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Stem Cell Research Jun 2024Marfan Syndrome, a connective tissue disorder caused by Fibrillin-1 (FBN1) gene mutations, induces disease in the ocular, musculoskeletal, and cardiovascular systems and...
Marfan Syndrome, a connective tissue disorder caused by Fibrillin-1 (FBN1) gene mutations, induces disease in the ocular, musculoskeletal, and cardiovascular systems and increases aortic vulnerability to rupture associated with high mortality rates. We describe an induced pluripotent stem cell line (HFD1) generated from patient-derived human dermal fibroblasts harboring a heterozygous c.3338-2A>C intronic splice acceptor site variant preceding Exon 28 of FBN1. The clonal line, which produces abnormal FBN1 splice variants, has a normal karyotype, expresses appropriate stemness markers, and maintains trilineage differentiation potential. This line represents a valuable resource for studying how abnormal splicing variants contribute to Marfan Syndrome.
PubMed: 38941881
DOI: 10.1016/j.scr.2024.103475 -
Cell Biology and Toxicology Jun 2024Structural cardiotoxicity (SCT) presents a high-impact risk that is poorly tolerated in drug discovery unless significant benefit is anticipated. Therefore, we aimed to...
Structural cardiotoxicity (SCT) presents a high-impact risk that is poorly tolerated in drug discovery unless significant benefit is anticipated. Therefore, we aimed to improve the mechanistic understanding of SCT. First, we combined machine learning methods with a modified calcium transient assay in human-induced pluripotent stem cell-derived cardiomyocytes to identify nine parameters that could predict SCT. Next, we applied transcriptomic profiling to human cardiac microtissues exposed to structural and non-structural cardiotoxins. Fifty-two genes expressed across the three main cell types in the heart (cardiomyocytes, endothelial cells, and fibroblasts) were prioritised in differential expression and network clustering analyses and could be linked to known mechanisms of SCT. This transcriptomic fingerprint may prove useful for generating strategies to mitigate SCT risk in early drug discovery.
Topics: Humans; Cardiotoxicity; Transcriptome; Myocytes, Cardiac; Induced Pluripotent Stem Cells; Gene Expression Profiling; Computational Biology; Machine Learning; Cardiotoxins; Fibroblasts; Endothelial Cells
PubMed: 38940987
DOI: 10.1007/s10565-024-09880-7 -
Development (Cambridge, England) Jul 2024Generation of hematopoietic stem and progenitor cells (HSPCs) ex vivo and in vivo, especially the generation of safe therapeutic HSPCs, still remains inefficient. In...
Generation of hematopoietic stem and progenitor cells (HSPCs) ex vivo and in vivo, especially the generation of safe therapeutic HSPCs, still remains inefficient. In this study, we have identified compound BF170 hydrochloride as a previously unreported pro-hematopoiesis molecule, using the differentiation assays of primary zebrafish blastomere cell culture and mouse embryoid bodies (EBs), and we demonstrate that BF170 hydrochloride promoted definitive hematopoiesis in vivo. During zebrafish definitive hematopoiesis, BF170 hydrochloride increases blood flow, expands hemogenic endothelium (HE) cells and promotes HSPC emergence. Mechanistically, the primary cilia-Ca2+-Notch/NO signaling pathway, which is downstream of the blood flow, mediated the effects of BF170 hydrochloride on HSPC induction in vivo. Our findings, for the first time, reveal that BF170 hydrochloride is a compound that enhances HSPC induction and may be applied to the ex vivo expansion of HSPCs.
Topics: Animals; Zebrafish; Hematopoietic Stem Cells; Mice; Cell Differentiation; Hematopoiesis; Receptors, Notch; Signal Transduction; Embryoid Bodies; Cilia; Blastomeres; Cells, Cultured
PubMed: 38940293
DOI: 10.1242/dev.202476 -
Current Neuropharmacology Jun 2024The human central nervous system (CNS) has a limited capacity for regeneration and repair, as many other organs do. Partly as a result, neurological diseases are the...
The human central nervous system (CNS) has a limited capacity for regeneration and repair, as many other organs do. Partly as a result, neurological diseases are the leading cause of medical burden globally. Most neurological disorders cannot be cured, and primary treatments focus on managing their symptoms and slowing down their progression. Cell therapy for neurological disorders offers several therapeutic potentials and provides hope for many patients. Here we provide a general overview of cell therapy in neurological disorders such as Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Wilson's disease (WD), stroke and traumatic brain injury (TBI), involving many forms of stem cells, including embryonic stem cells and induced pluripotent stem cells. We also address the current concerns and perspectives for the future. Most studies for cell therapy in neurological diseases are in the pre-clinical stage, and there is still a great need for further research to translate neural replacement and regenerative therapies into clinical settings.
