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BioRxiv : the Preprint Server For... Jun 2024PARP1 (ARTD1) and Tankyrases (TNKS1/TNKS2; PARP5a/5b) are poly-ADP-ribose polymerases (PARPs) with catalytic and non-catalytic functions that regulate both the genome...
PARP1 (ARTD1) and Tankyrases (TNKS1/TNKS2; PARP5a/5b) are poly-ADP-ribose polymerases (PARPs) with catalytic and non-catalytic functions that regulate both the genome and proteome during zygotic genome activation (ZGA), totipotent, and pluripotent embryonic stages. Here, we show that primed, conventional human pluripotent stem cells (hPSC) cultured continuously under non-specific TNKS1/TNKS2/PARP1-inhibited chemical naive reversion conditions underwent epigenetic reprogramming to clonal blastomere-like stem cells. TIRN stem cells concurrently expressed hundreds of gene targets of the ZGA-priming pioneer factor DUX4, as well as a panoply of four-cell (4C)-specific (e.g., TPRXL, HOX clusters), eight-cell (8C)-specific (e.g., DUXA, GSC, GATA6), primitive endoderm-specific (e.g., GATA4, SOX17), trophectoderm-specific (e.g., CDX2, TFAP2C), and naive epiblast-specific (e.g., DNMT3L, NANOG, POU5F1(OCT4)) factors; all in a hybrid, combinatorial single-cell manner. Mapping of proteomic and single-cell expressions of TIRN cells against human preimplantation embryo references identified them as relatively homogenous 4C-8C stage populations. Injection of TIRN cells into murine 8C-16C-staged embryos resulted in efficient totipotent-like single cell contributions of human cells to both extra-embryonic (trophectoderm, placenta) and embryonic (neural, fetal liver, hematopoietic) lineages in human-murine blastocyst and fetal chimeras. Pairing of proteome with ubiquitinome analyses of TIRN cells revealed a global shutdown of ADP-ribosylation, and a perturbed TNKS/PARP1 equilibrium which not only impacted the protein levels of hundreds of TNKS/PARP1 substrates via a rewiring of the ubiquitin-proteosome system (UPS), but also de-repressed expression of hundreds of developmental genes associated with PARP1 suppression. ChIP-Seq analysis of core NANOG-SOX2-OCT4 (NSO) pluripotency factors in TIRN cells identified reprogrammed DUX4-accessible distal and cis-regulatory enhancer regions that were co-bound by PARP1 (NSOP). These NSOP enhancer regions possessed co-binding motifs for hundreds of the same ZGA-associated, embryonic, and extraembryonic lineage-specifying pioneer factors (e.g., HOX, FOX, GATA, SOX, TBX, CDX families) that were concurrently co-expressed in TIRN cells; suggesting that PARP1 and DUX4 cooperate with NSO pluripotency core factors to regulate the epigenetic plasticity of a human totipotency program. These findings provide the first demonstration that global, proteome-wide perturbations of post-translational modifications (i.e., ADP-ribosylation, ubiquitination) can regulate epigenetic reprogramming during human embryogenesis. Totipotent TIRN stem cells will provide a valuable cell culture model for studying the proteogenomic regulation of lineage specification from human blastomere stages and may facilitate the efficient generation of human organs in interspecies chimeras.
PubMed: 38915486
DOI: 10.1101/2024.06.14.598510 -
Frontiers in Cardiovascular Medicine 2024There have been conflicting reports about the proarrhythmic risk of -synephrine (SYN). To address this, human induced pluripotent stem cell-derived cardiomyocytes...
Non-invasive assessment of proarrhythmic risks associated with isoprenaline and the dietary supplement ingredient synephrine using human induced pluripotent stem cell-derived cardiomyocytes.
BACKGROUND
There have been conflicting reports about the proarrhythmic risk of -synephrine (SYN). To address this, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combined with the microelectrode array (MEA) system have been utilized to assess arrhythmia risks, particularly in the context of adrenomimetic drugs.
