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Abstract Autologous skin grafting is a standard treatment for skin defects such as burns. No artificial skin substitutes are functionally equivalent to autologous skin grafts. The cultured epidermis lacks the dermis and does not engraft deep wounds. Although reconstituted skin, which consists of cultured epidermal cells on a synthetic dermal substitute, can engraft deep wounds, it requires the wound bed to be well-vascularized and lacks skin appendages. In this study, we successfully generate complete skin grafts with pluripotent stem cell-derived epidermis with appendages on p63 knockout embryos’ dermis. Donor pluripotent stem cell-derived keratinocytes encroach the embryos’ dermis by eliminating p63 knockout keratinocytes based on cell-extracellular matrix adhesion mediated cell competition. Although the chimeric skin contains allogenic dermis, it is engraftable as long as autologous grafts. Furthermore, we could generate semi-humanized skin segments by human keratinocytes injection into the amnionic cavity of p63 knockout mice embryos. Niche encroachment opens the possibility of human skin graft production in livestock animals.

Abstract Background and Aims Burning mouth syndrome (BMS) causes burning or uncomfortable feelings in the oral cavity without any obvious injuries. This condition's etiopathogenesis is still unknown, consequently, BMS management is very challenging. Alpha‐lipoic acid (ALA) is a naturally occurring potent bioactive compound that has been found to be useful in the management of BMS in many studies. Therefore, we conducted a comprehensive systematic review to investigate the usefulness of ALA in the management of BMS based on randomized controlled trials (RCTs). Methods Different electronic databases, including PubMed, Scopus, Embase, Web of Science, and Google Scholar, were extensively searched to find relevant studies. Results This study included nine RCTs that matched the inclusion criteria. In most studies, ALA was given at a dose of 600–800 mg/day, with up to two months of follow‐up. The majority of studies (six out of nine studies) indicated that ALA was more effective in BMS patients than in the placebo‐controlled group. Conclusions This comprehensive systematic review provides evidence of the positive outcomes of the treatment of BMS with ALA. However, more research might be needed before ALA can be considered the first‐line therapy for BMS.

Oral administration of active pharmaceutical ingredients is desirable because it is easy, safe, painless, and can be performed by patients, resulting in good medication adherence. The mucus layer in the gastrointestinal (GI) tract generally acts as a barrier to protect the epithelial membrane from foreign substances; however, in the absorption process after oral administration, it can also disturb effective drug absorption by trapping it in the biological sieve structured by mucin, a major component of mucus, and eliminating it by mucus turnover. Recently, functional nanocarriers (NCs) have attracted much attention due to their immense potential and effectiveness in the field of oral drug delivery. Among them, NCs with mucopenetrating and mucoadhesive properties are promising dosage options for controlling drug absorption from the GI tracts. Mucopenetrating and mucoadhesive NCs can rapidly deliver encapsulated drugs to the absorption site and/or prolong the residence time of NCs close to the absorption membrane, providing better medications than conventional approaches. The surface characteristics of NCs are important factors that determine their functionality, owing to the formation of various kinds of interactions between the particle surface and mucosal components. Thus, a deeper understanding of surface modifications on the biopharmaceutical characteristics of NCs is necessary to develop the appropriate mucosal drug delivery systems (mDDS) for the treatment of target diseases. This review summarizes the basic information and functions of the mucosal layer, highlights the recent progress in designing functional NCs for mDDS, and discusses their performance in the GI tract.

In this study, we developed stabilized astaxanthin (AX) nanoparticles (sNP/AX) to improve the physicochemical properties, oral bioavailability, and hepatoprotection of AX. A flash nanoprecipitation technique was used with a multi-inlet vortex mixer to prepare the sNP/AX. Vitamins E (VE) and C (VC) were used as co-stabilizers with poloxamer 407 as a stabilizer to inhibit the oxidative degradation of AX during sNP/AX formation and storage. VC stabilized AX in the aqueous phase during the preparation, whereas VE markedly improved the storage stability of sNP/AX, as evidenced by the AX contents remaining at 94 and 81% after 12 weeks of storage at 4 °C and 25 °C, respectively. The mean sNP/AX diameter was 215 nm, which resulted in higher AX release properties than those of crystalline AX. Rats, orally administered sNP/AX (33.2 mg AX/kg), exhibited higher systemic exposure to AX, whereas oral absorption in the crystalline AX group was negligible. In the rat hepatic injury model, oral pretreatment with sNP/AX (33.2 mg AX/kg) markedly attenuated hepatic damage, as shown by the histopathological analysis and reduced levels of plasma biomarkers for hepatic injury. These findings suggest that strategically including antioxidative additives in the sNP/AX has the potential to improve the physicochemical and nutraceutical properties of AX.

