CRISPR/Cas9-mediated knock-in of a fluorescent reporter into the target locus of interest in human pluripotent stem cells.

The method presented herein is associated with the Lab Resource article titled "Generation of αMHC-EGFP knock-in in human pluripotent stem cell line, SNUe003-A-3, using CRISPR/CAS9-based gene targeting" [1]. The cardiac muscle-specific protein, α-myosin heavy chain (αMHC), is encoded by the human MYH6 gene, which is expressed in both the atria and ventricles during embryonic development and is predominantly expressed in the atria after birth [2]. Herein, the methods used to achieve CRISPR/SpCas9-mediated introduction of an EGFP reporter into αMHC, the target locus in human pluripotent stem cells (hPSCs) for cardiac lineage tracing and clinical cell sorting are described. The CRISPR-Cas9 system enables efficient replacement of the stop codon in the last exon of αMHC with a 2A non-joining peptide (T2A)-EGFP cassette. First, hPSCs are transfected with the donor construct and Cas9/sgRNA plasmids via electroporation and selected with neomycin for approximately 3 weeks. Thereafter, the established cell line exhibits typical characteristics of human embryonic stem cells (hESCs). When these cells differentiate into cardiomyocytes, the expression of EGFP is confirmed using confocal microscopy, flow cytometry analysis, and immunostaining.•The line enables monitoring of cell maturation events during human cardiac development.•The line is a valuable platform for cardiotoxicity tests and drug screening.•This method has already been employed in two original studies, as previously reported for reporter cell line generation using CRISPR/Cas9.

New health index derived from oxidative potential and cell toxicity of fine particulate matter to assess its potential health effect.

Toxicological data and exposure levels of fine particulate matters (PM2.5) are necessary to better understand their health effects. Simultaneous measurements of PM2.5 oxidative potential (OP) and cell toxicity in urban areas (Beijing, China and Gwangju, Korea) reveal their dependence on chemical composition. Notably, acids (Polar), benzocarboxylic acids, and Pb were the chemical components that affected both OP and cell toxicity. OP varied more significantly among different locations and seasons (winter and summer) than cell toxicity. Using the measured OP, cell toxicity, and PM2.5 concentration, a health index was developed to better assess the potential health effects of PM2.5. The health index was related to the sources of PM2.5 derived from the measured chemical components. The contributions of secondary organic aerosols and dust to the proposed health index were more significant than their contributions to PM2.5 mass. The developed regression equation was used to predict the health effect of PM2.5 without further toxicity measurements. This new index could be a valuable health metric that provides information beyond just the PM2.5 concentration level.

Optical Transitions of a Single Nodal Ring in SrAs_{3}: Radially and Axially Resolved Characterization.

SrAs_{3} is a unique nodal-line semimetal that contains only a single nodal ring in the Brillouin zone, uninterrupted by any trivial bands near the Fermi energy. We performed axis-resolved optical reflection measurements on SrAs_{3} and observed that the optical conductivity exhibits flat absorption up to 129 meV in both the radial and axial directions, confirming the robustness of the universal power-law behavior of the nodal ring. The axis-resolved optical conductivity, in combination with theoretical calculations, further reveals fundamental properties beyond the flat absorption, including the overlap energy of the topological bands, the spin-orbit coupling gap along the nodal ring, and the geometric properties of the nodal ring such as the average ring radius, ring ellipticity, and velocity anisotropy. In addition, our temperature-dependent measurements revealed a spectral weight transfer between intraband and interband transitions, indicating a possible violation of the optical sum rule within the measured energy range.

Establishment of TUBB3-mCherry knock-in human pluripotent stem cell line using CRISPR/Cas9 (SNUe003-A-4).

TUBB3 is a structural neuronal protein important for multiple neuronal functions including axonal guidance and maturation. This study aimed to generate a human pluripotent stem cell (hPSC) line with a TUBB3-mCherry reporter using CRISPR/SpCas9 nuclease. The stop codon in the last exon of TUBB3 was replaced with a T2A-mCherry cassette using CRISPR/SpCas9-mediated homologous recombination. The established TUBB3-mCherry knock-in cell line exhibited typical pluripotent characteristics. The mCherry reporter faithfully replicated the endogenous level of TUBB3 upon induction of neuronal differentiation. The reporter cell line could contribute to the investigation of neuronal differentiation, neuronal toxicity, and neuronal tracing.