PubMed: 38939990
DOI: 10.2174/1570159X22666240509092903 -
Circulation Jun 2024Despite major advances in the clinical management of long QT syndrome, some patients are not fully protected by beta-blocker therapy. Mexiletine is a well-known sodium...
Therapeutic Efficacy of Mexiletine for Long QT Syndrome Type 2: Evidence From Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes, Transgenic Rabbits, and Patients.
BACKGROUND
Despite major advances in the clinical management of long QT syndrome, some patients are not fully protected by beta-blocker therapy. Mexiletine is a well-known sodium channel blocker, with proven efficacy in patients with sodium channel-mediated long QT syndrome type 3. Our aim was to evaluate the efficacy of mexiletine in long QT syndrome type 2 (LQT2) using cardiomyocytes derived from patient-specific human induced pluripotent stem cells, a transgenic LQT2 rabbit model, and patients with LQT2.
METHODS
Heart rate-corrected field potential duration, a surrogate for QTc, was measured in human induced pluripotent stem cells from 2 patients with LQT2 (KCNH2-p.A561V, KCNH2-p.R366X) before and after mexiletine using a multiwell multi-electrode array system. Action potential duration at 90% repolarization (APD) was evaluated in cardiomyocytes isolated from transgenic LQT2 rabbits (KCNH2-p.G628S) at baseline and after mexiletine application. Mexiletine was given to 96 patients with LQT2. Patients were defined as responders in the presence of a QTc shortening ≥40 ms. Antiarrhythmic efficacy of mexiletine was evaluated by a Poisson regression model.
RESULTS
After acute treatment with mexiletine, human induced pluripotent stem cells from both patients with LQT2 showed a significant shortening of heart rate-corrected field potential duration compared with dimethyl sulfoxide control. In cardiomyocytes isolated from LQT2 rabbits, acute mexiletine significantly shortened APD (∆APD shortening 113 ms), indicating a strong mexiletine-mediated shortening across different LQT2 model systems. Mexiletine was given to 96 patients with LQT2 either chronically (n=60) or after the acute oral drug test (n=36): 65% of the patients taking mexiletine only chronically and 75% of the patients who performed the acute oral test were responders. There was a significant correlation between basal QTc and ∆QTc during the test (= -0.8; <0.001). The oral drug test correctly predicted long-term effect in 93% of the patients. Mexiletine reduced the mean yearly event rate from 0.10 (95% CI, 0.07-0.14) to 0.04 (95% CI, 0.02-0.08), with an incidence rate ratio of 0.40 (95% CI, 0.16-0.84), reflecting a 60% reduction in the event rate (=0.01).
CONCLUSIONS
Mexiletine significantly shortens cardiac repolarization in LQT2 human induced pluripotent stem cells, in the LQT2 rabbit model, and in the majority of patients with LQT2. Furthermore, mexiletine showed antiarrhythmic efficacy. Mexiletine should therefore be considered a valid therapeutic option to be added to conventional therapies in higher-risk patients with LQT2.
PubMed: 38939955
DOI: 10.1161/CIRCULATIONAHA.124.068959 -
Journal of Extracellular Biology Jan 2024Extracellular vesicles (EVs) secreted by human-induced pluripotent stem cells (hiPSCs) have great potential as cell-free therapies in various diseases, including...
