Bone morphogenetic protein signaling inhibitor improves differentiation and function of 3D muscle construct fabricated using C2C12.

Functional tissue-engineered artificial skeletal muscle tissue has great potential for pharmacological and academic applications. This study demonstrates an in vitro tissue engineering system to construct functional artificial skeletal muscle tissues using self-organization and signal inhibitors. To induce efficient self-organization, we optimized the substrate stiffness and extracellular matrix (ECM) coatings. We modified the tissue morphology to be ring-shaped under optimized self-organization conditions. A bone morphogenetic protein (BMP) inhibitor was added to improve overall myogenic differentiation. This supplementation enhanced the myogenic differentiation ratio and myotube hypertrophy in two-dimensional cell cultures. Finally, we found that myotube hypertrophy was enhanced by a combination of self-organization with ring-shaped tissue and a BMP inhibitor. BMP inhibitor treatment significantly improved myogenic marker expression and contractile force generation in the self-organized tissue. These observations indicated that this procedure may provide a novel and functional artificial skeletal muscle for pharmacological studies.

Nanowarming of vitrified pancreatic islets as a cryopreservation technology for transplantation.

Biobanking of pancreatic islets for transplantation could solve the shortage of donors, and cryopreservation of vitrified islets is a possible approach. However, a technological barrier is rewarming of large volumes both uniformly and rapidly to prevent ice formation due to devitrification. Here, we describe successful recovery of islets from the vitrified state using a volumetric rewarming technology called "nanowarming," which is inductive heating of magnetic nanoparticles under an alternating magnetic field. Convective warming using a 37°C water bath as the gold standard for rewarming of vitrified samples resulted in a decrease in the viability of mouse islets in large volumes (>1 ml) owing to devitrification caused by slow warming. Nanowarming showed uniform and rapid rewarming of vitrified islets in large volumes. The viability of nanowarmed islets was significantly improved and islets transplanted into streptozotocin-induced diabetic mice successfully lowered serum glucose. The results suggest that nanowarming will lead to a breakthrough in biobanking of islets for transplantation.

Effective magnetic hyperthermia induced by mitochondria-targeted nanoparticles modified with triphenylphosphonium-containing phospholipid polymers.

Magnetic hyperthermia (MHT) is a promising cancer treatment because tumor tissue can be specifically damaged by utilizing the heat generated by nano-heaters such as magnetite nanoparticles (MNPs) under an alternating magnetic field. MNPs are taken up by cancer cells, enabling intracellular MHT. Subcellular localization of MNPs can affect the efficiency of intracellular MHT. In this study, we attempted to improve the therapeutic efficacy of MHT by using mitochondria-targeting MNPs. Mitochondria-targeting MNPs were prepared by the modification of carboxyl phospholipid polymers containing triphenylphosphonium (TPP) moieties that accumulate in mitochondria. The mitochondrial localization of polymer-modified MNPs was supported by transmission electron microscopy observations of murine colon cancer CT26 cells treated with polymer-modified MNPs. In vitro and in vivo MHT using polymer-modified MNPs revealed that the therapeutic effects were enhanced by introducing TPP. Our results indicate the validity of mitochondria targeting in enhancing the therapeutic outcome of MHT. These findings will pave the way for developing a new strategy for the surface design of MNPs and therapeutic strategies for MHT.

Progress of Endogenous and Exogenous Nanoparticles for Cancer Therapy and Diagnostics.

The focus of this brief review is to describe the application of nanoparticles, including endogenous nanoparticles (e.g., extracellular vesicles, EVs, and virus capsids) and exogenous nanoparticles (e.g., organic and inorganic materials) in cancer therapy and diagnostics. In this review, we mainly focused on EVs, where a recent study demonstrated that EVs secreted from cancer cells are associated with malignant alterations in cancer. EVs are expected to be used for cancer diagnostics by analyzing their informative cargo. Exogenous nanoparticles are also used in cancer diagnostics as imaging probes because they can be easily functionalized. Nanoparticles are promising targets for drug delivery system (DDS) development and have recently been actively studied. In this review, we introduce nanoparticles as a powerful tool in the field of cancer therapy and diagnostics and discuss issues and future prospects.

CALN1 hypomethylation as a biomarker for high-risk bladder cancer.

