derived from pluripotent stem cells. These results suggest that our protocol may promote advanced photoreceptor maturation within a relatively shortened culture period. Thus, this method could serve as a useful model for investigating retinal development and related pathologies, building upon previous protocols. Full article

Mitochondrial dysfunction is a key contributor to cardiac injury and heart failure, and extracellular vesicles (EVs) have emerged as promising therapeutic agents due to their ability to deliver mitochondrial-targeted cargo. This review systematically maps the evidence on how EVs modulate mitochondrial dynamics—including fusion, fission, mitophagy, and biogenesis—in regenerative cardiology. We comprehensively searched PubMed, Scopus, and Web of Science up to September 2025 for original studies. A total of 48 studies were included, with most utilizing EVs from mesenchymal stem cells, induced pluripotent stem cells, or cardiac progenitors. The review found that EV cargo influences key pathways such as DRP1 and MFN2, restores mitochondrial membrane potential, reduces ROS accumulation, and improves cardiomyocyte survival. While engineered EVs showed enhanced specificity, a lack of standardized preparation and quantitative assessment methods remains a significant challenge. We conclude that EV-mediated mitochondrial modulation is a promising strategy for cardiac repair, but the field needs harmonized protocols, deeper mechanistic understanding, and improved translational readiness to advance beyond preclinical research. The future of this research lies in integrating systems biology and precision targeting. Full article

The fields of regenerative medicine and stem cell-based tissue engineering hold great potential for treating a wide range of tissue and organ defects. Stem cells are ideal candidates for regenerative medicine because they are undifferentiated cells with the capacity for self-renewal, rapid proliferation, multilineage differentiation, and expression of pluripotency-associated genes. Human dental pulp stem cells (DPSCs) consist of various cell types (including stem cells) and possess multilineage differentiation potential. Owing to their easy isolation and rapid proliferation, DPSCs and their derivatives are considered promising candidates for repairing injured tissues. Recent advances in gene engineering have enabled cells to express specific genes of interest, leading to the secretion of medically important proteins or the alteration of cell behavior. For example, transient expression of Yamanaka’s factors in DPSCs can induce transdifferentiation into induced pluripotent stem cells (iPSCs). These gene-engineered cells represent valuable candidates for regenerative medicine, including stem cell therapies and tissue engineering. However, challenges remain in their development and application, particularly regarding safety, efficacy, and scalability. This review summarizes current knowledge on gene-engineered DPSCs and their derivatives and explores possible clinical applications, with a special focus on oral regeneration. Full article

Amyloid cardiomyopathy (ACM), driven by transthyretin (TTR) and immunoglobulin light chain (LC) amyloid fibrils, remains a major clinical challenge due to limited mechanistic understanding and insufficient preclinical models. Human-induced pluripotent stem cells (iPSCs) have emerged as a transformative platform to model ACM, offering patient-specific and genetically controlled systems. In this review, we summarize recent advances in the use of iPSC-derived cardiomyocytes (iPSC-CMs) in both two-dimensional (2D) monolayer cultures and three-dimensional (3D) constructs—including spheroids, organoids, cardiac microtissues, and engineered heart tissues (EHTs)—for disease modeling, mechanistic research, and drug discovery. While 2D culture of iPSC-CMs reproduces hallmark proteotoxic phenotypes such as sarcomeric disorganization, oxidative stress, and apoptosis in ACM, 3D models provide enhanced physiological relevance through incorporating multicellularity, extracellular matrix interactions, and mechanical load-related features. Genome editing with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 further broadens the scope of iPSC-based models, enabling isogenic comparisons and the dissection of mutation-specific effects, particularly in transthyretin-related amyloidosis (ATTR). Despite limitations such as cellular immaturity and challenges in recapitulating aging-associated phenotypes, ongoing refinements in differentiation, maturation, and dynamic training of iPSC-cardiac models hold great promise for overcoming these barriers. Together, these advances position iPSC-based systems as powerful human-relevant platforms for modeling and elucidating disease mechanisms and accelerating therapeutic development to prevent ACM. Full article