Abstract Background Postural abnormality is one of the main symptoms of Parkinson’s disease (PD). The erector spinae muscles play an important role in maintaining an upright posture, but the fatigability of the erector spinae in patients with PD is unknown. The purpose of this study was to compare the trunk extension maximum voluntary contraction (MVC) and the fatigability of the erector spinae between female patients with PD and healthy volunteers. Methods Th participants of this cross-sectional pilot study comprised 19 patients with PD and nine healthy volunteers matched for sex, age, and physical characteristics as a control group. The MVC of all participants was measured, and after sufficient rest, the Sørensen back endurance test was conducted to the point of exhaustion. The muscle activity of the erector spinae during the Sørensen back endurance test was measured using surface electromyography. The median frequency (MF) slope, which is an index of fatigability, was calculated from the recorded surface muscle activity by means of power spectrum analysis using a Fast Fourier transformation. Results Nine of the 19 patients with PD were unable to perform the Sørensen back endurance test, and a lower proportion of the PD group were able to perform it compared with the control group. The MVC of those patients with PD who were able to perform the Sørensen back endurance test was lower than that of the control group, and the time for which the pose could be maintained was shorter. There was no significant difference between the MF slope on the left and right side in the PD group, and it was higher on both sides than in the control group. Conclusion This is the first study to demonstrate a reduction of maximum muscle strength and great fatigability of the erector spinae in patients with PD. This discovery strongly underlines the need for paraspinal muscle training from an early stage with the aim of preventing the progression of postural abnormality in patients with PD.

Abstract Blastocyst complementation is an intriguing way of generating humanized animals for organ preparation in regenerative medicine and establishing novel models for drug development. Confirming that complemented organs and cells work normally in chimeric animals is critical to demonstrating the feasibility of blastocyst complementation. Here, we generated thymus-complemented chimeric mice, assessed the efficacy of anti-PD-L1 antibody in tumor-bearing chimeric mice, and then investigated T-cell function. Thymus-complemented chimeric mice were generated by injecting C57BL/6 (B6) embryonic stem cells into Foxn1 nu/nu morulae or blastocysts. Flow cytometry data showed that the chimeric mouse thymic epithelial cells (TECs) were derived from the B6 cells. T cells appeared outside the thymi. Single-cell RNA-sequencing analysis revealed that the TEC gene-expression profile was comparable to that in B6 mice. Splenic T cells of chimeric mice responded very well to anti-CD3 stimulation in vitro; CD4+ and CD8+ T cells proliferated and produced IFNγ, IL-2, and granzyme B, as in B6 mice. Anti-PD-L1 antibody treatment inhibited MC38 tumor growth in chimeric mice. Moreover, in the chimeras, anti-PD-L1 antibody restored T-cell activation by significantly decreasing PD-1 expression on T cells and increasing IFNγ-producing T cells in the draining lymph nodes and tumors. T cells produced by complemented thymi thus functioned normally in vitro and in vivo. To successfully generate humanized animals by blastocyst complementation, both verification of the function and gene expression profiling of complemented organs/cells in interspecific chimeras will be important in the near future.