BACKGROUND: DNA methylation in cancer is considered a diagnostic and predictive biomarker. We investigated the usefulness of the methylation status of CALN1 as a biomarker for bladder cancer using methylation-sensitive restriction enzyme (MSRE)-quantitative polymerase chain reaction (qPCR). METHODS: Eighty-two bladder cancer fresh samples were collected via transurethral resection of bladder tumors. Genomic DNA was extracted from the samples, and MSRE-qPCR was performed to determine the CALN1 methylation percentage. Reverse transcription-qPCR was performed to assess the correlation between CALN1 methylation and mRNA expression. The association between CALN1 methylation percentage and clinicopathological variables of all cases and intravesical recurrence of non-muscle-invasive bladder cancer (non-MIBC) cases were analyzed. RESULTS: Of the 82 patients, nine had MIBC and 71 had non-MIBC who had not undergone total cystectomy. The median CALN1 methylation percentage was 79.5% (interquartile range: 51.1-92.6%). The CALN1 methylation percentage had a negative relationship with CALN1 mRNA expression (Spearman's ρ = - 0.563 and P = 0.012). Hypomethylation of CALN1 was associated with advanced tumor stage (P = 0.0007) and histologically high grade (P = 0.018). Furthermore, multivariate analysis revealed that CALN1 hypomethylation was an independent risk factor for intravesical recurrence in non-MIBC patients (hazard ratio 3.83, 95% confidence interval; 1.14-13.0, P = 0.031). CONCLUSION: Our findings suggest that CALN1 methylation percentage could be a useful molecular biomarker for bladder cancer.

Recent advances in animal cell technologies for industrial and medical applications.

The industrial use of living organisms for bioproduction of valued substances has been accomplished mostly using microorganisms. To produce high-value bioproducts such as antibodies that require glycosylation modification for better performance, animal cells have been recently gaining attention in bioengineering because microorganisms are unsuitable for producing such substances. Furthermore, animal cells are now classified as products because a large number of cells are required for use in regenerative medicine. In this article, we review animal cell technologies and the use of animal cells, focusing on useable cell generation and large-scale production of animal cells. We review recent advance in mammalian cell line development because this is the first step in the production of recombinant proteins, and it largely affects the efficacy of the production. We next review genetic engineering technology focusing on CRISPR-Cas system as well as surrounding technologies as these methods have been gaining increasing attention in areas that use animal cells. We further review technologies relating to bioreactors used in the context of animal cells because they are essential for the mass production of target products. We also review tissue engineering technology because tissue engineering is one of the main exits for mass-produced cells; in combination with genetic engineering technology, it can prove to be a promising treatment for patients with genetic diseases after the establishment of induced pluripotent stem cell technology. The technologies highlighted in this review cover brief outline of the recent animal cell technologies related to industrial and medical applications.

Contractile Activity of Myotubes Derived from Human Induced Pluripotent Stem Cells: A Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a genetic disorder that results from deficiency of the dystrophin protein. In recent years, DMD pathological models have been created using induced pluripotent stem (iPS) cells derived from DMD patients. In addition, gene therapy using CRISPR-Cas9 technology to repair the dystrophin gene has been proposed as a new treatment method for DMD. However, it is not known whether the contractile function of myotubes derived from gene-repaired iPS cells can be restored. We therefore investigated the maturation of myotubes in electrical pulse stimulation culture and examined the effect of gene repair by observing the contractile behaviour of myotubes. The contraction activity of myotubes derived from dystrophin-gene repaired iPS cells was improved by electrical pulse stimulation culture. The iPS cell method used in this study for evaluating muscle contractile activity is a useful technique for analysing the mechanism of hereditary muscular disease pathogenesis and for evaluating the efficacy of new drugs and gene therapy.

Microarray profiling of gene expression in C2C12 myotubes trained by electric pulse stimulation.

Electric pulse-stimulated C2C12 myotubes are gaining interest in the field of muscle physiology and biotechnology because electric pulse stimulation (EPS) enhances sarcomere structure development and active tension generation capability. Recently, we found that termination of EPS results in the rapid loss of active tension generation accompanied by disassembly of the sarcomere structure, which may represent an in vitro muscle atrophy model. To elucidate the molecular mechanism underlying this rapid loss of active tension generation and sarcomere structure disassembly after termination of EPS, we performed transcriptomic analysis using microarray. After termination of EPS, 74 genes were upregulated and 120 genes were downregulated after 30 min; however, atrophy-related genes were not found among these genes. To further assess the effect of EPS on gene expression, we re-applied EPS after its termination for 8 h and searched for genes whose expression was reversed. Four genes were upregulated by termination of EPS and downregulated by the re-application of EPS, whereas two genes were downregulated by termination of EPS and upregulated by the re-application of EPS. Although none of these genes were atrophy- or hypertrophy-related, the results presented in this study will contribute to the understanding of gene expression changes that mediate rapid loss of active tension generation and sarcomere structure disassembly following termination of EPS in C2C12 myotubes.

