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 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 , the target locus in human pluripotent stem cells (hPSCs) for cardiac lineage tracing and clinical cell sorting are described.

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.

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.

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.

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).

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.

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.

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 () 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

Unlabelled: 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.

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.

Bio-inspired oligovitronectin-grafted surface for enhanced self-renewal and long-term maintenance of human pluripotent stem cells under feeder-free conditions.

Current protocols for human pluripotent stem cell (hPSC) expansion require feeder cells or matrices from animal sources that have been the major obstacle to obtain clinical grade hPSCs due to safety issues, difficulty in quality control, and high expense. Thus, feeder-free, chemically defined synthetic platforms have been developed, but are mostly confined to typical polystyrene culture plates. Here, we report a chemically defined, material-independent, bio-inspired surface coating allowing for feeder-free expansion and maintenance of self-renewal and pluripotency of hPSCs on various polymer substrates and devices.

Induced pluripotent stem cells for modeling of pediatric neurological disorders.

The pathophysiological mechanisms underlying childhood neurological disorders have remained obscure due to a lack of suitable disease models reflecting human pathogenesis. Using induced pluripotent stem cell (iPSC) technology, various neurological disorders can now be extensively modeled. Specifically, iPSC technology has aided the study and treatment of early-onset pediatric neurodegenerative diseases such as Rett syndrome, Down syndrome, Angelman syndrome.

Mesenchymal stem cell-conditioned medium enhances osteogenic and chondrogenic differentiation of human embryonic stem cells and human induced pluripotent stem cells by mesodermal lineage induction.

Human mesenchymal stem cells (hMSCs) have the ability to differentiate into mesenchymal lineages. In this study, we hypothesized that treatment of embryoid bodies (EBs) composed of either human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) with a hMSC-conditioned medium (CM) can stimulate the induction of the mesodermal lineage and subsequent differentiation toward the osteogenic and chondrogenic lineage. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) analysis indicated that the hMSC-CM treatment increased gene expression related to the mesodermal lineage and decreased gene expression related to the endodermal and ectodermal lineage in EBs.

ABCD2 is a direct target of β-catenin and TCF-4: implications for X-linked adrenoleukodystrophy therapy.

X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder caused by mutations in the ABCD1 gene that encodes the peroxisomal ATP-binding cassette (ABC) transporter subfamily D member 1 protein (ABCD1), which is referred to as the adrenoleukodystrophy protein (ALDP). Induction of the ABCD2 gene, the closest homolog of ABCD1, has been mentioned as a possible therapeutic option for the defective ABCD1 protein in X-ALD. However, little is known about the transcriptional regulation of ABCD2 gene expression.

The genetically modified polysialylated form of neural cell adhesion molecule-positive cells for potential treatment of X-linked adrenoleukodystrophy.

Purpose: Cell transplantation of myelin-producing exogenous cells is being extensively explored as a means of remyelinating axons in X-linked adrenoleukodystrophy. We determined whether 3,3',5-Triiodo-L-thyronine (T3) overexpresses the ABCD2 gene in the polysialylated (PSA) form of neural cell adhesion molecule (NCAM)-positive cells and promotes cell proliferation and favors oligodendrocyte lineage differentiation.

Materials And Methods: PSA-NCAM+ cells from newborn Sprague-Dawley rats were grown for five days on uncoated dishes in defined medium with or without supplementation of basic fibroblast growth factor (bFGF) and/or T3.