The aim of the present study was to develop an injectable hydrogel (HG) formulation of fuzapladib sodium (FZP), an animal drug for acute pancreatitis (AP), with the use of polyethyleneoxide (PEO) and polylysine (pLys), a cationic polymer. A mixture of pLys and FZP was added to PEO to prepare an HG formulation, and the formulation was optimized by release test and viscosity measurements. Circular dichroism (CD) and infrared absorption (IR) spectral analyses were applied to clarify the intermolecular interactions between FZP and pLys. The pharmacokinetic behavior of FZP was evaluated after a subcutaneous administration of FZP samples (2.0 mg-FZP/kg) to rats. Although the immediate release of FZP was observed for the HG formulation, the addition of pLys at a 20-fold amount of FZP or higher led to the sustained release of FZP. Considering release behavior, the concentration of pLys was optimized as 100-fold that of FZP in the HG formulation. CD and IR spectroscopic analyses of FZP and/or pLys demonstrated an intermolecular interaction between FZP and pLys, as evidenced by the slight spectral transition. After a subcutaneous administration of HG formulation containing pLys to rats, compared with FZP alone, significant differences were observed in the pharmacokinetic behavior with a decrease of Cmax from 2.3 to 0.9 mg/mL and slower elimination kinetics. HG formulation using pLys might be a viable dosage option for FZP for the treatment of AP in animals.

Lisinopril, a highly hydrophilic long‐acting angiotensin‐converting enzyme inhibitor, is frequently prescribed for the treatment of hypertension and congestive heart failure. Green tea consumption may reduce the risk of cardiovascular outcomes and total mortality, whereas green tea or its catechin components has been reported to decrease plasma concentrations of a hydrophilic β blocker, nadolol, in humans. The aim of this study was to evaluate possible effects of green tea extract (GTE) on the lisinopril pharmacokinetics. In an open‐label, randomized, single‐center, 2‐phase crossover study, 10 healthy subjects ingested 200 mL of an aqueous solution of GTE containing ~ 300 mg of (–)‐epigallocatechin gallate, a major catechin component in green tea, or water (control) when receiving 10 mg of lisinopril after overnight fasting. The geometric mean ratio (GTE/control) for maximum plasma concentration and the area under the plasma concentration‐time curve of lisinopril were 0.289 (90% confidence interval (CI) 0.226–0.352) and 0.337 (90% CI 0.269–0.405), respectively. In contrast, there were no significant differences in time to reach maximum lisinopril concentration (6 hours in both phases) and renal clearance of lisinopril (57.7 mL/minute in control vs. 56.9 mL/minute in GTE). These results suggest that the extent of intestinal absorption of lisinopril was significantly impaired in the presence of GTE, whereas it had no major effect on the absorption rate and renal excretion of lisinopril. Concomitant use of lisinopril and green tea may decrease oral exposure to lisinopril, and therefore result in reduced therapeutic efficacy.

This study aimed to develop a cyclosporine A (CsA)-loaded ternary solid dispersion (tSD/CsA) to improve the storage stability of a solid dispersion (SD) system and the oral absorbability of CsA. Hydroxypropyl cellulose (HPC) and hydroxypropyl methylcellulose acetate succinate (HPMCAS) were selected as carrier materials of tSD, and tSD/CsA was prepared with a fine droplet drying process, a powderization technology that employs an inkjet head. The physicochemical properties of tSD/CsA were evaluated in terms of morphology, storage stability, dissolution behavior, and mucoadhesive property. After the oral administration of CsA samples (10 mg-CsA/kg) to rats, the plasma concentration of CsA was monitored to estimate oral absorbability. tSD/CsA comprised uniform shriveled particles with a diameter of 3.4 mm and span factor of 0.4, which is a parameter to estimate the particle size distribution. Although HPC-based binary SD showed marked aggregation of the particles after storage under 40 °C/75% relative humidity, there were no significant aggregations of tSD/CsA, due to the relatively low hygroscopic property of HPMCAS. The pH-dependent release of CsA with improved dissolution was observed in tSD/CsA. In the in vitro mucoadhesive evaluation using a mucin disk, tSD/CsA exhibited a better mucoadhesive property than HPC-based SD, possibly leading to prolonged retention of tSD particles in the gastrointestinal tract after oral administration. Orally-dosed tSD/CsA in rats resulted in significantly improved oral absorption of CsA, as evidenced by a 27-fold higher bioavailability than amorphous CsA. tSD/CsA may be a promising dosage option to improve the storage stability of a SD system and the biopharmaceutical properties of CsA.

The author wishes to make the following correction to this paper [...]