AIM
This study aims to determine whether MEA recordings from hiPSC-CMs could predict the proarrhythmic risk of adrenomimetic drugs and to investigate the cardiovascular effects and mechanisms of SYN.
MATERIALS AND METHODS
We employed MEA recordings to assess the electrophysiological properties of hiPSC-CMs and conducted concentration-response analyses to evaluate the effects of SYN and Isoprenaline (ISO) on beating rate and contractility. A risk scoring system for proarrhythmic risks was established based on hiPSC-CMs in this study. ISO, a classic beta-adrenergic drug, was also evaluated. Furthermore, the study evaluated the risk of SYN and recorded the concentration-response of beating rate, contractility and the change in the presence or absence of selective β1, β2 and β3 adrenergic blockers.
RESULTS
Our results suggested that ISO carries a high risk of inducing arrhythmias, aligning with existing literature. SYN caused a 30% prolongation of the field potential duration (FPD) at a concentration of 206.326 μM, a change significantly different from baseline measurements and control treatments. The half maximal effective concentration (EC50) of SYN (3.31 μM) to affect hiPSC-CM beating rate is much higher than that of ISO (18.00 nM). The effect of SYN at an EC50 of 3.31 μM is about ten times more potent in hiPSC-CMs compared to neonatal rat cardiomyocytes (34.12 μM). SYN increased the contractility of cardiomyocytes by 29.97 ± 11.65%, compared to ISO's increase of 50.56 ± 24.15%. β1 receptor blockers almost eliminated the beating rate increase induced by both ISO and SYN, while neither β2 nor β3 blockers had a complete inhibitory effect.
CONCLUSION
The MEA and hiPSC-CM system could effectively predict the risk of adrenomimetic drugs. The study concludes that the proarrhythmia risk of SYN at conventional doses is low. SYN is more sensitive in increasing beating rate and contractility in human cardiomyocytes compared to rats, primarily activating β1 receptor.
PubMed: 38911513
DOI: 10.3389/fcvm.2024.1407138 -
Frontiers in Physiology 2024Pulsed Field Ablation (PFA) is a novel non-thermal method for cardiac ablation, relying on irreversible electroporation induced by high-energy pulsed electric fields...
INTRODUCTION
Pulsed Field Ablation (PFA) is a novel non-thermal method for cardiac ablation, relying on irreversible electroporation induced by high-energy pulsed electric fields (PEFs) to create localized lesions in the heart atria. A significant challenge in optimizing PFA treatments is determining the lethal electric field threshold (EFT), which governs ablation volume and varies with PEF waveform parameters. However, the proprietary nature of device developer's waveform characteristics and the lack of standardized nonclinical testing methods have left optimal EFTs for cardiac ablation uncertain.
METHODS
To address this gap, we introduced a laboratory protocol employing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in monolayer format to evaluate the impact of a range of clinically relevant biphasic pulse parameters on lethal EFT and adiabatic heating (AH). Cell death areas were assessed using fluorescent dyes and confocal microscopy, while lethal EFTs were quantified through comparison with electric field numerical simulations.
RESULTS AND CONCLUSION
Our study confirmed a strong correlation between cell death in hiPSC-CMs and the number and duration of pulses in each train, with pulse repetition frequency exerting a comparatively weaker influence. Fitting of these results through machine learning algorithms were used to develop an open-source online calculator. By estimating lethal EFT and associated temperature increases for diverse pulse parameter combinations, this tool, once validated, has the potential to significantly reduce reliance on animal models during early-stage device de-risking and performance assessment. This tool also offers a promising avenue for advancing PFA technology for cardiac ablation medical devices to enhance patient outcomes.
PubMed: 38911328
DOI: 10.3389/fphys.2024.1395923 -
Frontiers in Cell and Developmental... 2024Mammalian germ cells are derived from primordial germ cells (PGCs) and ensure species continuity through generations. Unlike irreversible committed mature germ cells,...