Generation of αMHC-EGFP knock-in in human pluripotent stem cell line, SNUe003-A-3 using CRISPR/Cas9-based gene targeting.

The cardiac muscle-specific protein, α-myosin heavy chain (αMHC), is a major component of cardiac muscle filaments involved in cardiac muscle contraction. Here, we established an αMHC-enhanced fluorescent protein (EGFP) knock-in human pluripotent stem cell (hPSC) line by linking the EGFP gene to the C-terminal region of αMHC via a 2A non-joining peptide using CRISPR/Cas9 nuclease. The EGFP reporter precisely reflected the endogenous level of αMHC upon the induction of cardiac differentiation. This reporter cell line will be a valuable platform for cardiotoxicity tests, drug screening, and investigating the pathological mechanisms of cardiomyocytes.

Generation of mutation-corrected induced pluripotent stem cell lines derived from adrenoleukodystrophy patient by using homology directed repair.

X-linked adrenoleukodystrophy (ALD) caused by the ABCD1 mutation, is the most common inherited peroxisomal disease. Previously, we generated an ALD patient-derived SCHi001-A iPSC model. In this study, we have performed the first genome editing of ALD patient-derived SCHi001-A iPSCs using homology-directed repair (HDR). The mutation site, c.1534G > A [GenBank: NM_000033.4], was corrected by introducing ssODN and the CRISPR/Cas9 system. The cell line exhibited normal iPSC plulipotency marker expression following genome editing. Mutation-corrected iPSCs from SCHi001-A iPSC line can be used in research into the pathophysiology of and therapeutics for ALD.

Generation of Brachyury-mCherry knock-in reporter human pluripotent stem cell line (SNUe003-A-2) using CRISPR/CAS9 nuclease.

Brachyury is an embryonic nuclear transcription factor required for mesoderm formation and differentiation. Here, we introduced an mCherry reporter into the C-terminus of Brachyury in the human pluripotent stem cell line SNUhES3 using the CRISPR/Cas9 nuclease approach. Successful gene editing was verified by DNA sequencing. SNUhES3-Brachyury-mCherry cells expressed pluripotent stem cell markers, exhibited a normal karyotype, and could generate all three germ layers. This cell line expressed the red fluorescence protein mCherry upon the induction of mesoderm differentiation. This reporter cell line could be used to monitor mesodermal population enrichment during mesodermal differentiation.

Establishment of PITX3-mCherry knock-in reporter human embryonic stem cell line (WAe009-A-23).

Pituitary homeobox 3 (Pitx3) is a key transcription factor that plays an important role in the development and maintenance of midbrain dopaminergic (mDA) neurons. Here, we established a PITX3-mCherry knock-in reporter human embryonic stem cell (hESC) line using the CRISPR/Cas9 system. PITX3-mCherry hESCs maintained pluripotency marker expression and exhibited the capacity to generate all 3 germ layers and a normal karyotype. After differentiation into mDA neurons, most PITX3 immunoreactivity overlapped with the red fluorescence of mCherry. This reporter cell line may be used to study the development of mDA neurons or to enrich mDA populations for transplantation.

Exogenous Addition of 25-Hydroxycholesterol Reduces Level of Very Long-Chain Fatty Acids in X-Linked Adrenoleukodystrophy.

X-Linked adrenoleukodystrophy (X-ALD) is a severe metabolic disorder characterized by the accumulation of very long-chain fatty acids (VLCFAs). Recently, we demonstrated that levels of 25-hydroxycholesterol (25-HC) and cholesterol 25-hydroxylase (CH25H) were found to be elevated in X-ALD. Herein, we report that the exogenous addition of 25-HC significantly reduces C26:0 levels in X-ALD patient-derived fibroblasts and oligodendrocytes differentiated from induced pluripotent stem cells (iPSCs) derived from X-ALD patients. Moreover, 25-HC treatment was found to down-regulate the expression of ELOVL1, a key enzyme for the synthesis of C26. In addition, activation of liver X receptor (LXR), a molecular target of endogenous 25-HC, also reduced C26:0 level. The reduction of C26:0 levels by 25-HC treatment might result, at least partially, from the decrease of ELOVL1 expression as well as the activation of LXR. Our findings could provide a better understanding of the role of 25-HC in X-ALD and useful information to find therapeutic agents to treat X-ALD.