The use of pluripotent-stem cell derived organs for transplantation would be promising, if organs can be grown in a suitable host. Here, the authors use interspecific blastocyst complementation to generate a mouse pluripotent stem cell-derived kidney in anephric Sall1 mutant rats.

Summary: Intra-embryo genome editing by CRISPR/Cas9 enables easy generation of gene-modified animals by non-homologous end joining (NHEJ)-mediated frameshift mutations or homology-directed repair (HDR)-mediated point mutations. However, large modifications, such as gene replacement or gene fusions, are still difficult to introduce in embryos without costly micromanipulators. Moreover, micromanipulation techniques for intra-embryo genome editing have been established in only a small set of animals. To overcome these issues, we developed a method of large-fragment DNA knockin without micromanipulation. In this study, we successfully delivered the knockin donor DNA into zygotes by adeno-associated virus (AAV) without removing the zona pellucida, and we succeeded in both large-DNA fragment knockin and whole exon exchange with electroporation of CRISPR/Cas9 ribonucleoprotein. By this method, we can exchange large DNA fragments conveniently in various animal species without micromanipulation. : Techniques in Genetics; Genetic Engineering Subject Areas: Techniques in Genetics, Genetic Engineering

Abstract Helicobacter pylori cagA-positive strains are critically involved in the development of gastric cancer. Upon delivery into gastric epithelial cells via type IV secretion, the cagA-encoded CagA interacts with and thereby perturbs the pro-oncogenic phosphatase SHP2 and the polarity-regulating kinase PAR1b via the tyrosine-phosphorylated EPIYA-C/D segment and the CM sequence, respectively. Importantly, sequences spanning these binding regions exhibit variations among CagA proteins, which influence the pathobiological/oncogenic potential of individual CagA. Here we isolated an H. pylori strain (Hp_TH2099) naturally infecting the stomach of a housed macaque, indicating a zoonotic feature of H. pylori infection. Whole genome sequence analysis revealed that Hp_TH2099 belongs to the hpAsia2 cluster and possesses ABC-type Western CagA, which contains hitherto unreported variations in both EPIYA-C and CM sequences. The CM variations almost totally abolished PAR1b binding. Whereas pTyr + 5 variation in the EPIYA-C segment potentiated SHP2-binding affinity, pTyr-2 variation dampened CagA tyrosine phosphorylation and thus impeded CagA-SHP2 complex formation. As opposed to the H. pylori standard strain, infection of mouse ES cell-derived gastric organoids with Hp_TH2099 failed to elicit CagA-dependent epithelial destruction. Thus, the macaque-isolated H. pylori showed low virulence due to attenuated CagA activity through multiple substitutions in the sequences involved in binding with SHP2 and PAR1b.

Summary: In the case of organ transplantation accompanied by vascular anastomosis, major histocompatibility complex mismatched vascular endothelial cells become a target for graft rejection. Production of a rejection-free, transplantable organ, therefore, requires simultaneous generation of vascular endothelial cells within the organ. To generate pluripotent stem cell (PSC)-derived vascular endothelial cells, we performed blastocyst complementation with a vascular endothelial growth factor receptor-2 homozygous mutant blastocyst. This mutation is embryonic lethal at embryonic (E) day 8.5–9.5 due to an early defect in endothelial and hematopoietic cells. The Flk-1 homozygous knockout chimeric mice survived to adulthood for over 1 year without any abnormality, and all vascular endothelial cells and hematopoietic cells were derived from the injected PSCs. This approach could be used in conjunction with other gene knockouts which induce organ deficiency to produce a rejection-free, transplantable organ in which all the organ's cells and vasculature are PSC derived. : In this article, Yamaguchi and colleagues generated vascular endothelial cells and hematopoietic cells from pluripotent stem cells in vivo by blastocyst complementation technique. This approach could be used to produce a rejection-free, transplantable organ in which all the organ's cells and vasculature are pluripotent stem cell derived. Keywords: tissue regeneration, vascular endothelial cells, hematopoietic cells, blastocyst complementation, pluripotent stem cells