Mammalian germ cells are derived from primordial germ cells (PGCs) and ensure species continuity through generations. Unlike irreversible committed mature germ cells, migratory PGCs exhibit a latent pluripotency characterized by the ability to derive embryonic germ cells (EGCs) and form teratoma. Here, we show that inhibition of p38 mitogen-activated protein kinase (MAPK) by chemical compounds in mouse migratory PGCs enables derivation of chemically induced Embryonic Germ-like Cells (cEGLCs) that do not require conventional growth factors like LIF and FGF2/Activin-A, and possess unique naïve pluripotent-like characteristics with epiblast features and chimera formation potential. Furthermore, cEGLCs are regulated by a unique PI3K-Akt signaling pathway, distinct from conventional naïve pluripotent stem cells described previously. Consistent with this notion, we show by performing analysis that inhibition of p38 MAPK in organ culture supports the survival and proliferation of PGCs and also potentially reprograms PGCs to acquire indefinite proliferative capabilities, marking these cells as putative teratoma-producing cells. These findings highlight the utility of our model in mimicking teratoma formation, thereby providing valuable insights into the cellular mechanisms underlying tumorigenesis. Taken together, our research underscores a key role of p38 MAPK in germ cell development, maintaining proper cell fate by preventing unscheduled pluripotency and teratoma formation with a balance between proliferation and differentiation.
PubMed: 38911025
DOI: 10.3389/fcell.2024.1410177 -
APL Bioengineering Jun 2024Previous lung-on-chip devices have facilitated significant advances in our understanding of lung biology and pathology. Here, we describe a novel lung-on-a-chip model in...
Previous lung-on-chip devices have facilitated significant advances in our understanding of lung biology and pathology. Here, we describe a novel lung-on-a-chip model in which human induced pluripotent stem cell-derived alveolar epithelial type II cells (iAT2s) form polarized duct-like lumens alongside engineered perfused vessels lined with human umbilical vein endothelium, all within a 3D, physiologically relevant microenvironment. Using this model, we investigated the morphologic and signaling consequences of the KRAS mutation, a commonly identified oncogene in human lung adenocarcinoma (LUAD). We show that expression of the mutant KRAS isoform in iAT2s leads to a hyperproliferative response and morphologic dysregulation in the epithelial monolayer. Interestingly, the mutant epithelia also drive an angiogenic response in the adjacent vasculature that is mediated by enhanced secretion of the pro-angiogenic factor soluble uPAR. These results demonstrate the functionality of a multi-cellular platform capable of modeling mutation-specific behavioral and signaling changes associated with lung adenocarcinoma.
PubMed: 38911024
DOI: 10.1063/5.0207228 -
Biochemistry and Biophysics Reports Sep 2024The three-dimensional (3D) kidney organoid is a breakthrough model for recapitulating renal morphology and function , which is grown from stem cells and resembles... (Review)
Review
The three-dimensional (3D) kidney organoid is a breakthrough model for recapitulating renal morphology and function , which is grown from stem cells and resembles mammalian kidney organogenesis. Currently, protocols for cultivating this model from induced pluripotent stem cells (iPSCs) and patient-derived adult stem cells (ASCs) have been widely reported. In recent years, scientists have focused on combining cutting-edge bioengineering and bioinformatics technologies to improve the developmental accuracy of kidney organoids and achieve high-throughput experimentation. As a remarkable tool for mechanistic research of the renal system, kidney organoid has both potential and challenges. In this review, we have described the evolution of kidney organoid establishment methods and highlighted the latest progress leading to a more sophisticated kidney transformation research model. Finally, we have summarized the main applications of renal organoids in exploring kidney disease.
PubMed: 38910872
DOI: 10.1016/j.bbrep.2024.101736 -
Neuroscience Jun 2024Mesenchymal stromal cells (MSCs) hold therapeutic potential for neurological disorders, but their impact on neuronal activity remains unclear. We investigated the...