Pressure-induced changes on the morphology and gene expression in mammalian cells.

We evaluated the effect of high hydrostatic pressure on mouse embryonic fibroblasts (MEFs) and mouse embryonic stem (ES) cells. Hydrostatic pressures of 15, 30, 60, and 90 MPa were applied for 10 min, and changes in gene expression were evaluated. Among genes related to mechanical stimuli, death-associated protein 3 was upregulated in MEF subjected to 90 MPa pressure; however, other genes known to be upregulated by mechanical stimuli did not change significantly. Genes related to cell differentiation did not show a large change in expression. On the other hand, genes related to pluripotency, such as Oct4 and Sox2, showed a twofold increase in expression upon application of 60 MPa hydrostatic pressure for 10 min. Although these changes did not persist after overnight culture, cells that were pressurized to 15 MPa showed an increase in pluripotency genes after overnight culture. When mouse ES cells were pressurized, they also showed an increase in the expression of pluripotency genes. These results show that hydrostatic pressure activates pluripotency genes in mammalian cells. This article has an associated First Person interview with the first author of the paper.

Effect of hydrogen peroxide concentration on the maintenance and differentiation of cultured skeletal muscle cells.

We have studied the effects of hydrogen peroxide (H2O2) on the differentiation and maintenance of C2C12 myoblasts. The effects of H2O2 were evaluated by cell viability, total protein concentration, the relative amount of muscle-related proteins, sarcomere structure, and active tension generation. Oxidative stress is one of the major causes of myopathy after exercise and thus establishing the method to evaluate the effects on muscle function is essential. The primary function of striated muscle is to generate force, thus, the measurement of active tension is important in assessing the effect of chemicals on muscle. Among the indices we tested, the sarcomere structure was the most sensitive to the H2O2 exposure while the cell viability was less sensitive. The effects of H2O2 on active tension correlated with a decrease in the amount of muscle proteins. In this study, our results showed that the effect of chemicals on muscle should be measured in multiple ways, including active tension generation, for a better understanding of its physiological impact.

A case of temporary metastatic pulmonary calcification in a patient with hyperparathyroidism on peritoneal dialysis.

INTRODUCTION: Ectopic calcification is associated with secondary hyperparathyroidism (HPT) in patients with end-stage renal failure (ESRD). Metastatic pulmonary calcification (MPC) is another rare type of ectopic calcification, and there are a few reports on MPC in dialysis patients. CASE PRESENTATION: We report the case of a 52-year-old woman admitted with general fatigue and appetite loss, who was on peritoneal dialysis (PD) for 7 years. Although she was initially suspected of having secondary HPT due to ESRD, we finally diagnosed ectopic HPT that was caused by a cystic mass behind her thyroid gland overlapping with secondary HPT. We carefully observed her under conservative therapy because she refused surgery. On admission, she was diagnosed as having MPC because she had ground-glass-like opacification in her lung fields on high-resolution computed tomography scan, which was caused by a parathyroid tumor complicated by secondary HPT associated with ESRD. After she began intravenous injection of etelcalcetide hydrochloride, serum calcium, and intact parathyroid hormone (iPTH), values were adjusted, and the opacification disappeared. CONCLUSION: In a patient on PD, this is the first case of MPC that developed due to acute hypercalcemia, hyperphosphatemia, and dehydration and in which the ectopic pulmonary calcification clearly decreased with optimization of iPTH.

Magnetic heating of nanoparticles as a scalable cryopreservation technology for human induced pluripotent stem cells.

Scale-up of production is needed for industrial applications and clinical translation of human induced pluripotent stem cells (hiPSCs). However, in cryopreservation of hiPSCs, successful rewarming of vitrified cells can only be achieved by convective warming of small volumes (generally 0.2 mL). Here, we present a scalable nano-warming technology for hiPSC cryopreservation employing inductive heating of magnetic nanoparticles under an alternating magnetic field. The conventional method by water bath heating at 37 °C resulted in a decrease of cell viability owing to devitrification caused by slow warming of samples with large volumes (≥ 20 mL). Nano-warming showed uniform and rapid rewarming of vitrified samples and improved viability of hiPSCs in the 20-mL system. In addition to single cells, hiPSC aggregates prepared using a bioreactor-based approach were successfully cryopreserved by the nano-warming technique. These results demonstrate that nano-warming is a promising methodology for cryopreservation in mass production of hiPSCs.

Impact on survival of urgent dialysis initiation in patients with end-stage renal disease: a case-control study.

