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The quality and size of liver grafts are critical factors that influence living‐donor liver transplantation (LDLT) function and safety. However, the biomarkers used for predicting graft quality are lacking. In this study, we sought to identify unique graft quality markers, aside from donor age, by using the livers of non‐human primates. Hepatic gene microarray expression data from young and elderly cynomolgus macaques revealed a total of 271 genes with significantly increased expression in the elderly. These candidate genes were then narrowed down to six through bioinformatics analyses. The expression patterns of these candidate genes in human donor liver tissues were subsequently examined. Importantly, we found that grafts exhibiting up‐regulated expression of these six candidate genes were associated with an increased incidence of liver graft failure. Multivariable analysis further revealed that up‐regulated expression of LRRN2 (encoding leucine‐rich repeat protein, neuronal 2) in donor liver tissue served as an independent risk factor for graft failure (odds ratio 4.50, confidence interval 2.08–9.72). Stratification based on graft expression of LRRN2 and donor age was also significantly associated with 6‐month graft survival rates. Conclusion: Up‐regulated LRRN2 expression of liver graft is significantly correlated with graft failure in LDLT. In addition, combination of graft LRRN2 expression and donor age may represent a promising marker for predicting LDLT graft quality. [ABSTRACT FROM AUTHOR]

Muscle satellite cells (SCs) are stem cells that reside in skeletal muscles and contribute to regeneration upon muscle injury. SCs arise from skeletal muscle progenitors expressing transcription factors Pax3 and/or Pax7 during embryogenesis in mice. However, it is unclear whether these fetal progenitors possess regenerative ability when transplanted in adult muscle. Here we address this question by investigating whether fetal skeletal muscle progenitors (FMPs) isolated from Pax3GFP/+ embryos have the capacity to regenerate muscle after engraftment into Dystrophin-deficient mice, a model of Duchenne muscular dystrophy. The capacity of FMPs to engraft and enter the myogenic program in regenerating muscle was compared with that of SCs derived from adult Pax3GFP/+ mice. Transplanted FMPs contributed to the reconstitution of damaged myofibers in Dystrophin-deficient mice. However, despite FMPs and SCs having similar myogenic ability in culture, the regenerative ability of FMPs was less than that of SCs in vivo. FMPs that had activated MyoD engrafted more efficiently to regenerate myofibers than MyoD-negative FMPs. Transcriptome and surface marker analyses of these cells suggest the importance of myogenic priming for the efficient myogenic engraftment. Our findings suggest the regenerative capability of FMPs in the context of muscle repair and cell therapy for degenerative muscle disease. [ABSTRACT FROM AUTHOR]

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70–90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs. [ABSTRACT FROM AUTHOR]

Background: For regenerative therapy using induced pluripotent stem cell (iPSC) technology, cell type of origin to be reprogrammed should be chosen based on accessibility and reprogramming efficiency. Some studies report that iPSCs exhibited a preference for differentiation into their original cell lineages, while others did not. Therefore, the type of cell which is most appropriate as a source for iPSCs needs to be clarified. Methodology/Principal Findings: Genetically matched human iPSCs from different origins were generated using bone marrow stromal cells (BMSCs) and dermal fibroblasts (DFs) of the same donor, and global gene expression profile, DNA methylation status, and differentiation properties into the chondrogenic and osteogenic lineage of each clone were analyzed. Although genome-wide profiling of DNA methylation suggested tissue memory in iPSCs, genes expressed differentially in BMSCs and DFs were equally silenced in our bona fide iPSCs. After cell-autonomous and induced differentiation, each iPSC clone exhibited various differentiation properties, which did not correlate with cell-of-origin. Conclusions/Significance: The reprogramming process may remove the difference between DFs and BMSCs at least for chondrogenic and osteogenic differentiation. Qualified and genetically matched human iPSC clone sets established in this study are valuable resources for further basic study of clonal differences. [ABSTRACT FROM AUTHOR]