A nanocrystal formulation of lutein (NC/LT) was prepared by particle size reduction approach with a bottom-up system to improve nutraceutical properties of LT, and physicochemical and pharmacokinetic characterizations of NC/LT were undertaken. In NC/LT, LT nanoparticles with a mean diameter of 230 nm existed in a crystalline state, and, in comparison with crystalline LT, NC/LT exhibited a marked improvement in dissolution/dispersion behavior of LT in water. Orally-administered NC/LT exhibited enhanced oral absorption of LT in male Sprague-Dawley rats, as evidenced by relative bioavailability of 15.2 to crystalline LT. After oral administration of LT samples (100 mg-LT/kg, 7 days) to male Sprague-Dawley rats, the ocular accumulation of LT in NC/LT was 3.4-fold higher than that in crystalline LT. Upon these findings, particle size reduction of LT would be efficacious to improve its dissolution and pharmacokinetic characteristics, and NC/LT may be an attractive dosage form to enhance the nutraceutical properties of LT.

The aim of present study was to enhance the nutraceutical properties of ginger extract (GE) by employing supersaturable self-emulsifying drug delivery systems (S-SEDDS). SEDDS of GE (SEDDS/GE), consisting of medium-chain triglyceride, lysolecithin and glycerin, was produced. To prepare S-SEDDS of GE (S-SEDDS/GE), hydroxypropyl methylcellulose was added to the SEDDS/GE as a precipitation inhibitor. Physicochemical, pharmacokinetic, and hepatoprotective properties of GE formulations were characterized. Both formulations improved the dissolution behavior of GE due to the formation of fine micelles with a median diameter of ca. 110 nm. After oral administration of GE samples in rats, the relative bioavailabilities of 6-gingerol and 8-gingerol in the S-SEDDS/GE-treated group were ca. 3-fold higher than those of GE-treated group, respectively. Repeated oral administration of GE/S-SEDDS (100 mg-GE/kg) provided a hepatoprotective effect in a rat model of carbon tetrachloride-induced hepatotoxicity. From these observations, the S-SEDDS approach might be efficacious for enhancing the nutraceutical properties of GE.

Purpose: While ultrasmall superparamagnetic iron oxide (USPIO) is useful for identifying atherosclerotic lesions as an MRI contrast medium, there are limitations in its power to quantitatively evaluate and resolve USPIO in atherosclerotic lesions of the heart. Computed tomography (CT) has a higher resolution than MRI, and Dual Energy CT is capable of visualizing iron atoms, the main component of USPIO. More recently, a new USPIO capable of achieving longer retention times in blood circulation compared to the previous USPIO has been developed. The objective of this study was to investigate the feasibility of visualizing and quantifying the new USPIO by dual energy CT. Materials and Methods: USPIO with iron concentrations adjusted in 5 steps from 2.5 to 50 mg/mL was visualized by dual energy CT to measure the contrast on virtual monochromatic imaging (40 and 70 keV). In parallel experiments, iodine contrast medium was diluted to the same concentrations and visualized by dual energy CT to measure the contrast at 70 keV. The linearity of the contrast against the iron and iodine concentrations was measured for the quantitative evaluation. Further, a vascular phantom simulating clinical cases (divided into 4 layers: meat alone, meat + USPIO, vascular lumen, and with or without calcification) was prepared. The iron density image was overlaid on the image at 70 keV to evaluate the visualization of the USPIO medium. Results: In the imaging of the medium with an iron concentration of 25 mg/mL, the CT numberat 70 keV was 117.0 HU, or about 17% of that of iodine (664.4 HU). The CT number rose to 319.9 HU at 40 keV, or to about 48% of that of iodine. The linearity of the contrast against the iron concentration in USPIO was R2 = 0.9996, indicating a strong correlation. In the simulated vascular phantom, the iron concentration significantly increased in the region containing USPIO, and the quantity could be visualized by overlaying the iron density image displayed with a color scale on the 70-keV image. Conclusion: Our results suggested that macrophages could be both quantified and visualized by USPIO on dual energy CT. Keywords: ultrasmall superparamagnetic iron oxide, plaque imaging, dual energy CT, macrophage

