Home blood pressure-lowering effect of a non-steroidal mineralocorticoid receptor blocker, esaxerenone, versus trichlormethiazide for uncontrolled hypertension: the EXCITE-HT randomized controlled study.

The EXCITE-HT study aimed to evaluate the efficacy and safety of esaxerenone versus thiazide diuretics (trichlormethiazide) as second-line treatment for Japanese patients with uncontrolled essential hypertension. This was a 12-week, multicenter, randomized, open-label, parallel-group study. The non-inferiority of esaxerenone to trichlormethiazide was confirmed if the upper limit of the two-sided 95% confidence interval (CI) for the difference in systolic blood pressure (SBP)/diastolic blood pressure (DBP) change between groups was below 3.9/2.1 mmHg. A total of 295 and 290 patients were included in the esaxerenone and trichlormethiazide groups, respectively. The non-inferiority of esaxerenone to trichlormethiazide was demonstrated: least squares mean change differences in morning home SBP/DBP at end of treatment (EOT) were -2.2 (95% CI, -3.6, -0.8) mmHg for SBP/-0.6 (-1.4, 0.2) mmHg for DBP. Morning home, bedtime home, and office BP significantly decreased (all p < 0.001) from baseline to EOT in both groups. The urinary albumin-to-creatinine ratio and N-terminal pro-brain natriuretic peptide level decreased from baseline to Week 12 in both groups, with no notable intergroup difference. Serum potassium elevations occurred more frequently with esaxerenone, while serum potassium reductions occurred more with trichlormethiazide. Uric acid elevations were observed in both groups, but more frequently with trichlormethiazide than esaxerenone. No cases of gout occurred in this study. Reductions in estimated glomerular filtration rate were similarly observed in both groups. EXCITE-HT is the first randomized controlled study to demonstrate evidence that esaxerenone is non-inferior to trichlormethiazide as second-line treatment for Japanese patients with uncontrolled essential hypertension, with no new safety concerns. The EXCITE-HT study demonstrated the non-inferiority of esaxerenone to trichlormethiazide in its morning home blood pressure lowering effect and safety profile in Japanese patients with uncontrolled essential hypertension who were previously treated with an angiotensin II receptor blocker or calcium channel blocker.

Exploratory study on the relationship between urinary sodium/potassium ratio, salt intake, and the antihypertensive effect of esaxerenone: the ENaK Study.

Excessive salt intake is one of the causes of hypertension, and reducing salt intake is important for managing the risk of hypertension and subsequent cardiovascular events. Esaxerenone, a mineralocorticoid receptor blocker, has the potential to exert an antihypertensive effect in hypertensive patients with excessive salt intake, but evidence is still lacking, especially in clinical settings. We aimed to determine if baseline sodium/potassium ratio and baseline estimated 24-h urinary sodium excretion can predict the antihypertensive effect of esaxerenone in patients with essential hypertension inadequately controlled with an angiotensin receptor blocker (ARB) or a calcium channel blocker (CCB). This was an exploratory, open-label, interventional study with a 4-week observation period and a 12-week treatment period. Esaxerenone was orally administered once daily in accordance with the Japanese package insert. In total, 126 patients met the eligibility criteria and were enrolled (ARB subcohort, 67; CCB subcohort, 59); all were included in the full analysis set (FAS) and safety analysis. In the FAS, morning home systolic blood pressure (SBP)/diastolic blood pressure (DBP) significantly decreased from baseline to end of treatment (primary efficacy endpoint) (-11.9 ± 10.9/ - 6.4 ± 6.8 mmHg, both p < 0.001); a similar trend was observed in both subcohorts. Significant reductions were also shown in bedtime home and office SBP/DBP (all p < 0.001). Each BP change was consistent regardless of the urinary sodium/potassium ratio or estimated 24-h urinary sodium excretion at baseline. The urinary albumin-creatinine ratio (UACR) and N-terminal pro-brain natriuretic peptide (NT-proBNP) significantly decreased from baseline to Week 12 in the total population and both subcohorts. No new safety concerns were raised. Esaxerenone significantly decreased morning home, bedtime home, and office BP; UACR; and NT-proBNP in this patient population, regardless of concomitant ARB or CCB use. The antihypertensive effect of esaxerenone was independent of the urinary sodium/potassium ratio and estimated 24-h urinary sodium excretion at baseline.

Intranasal Sendai virus-based SARS-CoV-2 vaccine using a mouse model.