Mammalian preimplantation development involves several crucial events, such as compaction and blastocyst formation, but little is known about essential genes that regulate this developmental process. Here, we have focused on MAP kinase signaling pathways as potential regulatory pathways for the process. Our results show that inhibition of the JNK pathway or of the p38 MAP kinase pathway, but not of the ERK pathway, results in inhibition of cavity formation, and that JNK and p38 are active during mouse preimplantation development. Our subsequent microarray analyses show that, of about 39,000 transcripts analyzed, the number of those genes whose expression level is sensitive to the inhibition of the JNK or the p38 pathway, but insensitive to the inhibition of the ERK pathway, is only 156. Moreover, of the 156 genes, expression of 10 genes (two genes upregulated and eight genes downregulated) is sensitive to either inhibition of the JNK or p38 pathways. These 10 genes include several genes known for their function in axis and pattern formation. Downregulation of some of the 10 genes simultaneously using siRNA leads to abnormality in cavity formation. Thus, this study has successfully narrowed down candidate genes of interest, detailed analysis of which will probably lead to elucidation of the molecular mechanism of preimplantation development. [ABSTRACT FROM AUTHOR]

Lipid metabolism is one of energy metabolic pathways that produce adenosine triphosphate (ATP). In this pathway, lysosomal acid lipase (LAL) encoded by Lipase A (LIPA), plays an important role in catalyzing lipids to fatty acids (FAs), which drive oxidative phosphorylation (OXPHOS) and generate ATP. Previously, we found that a LIPA single nucleotide polymorphism rs143793106, which decreases the LAL activity, suppressed the cytodifferentiation of human periodontal ligament (HPDL) cells. However, the mechanisms underlying that suppression are still not fully clarified. Thus, we aimed to investigate the mechanisms regulating the cytodifferentiation of HPDL cells by LAL in terms of energy metabolism. We performed the osteogenic induction of HPDL cells with or without Lalistat-2, a LAL inhibitor. To visualize lipid droplet (LD) utilization, we performed confocal microscopy on HPDL cells. We also performed real-time PCR to analyze the gene expression of calcification-related and metabolism-related genes. Furthermore, we measured the ATP production rate from two major energy production pathways, OXPHOS and glycolysis, and OXPHOS-related parameters of HPDL cells during their cytodifferentiation. We found that LDs were utilized during the cytodifferentiation of HPDL cells. Alkaline phosphatase (ALPL), collagen type 1 alpha 1 chain (COL1A1), ATP synthase F1 subunit alpha (ATP5F1A), and carnitine palmitoyltransferase 1A (CPT1A) mRNA expressions were upregulated, whereas lactate dehydrogenase A (LDHA) mRNA expression was downregulated. Additionally, total ATP production rate was significantly increased. In contrast, in the presence of Lalistat-2, LD utilization was inhibited and ALPL , COL1A1 , and ATP5F1A mRNA expression was downregulated. Additionally, ATP production rate and spare respiratory capacity of the OXPHOS pathway were decreased in HPDL cells during their cytodifferentiation. Collectively, the defect of LAL in HPDL cells decreased LD utilization and OXPHOS capacity, resulting in reduced energy to sustain the adequate ATP production required for the cytodifferentiation of HPDL cells. Thus, LAL is important for periodontal tissue homeostasis as a regulator of bioenergetic process of HPDL cells. [Display omitted] • LAL inhibition by Lalistat-2 suppressed the cytodifferentiation of HPDL cells. • LAL was involved in energy metabolism during HPDL cytodifferentiation. • LAL inhibition reduced spare respiratory capacity during HPDL cytodifferentiation. [ABSTRACT FROM AUTHOR]