Granulosa cells (GCs) and ovarian putative stem cells (oPSCs) represent distinct but complementary populations within the mammalian ovary. While GCs have long been considered terminally differentiated and hormonally specialized, emerging evidence indicates that they retain epigenetic plasticity and, under defined conditions, can be reprogrammed into cells exhibiting pluripotent-like features. In contrast, oPSCs, including oogonial stem cells (OSCs) and very small embryonic-like stem cells (VSELs), are naturally multipotent and capable of spontaneous or inducible differentiation into neural, endothelial, and other somatic lineages. Both cell types express stemness-related markers, such as OCT4, SOX2, and c-KIT, and demonstrate potential for self-renewal and lineage conversion. Recent advances in chemical modulation of epigenetic reprogramming, particularly with agents from the family of non-specific DNA methyltransferase (DNMT) inhibitors, such as 5-azacytidine (5-azaC), highlight the feasibility of generating functional, lineage-specific derivatives of GCs or oPSCs without genetic manipulation. Not without significance is also the fact that extended/high-dose 5-azaC-mediated modulation can induce cell senescence or apoptotic/necrotic death. Therefore, dosing must be carefully titrated, which strongly supports a dose- and/or time-dependent mechanism for 5-azaC-based epigenetic modification in treated cells. This study aims to summarize the molecular and functional properties of mammalian GCs and oPSCs, emphasizing their applicability in regenerative medicine and reproductive bioengineering, with a focus on safe, patient-specific cell-based therapies. Full article

Background: Point mutations in mitochondrial DNA (mtDNA) cause a range of neurometabolic disorders that currently have no curative treatments. The m.8993T>G mutation in the Homo sapiens MT-ATP6 gene leads to neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) when heteroplasmy exceeds approximately 70%. Methods: We engineered a split DddA-derived cytosine base editor (DdCBE), each half fused to programmable TALE DNA-binding domains and a mitochondrial targeting sequence, to correct the m.8993T>G mutation in patient-derived induced pluripotent stem cells (iPSCs). Seven days after plasmid delivery, deep amplicon sequencing showed 35 ± 3% on-target C•G→T•A conversion at position 8993, reducing mutant heteroplasmy from 80 ± 2% to 45 ± 3% with less than 0.5% editing at ten predicted off-target loci. Results: Edited cells exhibited a 25% increase in basal oxygen consumption rate, a 50% improvement in ATP-linked respiration, and a 2.3-fold restoration of ATP synthase activity. Directed neural differentiation yielded 85 ± 2% Nestin-positive progenitors compared to 60 ± 2% in unedited controls. Conclusions: Edits remained stable over 30 days in culture. These results establish mitochondrial base editing as a precise and durable strategy to ameliorate biochemical and cellular defects in NARP patient cells. Full article

Kidney organoids, derived from stem cells, including pluripotent stem cells and adult progenitor cells, have been reported as three-dimensional in vitro models that reflect key aspects of kidney development, structure, and function. Advances in differentiation protocols and tissue engineering have enabled the generation of organoids that exhibit nephron-like structures, including glomerular and tubular structures. Kidney organoids have been widely applied in several directions, including disease modeling and therapeutic screening, drug nephrotoxicity evaluation, and regenerative medicine. In particular, kidney organoids offer a promising platform for studying genetic kidney diseases, such as polycystic kidney disease and congenital anomalies of the kidney and urinary tract (CAKUT), by allowing patient-specific modeling for the analysis of pathophysiology and therapeutic screening. Despite several current limitations, such as incomplete maturation, lack of full nephron segmentation, and variability between protocols and cell conditions, further technological innovations such as microfluidics and bioengineering may refine kidney organoid systems. This review highlights recent advances in kidney organoid research, outlines major applications, and discusses future directions to enhance their physiological relevance, functional maturity, and translational integration into preclinical and clinical nephrology. Full article

Proteomics of dystrophic muscle samples is limited by the amount of protein that can be extracted from patient biopsies. Cells and tissues derived from patient-derived induced pluripotent stem cells (iPSCs) can be an expandable alternative source. We have patterned iPSCs from three Duchenne muscular dystrophy (DMD) patient lines into skeletal muscle cells using a two-dimensional as well as our three-dimensional organoid differentiation system. Probes with sufficient protein amounts could be extracted and prepared for mass spectrometry. In total, 3007 proteins in 2D and 2709 proteins in 3D were detected in DMD patient probes. A total of 83 proteins in 2D and 338 proteins in 3D can be described as differentially expressed between DMD and control patient probes in a post hoc test. We have identified and we propose Myosin-9, Collagen 18A, Tropomyosin 1, BASP1, RUVBL1, and NCAM1 as proteins specifically altered in their expression in DMD for further investigation. Proteomics of skeletal muscle organoids resulted in greater consistency of results between cell lines in comparison to the two-dimensional myogenic differentiation protocol. Full article