Extracellular vesicles (EVs) secreted by human-induced pluripotent stem cells (hiPSCs) have great potential as cell-free therapies in various diseases, including prevention of blood-brain barrier senescence and stroke. However, there are still challenges in pre-clinical and clinical use of hiPSC-EVs due to the need for large-scale production of a large quantity. Vertical-Wheel bioreactors (VWBRs) have design features that allow the biomanufacturing of hiPSC-EVs using a scalable aggregate or microcarrier-based culture system under low shear stress. EV secretion by undifferentiated hiPSCs expanded as 3-D aggregates and on Synthemax II microcarriers in VWBRs were investigated. Additionally, two types of EV collection media, mTeSR and HBM, were compared. The hiPSCs were characterized by metabolite and transcriptome analysis as well as EV biogenesis markers. Protein and microRNA cargo were analysed by proteomics and microRNA-seq, respectively. The functional assays of microglia stimulation and proliferation were conducted. HiPSCs expanded as 3-D aggregates and on microcarriers had comparable cell number, while microcarrier culture had higher glucose consumption, higher glycolysis and lower autophagy gene expression based on mRNA-seq. The microcarrier cultures had at least 17-23 fold higher EV secretion, and EV collection in mTeSR had 2.7-3.7 fold higher yield than HBM medium. Microcarrier culture with mTeSR EV collection had a smaller EV size than other groups, and the cargo was enriched with proteins (proteomics) and miRNAs (microRNA-seq) reducing apoptosis and promoting cell proliferation (e.g. Wnt-related pathways). hiPSC-EVs demonstrated the ability of stimulating proliferation and M2 polarization of microglia . HiPSC expansion on microcarriers produces much higher yields of EVs than hiPSC aggregates in VWBRs. EV collection in mTeSR increases yield compared to HBM. The biomanufactured EVs from microcarrier culture in mTeSR have exosomal characteristics and are functional in microglia stimulation, which paves the ways for future in vivo anti-aging study.
PubMed: 38938678
DOI: 10.1002/jex2.133 -
Frontiers in Cardiovascular Medicine 2024Vascular pathologies are prevalent in a broad spectrum of diseases, necessitating a deeper understanding of vascular biology, particularly in overcoming the oxygen and... (Review)
Review
Vascular pathologies are prevalent in a broad spectrum of diseases, necessitating a deeper understanding of vascular biology, particularly in overcoming the oxygen and nutrient diffusion limit in tissue constructs. The evolution of vascularized tissues signifies a convergence of multiple scientific disciplines, encompassing the differentiation of human pluripotent stem cells (hPSCs) into vascular cells, the development of advanced three-dimensional (3D) bioprinting techniques, and the refinement of bioinks. These technologies are instrumental in creating intricate vascular networks essential for tissue viability, especially in thick, complex constructs. This review provides broad perspectives on the past, current state, and advancements in key areas, including the differentiation of hPSCs into specific vascular lineages, the potential and challenges of 3D bioprinting methods, and the role of innovative bioinks mimicking the native extracellular matrix. We also explore the integration of biophysical cues in vascularized tissues , highlighting their importance in stimulating vessel maturation and functionality. In this review, we aim to synthesize these diverse yet interconnected domains, offering a broad, multidisciplinary perspective on tissue vascularization. Advancements in this field will help address the global organ shortage and transform patient care.
PubMed: 38938652
DOI: 10.3389/fcvm.2024.1336910 -
Cell & Bioscience Jun 2024Neurodevelopmental disorders (NDD), such as autism spectrum disorders (ASD) and intellectual disorders (ID), are highly debilitating childhood psychiatric conditions....
BACKGROUND
Neurodevelopmental disorders (NDD), such as autism spectrum disorders (ASD) and intellectual disorders (ID), are highly debilitating childhood psychiatric conditions. Genetic factors are recognized as playing a major role in NDD, with a multitude of genes and genomic regions implicated. While the functional validation of NDD-associated genes has predominantly been carried out using mouse models, the significant differences in brain structure and gene function between mice and humans have limited the effectiveness of mouse models in exploring the underlying mechanisms of NDD. Therefore, it is important to establish alternative animal models that are more evolutionarily aligned with humans.
RESULTS
In this study, we employed CRISPR/Cas9 and somatic cell nuclear transplantation technologies to successfully generate a knockout miniature pig model of the MIR137 gene, which encodes the neuropsychiatric disorder-associated microRNA miR-137. The homozygous knockout of MIR137 (MIR137) effectively suppressed the expression of mature miR-137 and led to the birth of stillborn or short-lived piglets. Transcriptomic analysis revealed significant changes in genes associated with neurodevelopment and synaptic signaling in the brains of MIR137 miniature pig, mirroring findings from human ASD transcriptomic data. In comparison to miR-137-deficient mouse and human induced pluripotent stem cell (hiPSC)-derived neuron models, the miniature pig model exhibited more consistent changes in critical neuronal genes relevant to humans following the loss of miR-137. Furthermore, a comparative analysis identified differentially expressed genes associated with ASD and ID risk genes in both miniature pig and hiPSC-derived neurons. Notably, human-specific miR-137 targets, such as CAMK2A, known to be linked to cognitive impairments and NDD, exhibited dysregulation in MIR137 miniature pigs. These findings suggest that the loss of miR-137 in miniature pigs affects genes crucial for neurodevelopment, potentially contributing to the development of NDD.