The coronavirus disease 2019 (COVID-19) epidemic remains worldwide. The usefulness of the intranasal vaccine and boost immunization against severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) has recently received much attention. We developed an intranasal SARS-CoV-2 vaccine by loading the receptor binding domain of the S protein (S-RBD) of SARS-CoV-2 as an antigen into an F-deficient Sendai virus vector. After the S-RBD-Fd antigen with trimer formation ability was intranasally administered to mice, S-RBD-specific IgM, IgG, IgA, and neutralizing antibody titers were increased in serum or bronchoalveolar lavage fluid for 12 weeks. Furthermore, in mice that received a booster dose at week 8, a marked increase in neutralizing antibodies in the serum and bronchoalveolar lavage fluid was observed at the final evaluation at week 12, which neutralized the pseudotyped lentivirus expressing the SARS-CoV-2 spike protein, indicating the usefulness of the Sendai virus-based SARS-CoV-2 intranasal vaccine.

Combination treatment with bevacizumab plus erlotinib for meningeal carcinomatosis of afatinib-resistant EGFR mutated lung cancer without T790M mutation: a case report.

Meningeal carcinomatosis in lung cancer is known to have a very poor prognosis. Here we report a case in which bevacizumab plus erlotinib (BE) was effective against meningeal carcinomatosis from afatinib-resistant EGFR mutation-positive lung cancer. A 61-year-old man started afatinib, a 2nd generation molecular targeting drug, as first-line treatment for lung adenocarcinoma cT1bN0M1a stage IVA harboring EGFR exon19 deletion mutation. This treatment shrank the tumor and allowed sustained control of tumor growth. After 19 months from the start of treatment, head MRI revealed brain metastasis in the cerebellum and meningeal carcinomatosis with loss of appetite and slurred speech, in response to which whole-brain irradiation was performed. Head MRI 1 month after whole-brain irradiation showed no change in the disseminated lesions of the cerebellum. In Japan, osimertinib treatment after failure of EGFR-TKI treatments requires the T790M mutation in the tumor, blood or body fluid, so BE treatment was started as second-line treatment. Brain MRI showed improvement in cerebellar disseminated lesions 1 month after the start of BE treatment. BE treatment controlled intrapulmonary metastases, pleural disseminated lesions and meningeal carcinomatosis for 6 months. BE treatment as second-line treatment should be considered as an option for meningeal carcinomatosis of EGFR tyrosine kinase inhibitor (TKI) -resistant EGFR mutated lung cancer.

Visualization of sequential conversion of human intermediately reprogrammed stem cells into iPS cells.

Analysis of gene expression in single cells is required to understand somatic cell reprogramming into human induced pluripotent stem cells (iPSCs). To facilitate this, we established intermediately reprogrammed stem cells (iRSCs), pre-iPSC lines. The iRSC-iPSC conversion system enables the reproducible monitoring of reprogramming events and the analysis of progressive gene expression profiles using single-cell microarray analysis and genome editing. Here, single-cell microarray analysis showed the stage-specific sequential gene activation during the conversion of iRSCs into iPSCs, using OCT4, TDGF1 and E-CADHERIN as marker genes. Out of 75 OCT4-related genes, which were significantly up-regulated after the activation of OCT4, and entry into the mesenchymal-to-epithelial transition (MET), LIN28 (LIN28A) and FOXO1 were selected for applying to gene expression visualization. Multicolored visualization was achieved by the genome editing of LIN28 or FOXO1 with mCherry into OCT4-GFP iRSCs. Fluorescent analysis of gene activity in individual cells showed that OCT4 was dispensable for maintenance, but required for activation, of the LIN28 and FOXO1 expression in reprogramming.

Closed-channel culture system for efficient and reproducible differentiation of human pluripotent stem cells into islet cells.

Human pluripotent stem cells (hPSCs) are thought to be a promising cell-source solution for regenerative medicine due to their indefinite proliferative potential and ability to differentiate to functional somatic cells. However, issues remain with regard to achieving reproducible differentiation of cells with the required functionality for realizing human transplantation therapies and with regard to reducing the potential for bacterial or fungal contamination. To meet these needs, we have developed a closed-channel culture device and corresponding control system. Uniformly-sized spheroidal hPSCs aggregates were formed inside wells within a closed-channel and maintained continuously throughout the culture process. Functional islet-like endocrine cell aggregates were reproducibly induced following a 30-day differentiation protocol. Our system shows an easily scalable, novel method for inducing PSC differentiation with both purity and functionality.

OCT4 activity during conversion of human intermediately reprogrammed stem cells to iPSCs through mesenchymal-epithelial transition.