Integrative lipidomic and transcriptomic analysis of X-linked adrenoleukodystrophy reveals distinct lipidome signatures between adrenomyeloneuropathy and childhood cerebral adrenoleukodystrophy.

Precise pathophysiology with respect to the phenotypic variations and severity of X-ALD, specifically between adrenomyeloneuropathy (AMN) and childhood cerebral adrenoleukodystrophy (CCALD), has not been fully discovered. Herein, a systematic analysis using multi-layered lipidomics and transcriptomics was conducted to elucidate distinctive metabolic biosignatures among healthy control, AMN, and CCALD. Significant alterations regarding the accumulation of very long chain fatty acids were found in various lipid species such as phospholipids, glycerolipids, and sphingolipids. Remarkably, TG and CER that are physiologically essential were markedly down-regulated in CCALD than AMN. Transcriptomic analysis further supported the robustness of our findings by providing valuable information on the gene expressions of the regulatory factors. For instance, regulators of sphingolipid catabolism (SMPD1, CERK, and SPHK1) and TG anabolism (GPAM, GPAT2, and MBOAT2) were more up-regulated in AMN than in CCALD. These observations, among others, were in line with the recognized alterations of the associated lipidomes. In conclusion, the homeostatic imbalance of the complex lipid networks may be pathogenically important in X-ALD and the particular dysregulations of TG and CER may further influence the severity of CCALD among X-ALD patients.

PGC1α is required for the induction of contact inhibition by suppressing ROS.

Contact inhibition (CI) is an important tumor-suppressive mechanism that arrests cell cycle when cells reach high density. Indeed, CI is aberrantly absent in cancer cells and the dysregulation of this can contribute to tumorigenesis. Previously, it has been shown that reactive oxygen species (ROS) levels are repressed at high cell density, which is required for CI, but no molecular mechanism of this ROS regulation has been reported. Here, we show that PGC1α regulates cell density-dependent CI. PGC1α is markedly induced in response to high cell density and suppresses ROS production. Although cellular ROS levels are progressively decreased with increasing cell density, knockdown of PGC1α results in a defect of density-dependent ROS suppression. Importantly, PGC1α knockdown cells become less sensitive to high cell density and exhibit loss of CI. Mechanistically, PGC1α represses ROS production by inducing mitochondrial SIRT3, and thus SIRT3 overexpression rescues the defects of CI by PGC1α knockdown. These results demonstrate that mitochondrial ROS production is a crucial regulator of cell proliferation and identify a new role of PGC1α in CI.

Differential effects on sodium current impairments by distinct SCN1A mutations in GABAergic neurons derived from Dravet syndrome patients.

BACKGROUND: We investigated how two distinct mutations in SCN1A differentially affect electrophysiological properties of the patient-derived GABAergic neurons and clinical severities in two Dravet syndrome (DS) patients. MATERIALS AND METHODS: We established induced pluripotent stem cells from two DS patients with different mutations in SCN1A and subsequently differentiated them into forebrain GABAergic neurons. Functionality of differentiated GABAergic neurons was examined by electrophysiological recordings. RESULTS: DS-1 patient had a missense mutation, c.4261G > T [GenBank: NM_006920.4] and DS-2 patient had a nonsense frameshift mutation, c.3576_3580 del TCAAA [GenBank: NM_006920.4]. Clinically, contrary to our expectations, DS-1 patient had more severe symptoms including frequency of seizure episodes and the extent of intellectual ability penetration than DS-2 patient. Electrophysiologic recordings showed significantly lower sodium current density and reduced action potential frequency at strong current injection (>60 pA) in GABAergic neurons derived from both. Intriguingly, unique genetic alterations of SCN1A differentially impacted electrophysiological impairment of the neurons, and the impairment's extent corresponded with the symptomatic severity of the donor from which the iPSCs were derived. CONCLUSION: Our results suggest the possibility that patient-derived iPSCs may provide a reliable in vitro system that reflects clinical severities in individuals with DS.