Mesenchymal stromal cells (MSCs) hold therapeutic potential for neurological disorders, but their impact on neuronal activity remains unclear. We investigated the effects of SB623 cells (Notch-1 intracellular domain-transfected MSCs) and parental MSCs on human induced pluripotent stem cell (iPSC)-derived neurons using multi-electrode arrays. SB623 cells significantly increased neuronal activity and oscillation in a dose-dependent manner, surpassing astrocytes in promoting network bursts. Strikingly, glutamatergic neurons showed a rapid increase in activity and bursts compared to GABAergic neurons, suggesting glutamate release from SB623 cells. We confirmed this by finding high glutamate levels in SB623 cell conditioned medium, which were reduced by glutaminase inhibition. Glutamate release was further implicated by the reduced excitability in co-cultures with astrocytes, known glutamate scavengers. Our findings reveal a novel mechanism for MSCs: promoting neuronal activity and network formation through tonic glutamate release, with potential implications for MSC-based therapies.
PubMed: 38909673
DOI: 10.1016/j.neuroscience.2024.06.015 -
Stem Cell Research Jun 2024Ten-Eleven Translocation methylcytosine dioxygenase 1 (TET1) is known to play a broad tumor suppressor role through demethylating and activating tumor suppressor genes....
Ten-Eleven Translocation methylcytosine dioxygenase 1 (TET1) is known to play a broad tumor suppressor role through demethylating and activating tumor suppressor genes. TET1 missense mutations are previously reported in many types of leukemia. Here, the human induced pluripotent stem cell line MURAi001-A was generated from skin fibroblasts derived from a 56-year-old female patient carrying the TET1 gene mutation c.4404A > G (p.I1468M), who had a history of ovarian germ cell tumor. The MURAi001-A cell line demonstrated embryonic-like characteristics as it expressed specific stemness markers, differentiated into the three germ layers, and retained normal karyotyping.
PubMed: 38909482
DOI: 10.1016/j.scr.2024.103474 -
Cell & Bioscience Jun 2024Clinical outcome after traumatic brain injury (TBI) is closely associated conditions of other organs, especially lungs as well as degree of brain injury. Even if there... (Review)
Review
Clinical outcome after traumatic brain injury (TBI) is closely associated conditions of other organs, especially lungs as well as degree of brain injury. Even if there is no direct lung damage, severe brain injury can enhance sympathetic tones on blood vessels and vascular resistance, resulting in neurogenic pulmonary edema. Conversely, lung damage can worsen brain damage by dysregulating immunity. These findings suggest the importance of brain-lung axis interactions in TBI. However, little research has been conducted on the topic. An advanced disease model using stem cell technology may be an alternative for investigating the brain and lungs simultaneously but separately, as they can be potential candidates for improving the clinical outcomes of TBI.In this review, we describe the importance of brain-lung axis interactions in TBI by focusing on the concepts and reproducibility of brain and lung organoids in vitro. We also summarize recent research using pluripotent stem cell-derived brain organoids and their preclinical applications in various brain disease conditions and explore how they mimic the brain-lung axis. Reviewing the current status and discussing the limitations and potential perspectives in organoid research may offer a better understanding of pathophysiological interactions between the brain and lung after TBI.
PubMed: 38909262
DOI: 10.1186/s13578-024-01252-2 -
Cell Reports Jun 2024Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids are valuable tools for researching developmental biology and personalized therapies, but their...
Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids are valuable tools for researching developmental biology and personalized therapies, but their closed topology and relative immature state limit applications. Here, we use organ-on-chip technology to develop a hiPSC-derived intestinal barrier with apical and basolateral access in a more physiological in vitro microenvironment. To replicate growth factor gradients along the crypt-villus axis, we locally expose the cells to expansion and differentiation media. In these conditions, intestinal epithelial cells self-organize into villus-like folds with physiological barrier integrity, and myofibroblasts and neurons emerge and form a subepithelial tissue in the bottom channel. The growth factor gradients efficiently balance dividing and mature cell types and induce an intestinal epithelial composition, including absorptive and secretory lineages, resembling the composition of the human small intestine. This well-characterized hiPSC-derived intestine-on-chip system can facilitate personalized studies on physiological processes and therapy development in the human small intestine.
PubMed: 38907996
DOI: 10.1016/j.celrep.2024.114247