The present study aimed to develop an amorphous solid dispersion of nobiletin (ASD/NOB) using hydroxypropyl cellulose-SSL (HPC-SSL) to improve the pharmacokinetic properties and hypouricemic effect of NOB. ASD/NOB was prepared by the freeze-drying method (ASD/NOB). ASD/NOB was characterized with a focus on crystallinity, dissolution, pharmacokinetic behavior, and hypouricemic action in a rat model of hyperuricemia. ASD/NOB showed significant improvement in dissolution behavior, as evidenced by a 4.4-fold higher dissolved NOB concentration than crystalline NOB at 2 h in distilled water. After the oral administration of ASD/NOB (50 mg NOB/kg) in rats, higher systemic exposure to NOB was observed with an 18-fold enhancement in oral bioavailability, and the Tmax value of orally administered ASD/NOB was 60% shorter than that of orally administered crystalline NOB. In a rat model of hyperuricemia, orally dosed ASD/NOB showed an improved hypouricemic effect by a 16% reduction in the plasma uric acid level compared with orally administered crystalline NOB. Based on these findings, ASD/NOB may be an efficacious dosage option to improve the nutraceutical potential of NOB for the treatment of hyperuricemia.

The present study aimed to enhance the nutraceutical properties of sesamin (SES) by solid dispersion (SD) approach using α-glycosylated stevia (Stevia-G) as a carrier compound with solubilizing effect. The SD formulations were characterized in terms of physicochemical and pharmacokinetic properties. The skin protective effect was evaluated in a rat model of drug-induced photodermatosis. The SES-loaded SD with Stevia-G (SES/Stevia-G-SD) prepared with spray-drying were uniform and spherical with a diameter of about 3 μm, and SES within the formulation was found to be an amorphous state, possibly leading to improved dissolution behavior of SES. SES/Stevia-G-SD provided 190-fold increase in the dissolution amount of SES compared with crystalline SES. The oral bioavailability of SES/Stevia-G-SD (20 mg-SES/kg) was 30-fold higher than that of crystalline SES. Orally-dosed SES/Stevia-G-SD (20 mg-SES/kg) protected rat skins from drug-induced photodermatosis. Based on these findings, the SES/Stevia-G-SD approach might be efficacious to enhance the nutraceutical potential of SES.

Background: Pilsicainide, a pure Na+ channel blocker, is a popular antiarrhythmic drug for the management of atrial tachyarrhythmias (AT), in Japan. However, serious drug-induced proarrhythmias (DIPs) may unexpectedly occur. We assessed the clinical background of AT patients presenting with DIPs caused by pilsicainide. Methods: This study retrospectively enrolled 874 consecutive patients (543 men, 63.6±15.3 years old, and 57.9±16.5 kg of body weight), who were orally administered pilsicainide for AT management. We evaluated the relationship between DIPs and serum pilsicainide concentration, renal dysfunction (estimate glomerular filtration rate, eGFR), and electrocardiogram (ECG) parameters. Results: Among the patients, 154 (17.6%) had renal dysfunction (eGFR

Intra-embryo genome editing by CRISPR/Cas9 has enabled rapid generation of gene knockout animals. However, large fragment knock-in directly into embryos’ genome is still difficult, especially without microinjection of donor DNA. Viral vectors are good transporters of knock-in donor DNA for cell lines, but seemed unsuitable for pre-implantation embryos with zona pellucida, glycoprotein membrane surrounding early embryos. We found adeno-associated virus (AAV) can infect zygotes of various mammals through intact zona pellucida. AAV-mediated donor DNA delivery following Cas9 ribonucleoprotein electroporation enables large fragment knock-in without micromanipulation.