SIRT1 Enhances the Survival of Human Embryonic Stem Cells by Promoting DNA Repair.

Human embryonic stem cells (hESCs) hold great promise for the treatment of many incurable diseases. Sirtuin1 (SIRT1), a class III histone deacetylase, is abundantly expressed in hESCs and is known to regulate early differentiation and telomere elongation. Here, we show that downregulation of SIRT1 promotes cell death in hESCs, but not in differentiated cells, and the SIRT1-inhibition-mediated cell death is preceded by increased DNA damage. This increased DNA damage is at least partially due to decreased levels of DNA repair enzymes such as MSH2, MSH6, and APEX1. Furthermore, SIRT1 inhibition causes p53 activation, which eventually leads to DNA damage-induced apoptosis of hESCs. This study provides valuable insights into the mechanism of SIRT1-mediated hESC survival and should contribute to the development of safe and effective cell therapies.

25-hydroxycholesterol contributes to cerebral inflammation of X-linked adrenoleukodystrophy through activation of the NLRP3 inflammasome.

X-linked adrenoleukodystrophy (X-ALD), caused by an ABCD1 mutation, is a progressive neurodegenerative disorder associated with the accumulation of very long-chain fatty acids (VLCFA). Cerebral inflammatory demyelination is the major feature of childhood cerebral ALD (CCALD), the most severe form of ALD, but its underlying mechanism remains poorly understood. Here, we identify the aberrant production of cholesterol 25-hydroxylase (CH25H) and 25-hydroxycholesterol (25-HC) in the cellular context of CCALD based on the analysis of ALD patient-derived induced pluripotent stem cells and ex vivo fibroblasts. Intriguingly, 25-HC, but not VLCFA, promotes robust NLRP3 inflammasome assembly and activation via potassium efflux-, mitochondrial reactive oxygen species (ROS)- and liver X receptor (LXR)-mediated pathways. Furthermore, stereotaxic injection of 25-HC into the corpus callosum of mouse brains induces microglial recruitment, interleukin-1ß production, and oligodendrocyte cell death in an NLRP3 inflammasome-dependent manner. Collectively, our results indicate that 25-HC mediates the neuroinflammation of X-ALD via activation of the NLRP3 inflammasome.

Heavy-ion injector based on an electron cyclotron ion source for the superconducting linear accelerator of the Rare Isotope Science Project.

The injector for the main driver linear accelerator of the Rare Isotope Science Project in Korea, has been developed to allow heavy ions up to uranium to be delivered to the inflight fragmentation system. The critical components of the injector are the superconducting electron cyclotron resonance (ECR) ion sources, the radio frequency quadrupole (RFQ), and matching systems for low and medium energy beams. We have built superconducting magnets for the ECR ion source, and a prototype with one segment of the RFQ structure, with the aim of developing a design that can satisfy our specifications, demonstrate stable operation, and prove results to compare the design simulation.

Emittance study of a 28 GHz electron cyclotron resonance ion source for the Rare Isotope Science Project superconducting linear accelerator.

A 28 GHz electron cyclotron resonance (ECR) ion source is being developed for use as an injector for the superconducting linear accelerator of the Rare Isotope Science Project. Beam extraction from the ECR ion source has been simulated using the KOBRA3-INP software. The simulation software can calculate charged particle trajectories in three dimensional complex magnetic field structures, which in this case are formed by the arrangement of five superconducting magnets. In this study, the beam emittance is simulated to understand the effects of plasma potential, mass-to-charge ratio, and spatial distribution. The results of these simulations and their comparison to experimental results are presented in this paper.

Beam dynamics simulations of post low energy beam transport section in RAON heavy ion accelerator.