To facilitate understanding the mechanisms of somatic reprogramming to human induced pluripotent stem cells (iPSCs), we have established intermediately reprogrammed stem cells (iRSCs), human mesenchymal cells that express exogenous Oct4, Sox2, Klf4 and c-Myc (OSKM) and endogenous SOX2 and NANOG. iRSCs can be stably maintained at low density. At high density, however, they are induced to enter mesenchymal-epithelial transition (MET), resulting in reprogramming to an iPSC state. Morphological changes through MET correlate with silencing of exogenous OSKM, and upregulation of endogenous OCT4. A CRISPR/Cas9-mediated GFP knock-in visualized the temporal regulation of endogenous OCT4 in cells converting from iRSC to iPSC state. OCT4 activation coincident with silencing of OSKM occurred prior to entering MET. Notably, OCT4 instability was frequently observed in cells of developing post-MET colonies until a late stage (>200 cells), demonstrating that OCT4-activated post-MET cells switched from asymmetric to symmetric cell division in late stage reprogramming.

Single-cell cloning and expansion of human induced pluripotent stem cells by a microfluidic culture device.

The microenvironment of cells, which includes basement proteins, shear stress, and extracellular stimuli, should be taken into consideration when examining physiological cell behavior. Although microfluidic devices allow cellular responses to be analyzed with ease at the single-cell level, few have been designed to recover cells. We herein demonstrated that a newly developed microfluidic device helped to improve culture conditions and establish a clonality-validated human pluripotent stem cell line after tracing its growth at the single-cell level. The device will be a helpful tool for capturing various cell types in the human body that have not yet been established in vitro.

Nanog co-regulated by Nodal/Smad2 and Oct4 is required for pluripotency in developing mouse epiblast.

Nanog, a core pluripotency factor, is required for stabilizing pluripotency of inner cell mass (ICM) and embryonic stem cells (ESCs), and survival of primordial germ cells in mice. Here, we have addressed function and regulation of Nanog in epiblasts of postimplantation mouse embryos by conditional knockdown (KD), chromatin immunoprecipitation (ChIP) using in vivo epiblasts, and protein interaction with the Nanog promoter in vitro. Differentiation of Nanog-KD epiblasts demonstrated requirement for Nanog in stabilization of pluripotency. Nanog expression in epiblast is directly regulated by Nodal/Smad2 pathway in a visceral endoderm-dependent manner. Notably, Nanog promoters switch from Oct4/Esrrb in ICM/ESCs to Oct4/Smad2 in epiblasts. Smad2 directly associates with Oct4 to form Nanog promoting protein complex. Collectively, these data demonstrate that Nanog plays a key role in stabilizing Epiblast pluripotency mediated by Nodal/Smad2 signaling, which is involved in Nanog promoter switching in early developing embryos.

Self-renewal and pluripotency acquired through somatic reprogramming to human cancer stem cells.

Human induced pluripotent stem cells (iPSCs) are reprogrammed by transient expression of transcription factors in somatic cells. Approximately 1% of somatic cells can be reprogrammed into iPSCs, while the remaining somatic cells are differentially reprogrammed. Here, we established induced pluripotent cancer stem-like cells (iCSCs) as self-renewing pluripotent cell clones. Stable iCSC lines were established from unstable induced epithelial stem cell (iESC) lines through re-plating followed by embryoid body formation and serial transplantation. iCSCs shared the expression of pluripotent marker genes with iPSCs, except for REX1 and LIN28, while exhibited the expression of somatic marker genes EMP1 and PPARγ. iESCs and iCSCs could generate teratomas with high efficiency by implantation into immunodeficient mice. The second iCSCs isolated from dissociated cells of teratoma from the first iCSCs were stably maintained, showing a gene expression profile similar to the first iCSCs. In the first and second iCSCs, transgene-derived Oct4, Sox2, Klf4, and c-Myc were expressed. Comparative global gene expression analyses demonstrated that the first iCSCs were similar to iESCs, and clearly different from human iPSCs and somatic cells. In iCSCs, gene expression kinetics of the core pluripotency factor and the Myc-related factor were pluripotent type, whereas the polycomb complex factor was somatic type. These findings indicate that pluripotent tumorigenicity can be conferred on somatic cells through up-regulation of the core pluripotency and Myc-related factors, prior to establishment of the iPSC molecular network by full reprogramming through down-regulation of the polycomb complex factor.

Cure of ADPKD by selection for spontaneous genetic repair events in Pkd1-mutated iPS cells.