The present study aimed to develop inhalable poly (lactic-co-glycolic acid) (PLGA)-based microparticles of salmon calcitonin (sCT) for sustained pharmacological action by the fine droplet drying (FDD) process, a novel powderization technique employing printing technologies. PLGA was selected as a biodegradable carrier polymer for sustained-release particles of sCT (sCT/SR), and physicochemical characterizations of sCT/SR were conducted. To estimate the in vivo efficacy of the sCT/SR respirable powder (sCT/SR-RP), plasma calcium levels were measured after intratracheal administration in rats. The particle size of sCT/SR was 3.6 µm, and the SPAN factor, one of the parameters to present the uniformity of particle size distribution, was calculated to be 0.65. In the evaluation of the conformational structure of sCT, no significant changes were observed in sCT/SR even after the FDD process. The drug release from sCT/SR showed a biphasic pattern with an initial burst and slow diffusion in simulated lung fluid. sCT/SR-RP showed fine inhalation performance, as evidenced by a fine particle fraction value of 28% in the cascade impactor analysis. After the insufflation of sCT samples (40 µg-sCT/kg) in rats, sCT/SR-RP could enhance and prolong the hypocalcemic action of sCT possibly due to the sustained release and pulmonary absorption of sCT. From these observations, the strategic application of the FDD process could be efficacious to provide PLGA-based inhalable formulations of sCT, as well as other therapeutic peptides, to enhance their biopharmaceutical potentials.

Summary: Epiblast stem cells (EpiSCs) in mice and rats are primed pluripotent stem cells (PSCs). They barely contribute to chimeric embryos when injected into blastocysts. Reprogramming of EpiSCs to embryonic stem cell (ESC)-like cells (rESCs) may occur in response to LIF-STAT3 signaling; however, low reprogramming efficiency hampers potential use of rESCs in generating chimeras. Here, we describe dramatic improvement of conversion efficiency from primed to naive-like PSCs through upregulation of E-cadherin in the presence of the cytokine LIF. Analysis revealed that blocking nuclear localization of β-CATENIN with small-molecule inhibitors significantly enhances reprogramming efficiency of mouse EpiSCs. Although activation of Wnt/β-catenin signals has been thought desirable for maintenance of naive PSCs, this study provides the evidence that inhibition of nuclear translocation of β-CATENIN enhances conversion of mouse EpiSCs to naive-like PSCs (rESCs). This affords better understanding of gene regulatory circuits underlying pluripotency and reprogramming of PSCs. : In this article, Nakauchi and colleagues show that dramatic improvement of conversion efficiency from primed to naive-like mouse pluripotent stem cells through blocking nuclear localization of β-CATENIN in the presence of the cytokine LIF. This affords better understanding of gene regulatory circuits underlying pluripotency and reprogramming of primed pluripotent stem cells.

Background: Class IC antiarrhythmic drugs, used to prevent paroxysmal atrial tachyarrhythmias (ATs), are well known to effectively unmask the electrocardiogram (ECG) pattern of Brugada syndrome. We used these drugs to investigate the prevalence and prognosis of patients with lone and paroxysmal ATs and Brugada-type ECGs. Methods and results: We enrolled 702 consecutive patients who were given class IC antiarrhythmic drugs (pilsicainide or propafenone) to inhibit paroxysmal ATs. We analyzed 12-lead ECGs before and after drug administration using a computerized ECG system. A Brugada-type ECG was defined as a coved pattern of ST segment elevation (>0.2 mV) in leads V1−V2. No patients had risk-stratifying factors of the syndrome. Forty-four patients (6.3%) had significant ST-segment elevation in leads V1−V2 after drug administration. Among these patients, a coved ECG pattern was observed in 10 (1.4%). The use of pilsicainide (n=9) was significantly (P=0.02) greater than that of propafenone (n=1). During the follow-up period of 85±19 months, none of the 10 patients had ventricular tachyarrhythmias. Conclusions: Manifestation of Brugada-type ECGs by class IC antiarrhythmic drugs is neither rare nor serious in patients with lone and paroxysmal ATs but without risk-stratifying factors for the syndrome.

The purpose of this study is to examine a normal conduction pathway of the left atrium. During pulmonary vein isolation using a three-dimensional mapping system, we observed the characteristics of the conduction pattern. Subjects consisted of 15 patients with paroxysmal atrial fibrillation (mean age, 62.8 ± 8.4 years). Left atrial activation conduction begins in the interatrial septum, and moves toward the peak of the ridge between the left atrial appendage and left pulmonary vein. In coronary sinus or appendage pacing, conduction was in the opposite direction at the same location. The mean conduction velocity in this area was 2.03 ± 0.43 m/s. Features of decrement conduction were not present. Anatomical position of the appendage differed greatly between patients. A detailed observation using the inner-cavity view of a three-dimensional mapping system also showed that the peak of the ridge reached the center of the roof.