CONCLUSIONS
Our study highlights the impact of miR-137 loss on critical genes involved in neurodevelopment and related disorders in MIR137 miniature pigs. It establishes the miniature pig model as a valuable tool for investigating neurodevelopmental disorders, providing valuable insights for potential applications in human research.
PubMed: 38937838
DOI: 10.1186/s13578-024-01268-8 -
Molecular Neurobiology Jun 2024Cyclin-dependent kinase 5 (CDK5) is a protein kinase involved in neuronal homeostasis and development critical for neuronal survival. Besides, its deregulation is linked...
Cyclin-dependent kinase 5 (CDK5) is a protein kinase involved in neuronal homeostasis and development critical for neuronal survival. Besides, its deregulation is linked to neurodegenerative pathologies such as Alzheimer's and Parkinson's diseases. For that reason, we aimed to generate a deficient CDK5 genetic model in neurons derived from human-induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 technology. We obtained a heterozygous CDK5 clone for the FN2.1 hiPSC line that retained hiPSC stemness and pluripotent potential. Then, neural stem cells (NSCs) and further neurons were derived from the CDK5 KO FN2.1 hiPSCs, and their phenotype was validated by immunofluorescence staining using antibodies that recognize lineage-specific markers (SOX-1, SOX-2, and NESTIN for NSCs and TUJ-1, MAP-5, and MAP-2 for neurons). We found that the proliferation rate increased in CDK5 KO hiPSC-derived neurons concomitantly with a reduction in NEUN and P35 expression levels. However, the morphometric analysis revealed that CDK5 deficiency caused an increase in the length of the main, primary, and secondary neurites and the neuronal soma area. As a whole, we found that a deficit in CDK5 does not impair hiPSC neuronal differentiation but deregulates proliferation and neurite outgrowth, favoring elongation. The misregulated activity of specific kinases leads to abnormalities such as impaired axonal connectivity in neurodegenerative diseases. Thus, therapeutic approaches aimed at normalizing the activity of kinases, such as CDK5, may help prevent the degeneration of vulnerable neurons.
PubMed: 38937422
DOI: 10.1007/s12035-024-04325-y -
Biochimica Et Biophysica Acta.... Jun 2024Three-dimensional (3D) organoids derived from human pluripotent stem cells (hPSCs) have revolutionized in vitro tissue modeling, offering a unique opportunity to...
BACKGROUND
Three-dimensional (3D) organoids derived from human pluripotent stem cells (hPSCs) have revolutionized in vitro tissue modeling, offering a unique opportunity to replicate physiological tissue organization and functionality. This study investigates the impact of radiation on skeletal muscle response using an innovative in vitro human 3D skeletal muscle organoids (hSMOs) model derived from hPSCs.
METHODS
The hSMOs model was established through a differentiation protocol faithfully recapitulating embryonic myogenesis and maturation via paraxial mesodermal differentiation of hPSCs. Key skeletal muscle characteristics were confirmed using immunofluorescent staining and RT-qPCR. Subsequently, the hSMOs were exposed to a clinically relevant dose of 2 Gy of radiation, and their response was analyzed using immunofluorescent staining and RNA-seq.
RESULTS
The hSMO model faithfully recapitulated embryonic myogenesis and maturation, maintaining key skeletal muscle characteristics. Following exposure to 2 Gy of radiation, histopathological analysis revealed deficits in hSMOs expansion, differentiation, and repair response across various cell types at early (30 min) and intermediate (18 h) time points post-radiation. Immunofluorescent staining targeting γH2AX and 53BP1 demonstrated elevated levels of foci per cell, particularly in PAX7 cells, during early and intermediate time points, with a distinct kinetic pattern showing a decrease at 72 h. RNA-seq data provided comprehensive insights into the DNA damage response within the hSMOs.
CONCLUSIONS
Our findings highlight deficits in expansion, differentiation, and repair response in hSMOs following radiation exposure, enhancing our understanding of radiation effects on skeletal muscle and contributing to strategies for mitigating radiation-induced damage in this context.
PubMed: 38936620
DOI: 10.1016/j.bbamcr.2024.119792