Induced pluripotent stem cells (iPSCs) generated by epigenetic reprogramming of personal somatic cells have limited therapeutic capacity for patients suffering from genetic disorders. Here we demonstrate restoration of a genomic mutation heterozygous for Pkd1 (polycystic kidney disease 1) deletion (Pkd1(+/-) to Pkd1(+/R+)) by spontaneous mitotic recombination. Notably, recombination between homologous chromosomes occurred at a frequency of 1~2 per 10,000 iPSCs. Southern blot hybridization and genomic PCR analyses demonstrated that the genotype of the mutation-restored iPSCs was indistinguishable from that of the wild-type cells. Importantly, the frequency of cyst generation in kidneys of adult chimeric mice containing Pkd1(+/R+) iPSCs was significantly lower than that of adult chimeric mice with parental Pkd1(+/-) iPSCs, and indistinguishable from that of wild-type mice. This repair step could be directly incorporated into iPSC development programmes prior to cell transplantation, offering an invaluable step forward for patients carrying a wide range of genetic disorders.

Human and mouse induced pluripotent stem cells are differentially reprogrammed in response to kinase inhibitors.

Conventional human induced pluripotent stem cells (hiPSCs), reprogrammed from somatic cells by induced expression of Oct4, Sox2, Klf4, and c-Myc, are phenotypically different from mouse embryonic stem cells (ESCs). In mice, culture in N2B27 serum-free 2i media (mitogen-activated protein kinase/extracellular signal-regulated kinase and glycogen synthase kinase 3 inhibitors; PD0325901 and CHIR99021) plus leukemia inhibitory factor (LIF) (2i+LIF medium) enriches for germline competent ESCs. Here, we demonstrate that flat-shaped hiPSC colonies can be reprogrammed into bowl-shaped multi-potent stem cells (2i-hiPSCs) by using 2i+LIF medium. Mechanical dissociation of 2i-hiPSC colonies enables stable maintenance for >20 passages. Importantly, gene expression profiling demonstrated that 2i-hiPSCs more closely resemble primitive neural stem cells (PNSCs). Notably, this 2i-induced phenotype was generated from conventional hiPSCs, but not human ESCs (hESCs), thus correlating with the observation of neuroectodermal SOX1-positive colonies in conventional hiPSCs, but not hESCs in 2i+LIF medium. Thus, 2i-hiPSCs, which are nonteratoma forming PNSCs, may represent a safe source of cells for neural research and regenerative medicine.

Sox2 expression effects on direct reprogramming efficiency as determined by alternative somatic cell fate.

Induced pluripotent stem cells (iPSCs) are generated by directly reprogramming somatic cells by forcing them to express the exogenous transcription factors, Oct4, Sox2, Klf4 and c-Myc (OSKM). These cells could potentially be used in clinical applications and basic research. Here, we explored the molecular role of Sox2 by generating iPSCs that expressed Sox2 at various levels. Low Sox2 (LS) expression increased the efficiency of generating partially reprogrammed iPSCs in combination with OKM. Notably, we detected a significant increase in the number of fully reprogrammed iPSCs with three factors of OK and LS. LS expression was linked with the reduced expression of ectoderm and mesoderm marker genes. This indicates that cell differentiation into the ectoderm and mesoderm lineages was impeded during reprogramming. The quality of the iPSCs that was generated by using OK and LS was comparable to that of iPSCs that were produced via conventional OSK as seen by pluripotent marker gene expression and chimera formation. We conclude that Sox2 plays a crucial role in a dose-dependent manner in direct reprogramming of somatic cells to iPSCs.

Efficient reprogramming of human and mouse primary extra-embryonic cells to pluripotent stem cells.

Practical clinical applications for current induced pluripotent stem cell (iPSC) technologies are hindered by very low generation efficiencies. Here, we demonstrate that newborn human (h) and mouse (m) extra-embryonic amnion (AM) and yolk-sac (YS) cells, in which endogenous KLF4/Klf4, c-MYC/c-Myc and RONIN/Ronin are expressed, can be reprogrammed to hiPSCs and miPSCs with efficiencies for AM cells of 0.02% and 0.1%, respectively. Both hiPSC and miPSCs are indistinguishable from embryonic stem cells in colony morphology, expression of pluripotency markers, global gene expression profile, DNA methylation status of OCT4 and NANOG, teratoma formation and, in the case of miPSCs, generation of germline transmissible chimeric mice. As copious amounts of human AM cells can be collected without invasion, and stored long term by conventional means without requirement for in vitro culture, they represent an ideal source for cell banking and subsequent 'on demand' generation of hiPSCs for personal regenerative and pharmaceutical applications.