Fair comparison of reprogramming efficiencies and in vitro differentiation capabilities among induced pluripotent stem cell (iPSC) lines has been hampered by the cellular and genetic heterogeneity of de novo infected somatic cells. In order to address this problem, we constructed a single cassette all-in-one inducible lentiviral vector (Ai-LV) for the expression of three reprogramming factors (Oct3/4, Klf4 and Sox2). To obtain multiple types of somatic cells having the same genetic background, we generated reprogrammable chimeric mice using iPSCs derived from Ai-LV infected somatic cells. Then, hepatic cells, hematopoietic cells and fibroblasts were isolated at different developmental stages from the chimeric mice, and reprogrammed again to generate 2nd iPSCs. The results revealed that somatic cells, especially fetal hepatoblasts were reprogrammed 1200 times more efficiently than adult hepatocytes with maximum reprogramming efficiency reaching 12.5%. However, we found that forced expression of c-Myc compensated for the reduced reprogramming efficiency in aged somatic cells without affecting cell proliferation. All these findings suggest that the Ai-LV system enables us to generate a panel of iPSC clones derived from various tissues with the same genetic background, and thus provides an invaluable tool for iPSC research.

BackgroundRecent progress in rat pluripotent stem cell technology has been remarkable. Particularly salient is the demonstration that embryonic stem cells (ESCs) in the rat (rESCs) can contribute to germline transmission, permitting generation of gene-modified rats as is now done using mouse ESCs (mESCs) or mouse induced pluripotent stem cells (iPSCs; miPSCs). However, determinations of whether rat iPSCs (riPSCs) can contribute to germ cells are not published. Here we report the germline competency of riPSCs.Methodology/principal findingsWe generated riPSCs by transducing three mouse reprogramming factors (Oct3/4, Klf4, and Sox2) into rat somatic cells, followed by culture in the presence of exogenous rat leukemia inhibitory factor (rLIF) and small molecules that specifically inhibit GSK3, MEK, and FGF receptor tyrosine kinases. We found that, like rESCs, our riPSCs can contribute to germline transmission. Furthermore we found, by immunostaining of testis from mouse-rat interspecific chimeras with antibody against mouse vasa homolog, that riPSCs can contribute to embryonic development with chimera formation in mice (rat-mouse interspecific chimeras) and to interspecific germlines.Conclusions/significanceOur data clearly demonstrate that using only three reprogramming factors (Oct3/4, Klf4, and Sox2) rat somatic cells can be reprogrammed into a ground state. Our generated riPSCs exhibited germline transmission in either rat-rat intraspecific or mouse-rat interspecific chimeras.

Patients with permanent hypoparathyroidism require lifelong replacement therapy to avoid life-threatening complications, The benefits of conventional treatment are lim- ited, however. Transplanting a functional parathyroid gland (PTG) would yield better results. Parathyroid gland cells generated from pluripotent stem cells in vitro to date cannot mimic the physiological responses to extracellular calcium that are essential for calcium homeostasis. We thus hypothesized that blastocyst complementation (BC) could be a better strategy for generating functional PTG cells and compensating loss of parathyroid function. We here describe generation of fully functional PTGs from mouse embryonic stem cells (mESCs) with single-step BC. Using CRISPR-Cas9 knockout of Glial cells missing2 (Gcm2), we efficiently produced aparathyroid embryos for BC. In these embryos, mESCs differentiated into endocrinologically mature PTGs that rescued Gcm2−/− mice from neonatal death. The mESC-derived PTGs responded to extracellular calcium, restoring calcium homeostasis on transplantation into mice surgically ren- dered hypoparathyroid. We also successfully generated functional interspecies PTGs in Gcm2−/− rat neonates, an accomplishment with potential for future human PTG therapy using xenogeneic animal BC. Our results demonstrate that BC can produce functional endocrine organs and constitute a concept in treatment of hypoparathyroidism. [ABSTRACT FROM AUTHOR]