The Hedgehog pathway is activated in various types of malignancies. We previously reported that inhibition of SMO or GLI prevents osteosarcoma growth in vitro and in vivo. Recently, it has been reported that arsenic trioxide (ATO) inhibits cancer growth by blocking GLI transcription. In this study, we analyzed the function of ATO in the pathogenesis of osteosarcoma. Real-time PCR showed that ATO decreased the expression of Hedgehog target genes, including PTCH1, GLI1, and GLI2, in human osteosarcoma cell lines. WST-1 assay and colony formation assay revealed that ATO prevented osteosarcoma growth. These findings show that ATO prevents GLI transcription and osteosarcoma growth in vitro. Flow cytometric analysis showed that ATO promoted apoptotic cell death. Comet assay showed that ATO treatment increased accumulation of DNA damage. Western blot analysis showed that ATO treatment increased the expression of γH2AX, cleaved PARP, and cleaved caspase-3. In addition, ATO treatment decreased the expression of Bcl-2 and Bcl-xL. These findings suggest that ATO treatment promoted apoptotic cell death caused by accumulation of DNA damage. In contrast, Sonic Hedgehog treatment decreased the expression of γH2AX induced by cisplatin treatment. ATO re-induced the accumulation of DNA damage attenuated by Sonic Hedgehog treatment. These findings suggest that ATO inhibits the activation of Hedgehog signaling and promotes apoptotic cell death in osteosarcoma cells by accumulation of DNA damage. Finally, examination of mouse xenograft models showed that ATO administration prevented the growth of osteosarcoma in nude mice. Because ATO is an FDA-approved drug for treatment of leukemia, our findings suggest that ATO is a new therapeutic option for treatment of patients with osteosarcoma. [ABSTRACT FROM AUTHOR]

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70–90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs. [ABSTRACT FROM AUTHOR]

Abstract: Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) regulates intestinal immunological homeostasis. However, precise expression patterns of CEACAM1 isoforms remain poorly understood in the intestinal epithelia. Focusing on the small intestinal epithelium of BALB/c mice, we identified three novel splice variants encoding CEACAM1a-2, -2C1, and -4C1 by RT-PCR. CEACAM1a-2, -2C1, and -4C1 demonstrated secretory properties by transfection experiments in vitro. Among them, CEACAM1a-4C1 was the major secreted isoform in vivo due to the soluble/secreted CEACAM1a with a frameshift sequence in the C-terminus, specific for CEACAM1a-2C1 and -4C1. CEACAM1a-4C1 was capable of binding murine hepatitis virus (MHV) and was detected at approximately 120kDa in the small intestinal secretions. Neutralizing effects of the soluble CEACAM1a on MHV infectivity in vitro were demonstrated by using recombinant CEACAM1a-4C1. Our data suggest an intrinsic mechanism operated by free CEACAM1 for surveillance of pathogens and maintenance of homeostasis in the intestine. [Copyright &y& Elsevier]

Measles virus (MV) infection in children harboring human immunodeficiency virus type 1 (HIV-1) is often fatal, even in the presence of neutralizing antibodies; however, the underlying mechanisms are unclear. Therefore, the aim of the present study was to examine the interaction between HIV-1 and wild-type MV (MVwt) or an MV vaccine strain (MVvac) during dual infection. The results showed that the frequencies of MVwt- and MVvac-infected CD4+ T cells within the resting peripheral blood mononuclear cells (PBMCs) were increased 3- to 4-fold after HIV-1 infection, and this was associated with a marked upregulation of signaling lymphocytic activation molecule (SLAM) expression on CD4+ T cells but not on CD8+ T cells. SLAM upregulation was induced by infection with a replication-competent HIV-1 isolate comprising both the X4 and R5 types and to a lesser extent by a pseudotyped HIV-1 infection. Notably, SLAM upregulation was observed in HIV-infected as well as -uninfected CD4+ T cells and was abrogated by the removal of HLA-DR+ cells from the PBMC culture. Furthermore, SLAM upregulation did not occur in uninfected PBMCs cultured together with HIV-infected PBMCs in compartments separated by a permeable membrane, indicating that no soluble factors were involved. Rather, CD4+ T cell activation mediated through direct contact with dendritic cells via leukocyte function-associated molecule 1 (LFA-1)/intercellular adhesion molecule 1 (ICAM-1) and LFA-3/CD2 was critical. Thus, HIV-1 infection induces a high level of SLAM expression on CD4+ T cells, which may enhance their susceptibility to MV and exacerbate measles in coinfected individuals. [ABSTRACT FROM AUTHOR]