RAON (Rare isotope Accelerator Of Newness) heavy ion accelerator of the rare isotope science project in Daejeon, Korea, has been designed to accelerate multiple-charge-state beams to be used for various science programs. In the RAON accelerator, the rare isotope beams which are generated by an isotope separation on-line system with a wide range of nuclei and charges will be transported through the post Low Energy Beam Transport (LEBT) section to the Radio Frequency Quadrupole (RFQ). In order to transport many kinds of rare isotope beams stably to the RFQ, the post LEBT should be devised to satisfy the requirement of the RFQ at the end of post LEBT, simultaneously with the twiss parameters small. We will present the recent lattice design of the post LEBT in the RAON accelerator and the results of the beam dynamics simulations from it. In addition, the error analysis and correction in the post LEBT will be also described.

Prototyping of beam position monitor for medium energy beam transport section of RAON heavy ion accelerator.

A heavy ion accelerator, RAON is going to be built by Rare Isotope Science Project in Korea. Its target is to accelerate various stable ions such as uranium, proton, and xenon from electron cyclotron resonance ion source and some rare isotopes from isotope separation on-line. The beam shaping, charge selection, and modulation should be applied to the ions from these ion sources because RAON adopts a superconducting linear accelerator structure for beam acceleration. For such treatment, low energy beam transport, radio frequency quadrupole, and medium energy beam transport (MEBT) will be installed in injector part of RAON accelerator. Recently, development of a prototype of stripline beam position monitor (BPM) to measure the position of ion beams in MEBT section is under way. In this presentation, design of stripline, electromagnetic (EM) simulation results, and RF measurement test results obtained from the prototyped BPM will be described.

Thermo-responsive polymeric nanoparticles for enhancing neuronal differentiation of human induced pluripotent stem cells.

We report thermo-responsive retinoic acid (RA)-loaded poly(N-isopropylacrylamide)-co-acrylamide (PNIPAM-co-Am) nanoparticles for directing human induced pluripotent stem cell (hiPSC) fate. Fourier transform infrared spectroscopy and (1)H nuclear magnetic resonance analysis confirmed that RA was efficiently incorporated into PNIAPM-co-Am nanoparticles (PCANs). The size of PCANs dropped with increasing temperatures (300-400 nm at room temperature, 80-90 nm at 37°C) due to its phase transition from hydrophilic to hydrophobic. Due to particle shrinkage caused by this thermo-responsive property of PCANs, RA could be released from nanoparticles in the cells upon cellular uptake. Immunocytochemistry and quantitative real-time polymerase chain reaction analysis demonstrated that neuronal differentiation of hiPSC-derived neuronal precursors was enhanced after treatment with 1-2 µg/ml RA-loaded PCANs. Therefore, we propose that this PCAN could be a potentially powerful carrier for effective RA delivery to direct hiPSC fate to neuronal lineage. FROM THE CLINICAL EDITOR: The use of induced pluripotent stem cells (iPSCs) has been at the forefront of research in the field of regenerative medicine, as these cells have the potential to differentiate into various terminal cell types. In this article, the authors utilized a thermo-responsive polymer, Poly(N-isopropylacrylamide) (PNIPAM), as a delivery platform for retinoic acid. It was shown that neuronal differentiation could be enhanced in hiPSC-derived neuronal precursor cells. This method may pave a way for future treatment of neuronal diseases.

Sixth-order resonance of high-intensity linear accelerators.

It is shown that the sixth-order 6σ=720° (or 6∶2) resonance is manifested for high-intensity beams of linear accelerators through the space charge potential when the depressed phase advance per cell σ is close to and below 120° but no resonance effect is observed for σ above 120°. Simulation studies show a clear emittance growth by this resonance and a characteristic sixfold resonance structure in phase space. To verify that this is a resonance, a frequency analysis was conducted and a study was performed of crossing the resonance from above and from below the resonance. Canonical perturbation is carried out to show that this resonance arises through perturbation of strong 2σ=360° (2∶1) and 4σ=360° (4∶1) space charge resonances. Simulations also show that the space charge 6σ=360° (or 6∶1) resonance is very weak.