BACKGROUND: Outcomes of patients with end-stage renal disease at urgent dialysis initiation are varied, but evidence of their long-term prognosis is limited. We aimed to characterize patients undergoing urgent dialysis initiation and analyse its effect on survival outcome. METHODS: We retrospectively identified 208 patients who began haemodialysis from 1 January 2012 to 31 December 2018 at our hospital. In this observational case-control study, the case group comprised patients starting urgent dialysis, and the control group comprised patients starting planned dialysis. We analysed laboratory data, sex, age, smoking history, comorbidities and presence of vascular access and nephrology care that potentially affected the outcome. Data were analysed with Kaplan-Meier curves of early and late period (3 years after dialysis initiation) survival and log-rank tests and with Cox regression analysis. RESULTS: Median age (range) at dialysis initiation was 73 (28-90) years, with 50 (24%) patients in the urgent initiation group. Five (10%) patients in this group had vascular access at dialysis initiation, whereas 21 (42%) had not received adequate pre-dialysis nephrology care. The estimated median overall survival rates of the urgent group and planned initiation group were 42 months and not reached, respectively (P = 0.0011). Multivariable analysis found urgent dialysis initiation to be an independent risk factor for survival (HR 2.36; 95% CI 1.36-4.00; P = 0.02). Survival was not significantly different between the groups for patients who continued chronic dialysis for > 3 years from dialysis initiation (P = 0.1339). CONCLUSION: The prognosis of patients starting dialysis in an urgent condition was poor compared with those who started planned dialysis.

[A Study on the Number of Cycles of First-Line Chemotherapy for Advanced Urothelial Carcinoma].

The first line chemotherapy for advanced urothelial carcinoma is combination chemotherapy based on platinum. The optimal number of cycles for first line chemotherapy has not been defined yet. While cumulative toxicity of cisplatin can be a problem, the approval of pembrolizumab has changed the aspect of secondary treatment. We investigated 39 patients who were diagnosed with advanced urothelial carcinoma and treated with platinum-based chemotherapy between August 2009 and October 2018 in our hospital. We evaluated the correlation between number of cycles of first line chemotherapy and the survival rate of patients with advanced urothelial carcinoma. The primary tumor site was found in the bladder in 22 patients and in the upper urinary tract in 17 patients. Thirty one patients received cisplatin and 8 received carboplatin. Twelve patients received 2 or less cycles, 16 received 3 to 5 cycles and 11 received 6 or more cycles. The median overall survival in those populations was 5 months, 18 months, and 20 months, respectively. Patients who received 2 or less cycles showed significantly lower response rates to chemotherapy and the overall survival worsened. There was no significant difference in overall survival between patients who received 3 to 5 cycles and those who received more than 6 cycles. These results suggested that it may be excessive to continue the first line chemotherapy for more than 6 cycles.

Effect of cell-extracellular matrix interaction on myogenic characteristics and artificial skeletal muscle tissue.

Although various types of artificial skeletal muscle tissue have been reported, the contractile forces generated by tissue-engineered artificial skeletal muscles remain to be improved for biological model and clinical applications. In this study, we investigated the effects of extracellular matrix (ECM) and supplementation of a small molecule, which has been reported to enhance α7ß1 integrin expression (SU9516), on cell migration speed, cell fusion rate, myoblast (mouse C2C12 cells) differentiation and contractile force generation of tissue-engineered artificial skeletal muscles. When cells were cultured on varying ECM coated-surfaces, we observed significant enhancement in the migration speed, while the myotube formation (differentiation ratio) decreased in all except for cells cultured on Matrigel coated-surfaces. In contrast, SU9516 supplementation resulted in an increase in both the myotube width and differentiation ratio. Following combined culture with a Matrigel-coated surface and SU9516 supplementation, myotube width was further increased. Additionally, contractile forces produced by the tissue-engineered artificial skeletal muscles was augmented following combined culture. These findings indicate that regulation of the cell-ECM interaction is a promising approach to improve the function of tissue-engineered artificial skeletal muscles.

Targeted suppression of metastasis regulatory transcription factor SOX2 in various cancer cell lines using a sequence-specific designer pyrrole-imidazole polyamide.

Metastasis, a deadly feature of cancer, compromises the prognosis and accounts for mortality in the majority of cancer patients. SOX2, a well-known pluripotency transcription factor, plays a central role in cell fate determination and has an overlapping role as a regulatory factor in tumorigenesis and metastasis. The demand is increasing for clinically useful strategies for artificial control of SOX2 expression and its complex transcription machinery in cancer cells. N-Methylpyrrole (Py) and N-methylimidazole (Im) polyamides are small programmable designer ligands that can be pre-programmed to selectively recognize DNA sequence and control endogenous gene expression. Herein, we evaluated the anticancer activity of a designer ligand (SOX2i). SOX2i remarkably altered the expression of SOX2 at the mRNA and protein level in human cancer cell lines such as SW620 (colorectal adenocarcinoma), MKN45 (gastric adenocarcinoma), MCF7 (breast carcinoma), U2OS (osteosarcoma) and other cancer cell lines of different origin and type. Genome-wide transcriptome analysis and cell-based assays showed SOX2 to be a downregulated upstream regulator that alters cell proliferation, cell cycle progression, metabolism and apoptotic pathway. Studies in the mouse model confirmed the anti-metastatic property of SOX2i. SOX2i inhibited the expression of genes associated with EMT and stemness. Moreover, Wnt-canonical signaling was found to be downregulated in the SOX2i-treated group. Our proof-of-concept study supports the potential of DNA-based programmable small molecules for controlling the key regulatory factors associated with tumorigenesis and metastasis.

Linking substrate and nucleus via actin cytoskeleton in pluripotency maintenance of mouse embryonic stem cells.

Pluripotency of mouse embryonic stem cells is regulated by transcription factor regulatory networks as well as mechanical stimuli sensed by the cells. It has been unclear how the mechanical strain applied to the plasma membrane is transferred to the nucleus in mouse embryonic stem cells (mESCs). We here investigated the machinery of the mechanotransduction based on the finding that spontaneous differentiation of mESCs was inhibited with the downregulation of ROCK2 in cells attached to soft substrates. On examining the effects of actin bindings to both focal adhesions and cell junctions in cells on soft substrates, co-localization of actin filaments and α-catenin, which links actin to E-cadherin, decreased after differentiation induction. Also, disrupting actin-nucleus mechanical link through dominant negative assay of Nesprins helps to sustain the pluripotency genes; thus, revealing that mechanical strain relayed by actin-Nesprin connection is required for the initiation of the differentiation process.

Inkjet micropatterning through horseradish peroxidase-mediated hydrogelation for controlled cell immobilization and microtissue fabrication.

A simple fabrication method for cell micropatterns on hydrogel substrates was developed using an inkjet printing system that induced hydrogel micropatterns. The hydrogel micropatterns were created from inks resulting in cell-adhesive and non-cell-adhesive printed regions by horseradish peroxidase-catalyzed reaction onto non-cell-adhesive and cell-adhesive hydrogel substrates, respectively, to obtain the cell micropatterns. Cell-adhesive and non-cell-adhesive regions were obtained from gelatin and alginate derivatives, respectively. The cells on the cell-adhesive regions were successfully aligned, resulting in recognizable patterns. Furthermore, the proposed system permits the patterning of multiple cell types by switching the non-cell-adhesive region to the cell-adhesive region in the presence of growing cells. Also, we could fabricate disc- and filament-shaped small tissues by degrading the non-cell-adhesive substrates having dot- and line-shaped cell-adhesive micropatterns using alginate-lyase. These results indicate that our system is useful for fabrication of tailor-made cell patterns and microtissues with the shape defined by the micropattern, and will be conducive to a diverse range of biological applications.

Retroperitoneal lipogranuloma mimicking recurrence of renal cell carcinoma after laparoscopic partial nephrectomy.

Lipogranuloma is a rare inflammatory reactive process often reported to occur in the dermis and subcutis in the cosmetic surgery field.1 It very rarely occurs in the retroperitoneum. We present a case of retroperitoneal lipogranuloma mimicking metastases of renal cell carcinoma (RCC) after laparoscopic partial nephrectomy. A 63-year-old man who underwent laparoscopic left partial nephrectomy for RCC one year earlier had developed a left retroperitoneal tumor during postoperative surveillance. The tumor looked identical to an implant or recurrence of RCC on contrast-enhanced computed tomography (CT) and 18F-fluorodeoxyglucose positron emission tomography/CT. We resected the tumor, and pathology showed a lipogranuloma.

Magnetically triggered transgene expression in mammalian cells by localized cellular heating of magnetic nanoparticles.

To develop a remote control system of transgene expression through localized cellular heating of magnetic nanoparticles, a heat-inducible transgene expression system was introduced into mammalian cells. Cells were labeled with magnetic nanoparticles and exposed to an alternating magnetic field. The magnetically labeled cells expressed the transgene in a monolayer and multilayered cell sheets in which cells were heated around the magnetic nanoparticles without an apparent temperature increase in the culture medium. Magnetic cells were also generated by genetically engineering with a ferritin gene, and transgene expression could be induced by exposure to an alternating magnetic field. This approach may be applicable to the development of novel gene therapies in cell-based medicine.