Azacitidine is a potential therapeutic drug for pyridoxine-refractory female X-linked sideroblastic anemia.

X-linked sideroblastic anemia (XLSA) is associated with mutations in the erythroid-specific δ-aminolevulinic acid synthase (ALAS2) gene. Treatment of XLSA is mainly supportive, except in patients who are pyridoxine responsive. Female XLSA often represents a late onset of severe anemia, mostly related to the acquired skewing of X chromosome inactivation. In this study, we successfully generated active wild-type and mutant ALAS2-induced pluripotent stem cell (iPSC) lines from the peripheral blood cells of an affected mother and 2 daughters in a family with pyridoxine-resistant XLSA related to a heterozygous ALAS2 missense mutation (R227C). The erythroid differentiation potential was severely impaired in active mutant iPSC lines compared with that in active wild-type iPSC lines. Most of the active mutant iPSC-derived erythroblasts revealed an immature morphological phenotype, and some showed dysplasia and perinuclear iron deposits. In addition, globin and HO-1 expression and heme biosynthesis in active mutant erythroblasts were severely impaired compared with that in active wild-type erythroblasts. Furthermore, genes associated with erythroblast maturation and karyopyknosis showed significantly reduced expression in active mutant erythroblasts, recapitulating the maturation defects. Notably, the erythroid differentiation ability and hemoglobin expression of active mutant iPSC-derived hematopoietic progenitor cells (HPCs) were improved by the administration of δ-aminolevulinic acid, verifying the suitability of the cells for drug testing. Administration of a DNA demethylating agent, azacitidine, reactivated the silent, wild-type ALAS2 allele in active mutant HPCs and ameliorated the erythroid differentiation defects, suggesting that azacitidine is a potential novel therapeutic drug for female XLSA. Our patient-specific iPSC platform provides novel biological and therapeutic insights for XLSA.

Expression dynamics of HAND1/2 in in vitro human cardiomyocyte differentiation.

Hand1 and Hand2 are transcriptional factors, and knockout mice of these genes show left and right ventricular hypoplasia, respectively. However, their function and expression in human cardiogenesis are not well studied. To delineate their expressions and assess their functions in human cardiomyocytes (CMs) in vitro, we established two triple-reporter human induced pluripotent stem cell lines that express HAND1mCherry, HAND2EGFP and either MYH6-driven iRFP670 or tagBFP constitutively and investigated their expression dynamics during cardiac differentiation. On day 5 of the differentiation, HAND1 expression marked cardiac progenitor cells. We profiled the CM subpopulations on day 20 with RNA sequencing and found that mCherry+ CMs showed higher proliferative ability than mCherry- CMs and identified a gene network of LEF1, HAND1, and HAND2 to regulate proliferation in CMs. Finally, we identified CD105 as a surface marker of highly proliferative CMs.

Erratum to "Optical Recording of Action Potentials in Human Induced Pluripotent Stem Cell-Derived Cardiac Single Cells and Monolayers Generated from Long QT Syndrome Type 1 Patients".

[This corrects the article DOI: 10.1155/2019/7532657.].

Optical Recording of Action Potentials in Human Induced Pluripotent Stem Cell-Derived Cardiac Single Cells and Monolayers Generated from Long QT Syndrome Type 1 Patients.

Induced pluripotent stem cells (iPSCs) from type 1 long QT (LQT1) patients can differentiate into cardiomyocytes (CMs) including ventricular cells to recapitulate the disease phenotype. Although optical recordings using membrane potential dyes to monitor action potentials (APs) were reported, no study has investigated the disease phenotypes of cardiac channelopathy in association with the cardiac subtype at the single-cell level. We induced iPSC-CMs from three control and three LQT1 patients. Single-cell analysis using a fast-responding dye confirmed that ventricular cells were the dominant subtype (control-iPSC-CMs: 98%, 88%, 91%; LQT1-iPSC-CMs: 95%, 79%, 92%). In addition, LQT1-iPSC-ventricular cells displayed an increased frequency of early afterdepolarizations (pvalue = 0.031). Cardiomyocyte monolayers constituted mostly of ventricular cells derived from LQT1-iPSCs showed prolonged AP duration (APD) (pvalue = 0.000096). High-throughput assays using cardiomyocyte monolayers in 96-well plates demonstrated that IKr inhibitors prolonged APDs in both control- and LQT1-iPSC-CM monolayers. We confirmed that the optical recordings of APs in single cells and monolayers derived from control- and LQT1-iPSC-CMs can be used to assess arrhythmogenicity, supporting the feasibility of membrane potential dye-based high-throughput screening to study ventricular arrhythmias caused by genetic channelopathy or cardiotoxic drugs.

Monitoring and visualizing microRNA dynamics during live cell differentiation using microRNA-responsive non-viral reporter vectors.

MicroRNA (miRNA) activity differs with cell type, suggesting it can be used as a cell marker. In this study, we developed novel miRNA-responsive non-viral reporter vectors to continuously monitor and visualize miRNA dynamics during differentiation and to efficiently purify target living cells. Each vector codes miRNA-responsive and reference reporter genes in a single mRNA. These two genes are independent modules but transcribed by a single promoter, which enables us to distinguish miRNA-mediated post-transcriptional repression from transcriptional repression. We generated stable, miRNA-responsive vector-containing human induced pluripotent stem cells (hiPSCs) using the piggyBac transposon or episomal vectors. We could continuously monitor the differentiation status of living hiPSCs by detecting the activity of hiPSC-specific miRNA (miR-302a*). In addition, we could selectively sort hiPSC-derived cardiomyocytes using cardiomyocyte-specific miRNA (miR-208a or miR-1)-reporter vectors. Our miRNA reporter system provides a simple way to quantitatively and continuously monitor and visualize changes in the cellular state and should facilitate a broad range of studies that depend on cellular changes including drug discovery and cell-fate conversion.

Epigenetic Variation between Human Induced Pluripotent Stem Cell Lines Is an Indicator of Differentiation Capacity.

Variation in the differentiation capacity of induced pluripotent stem cells (iPSCs) to specific lineages is a significant concern for their use in clinical applications and disease modeling. To identify factors that affect differentiation capacity, we performed integration analyses between hematopoietic differentiation performance and molecular signatures such as gene expression, DNA methylation, and chromatin status, using 35 human iPSC lines and four ESC lines. Our analyses revealed that hematopoietic commitment of PSCs to hematopoietic precursors correlates with IGF2 expression level, which in turn depends on signaling-dependent chromatin accessibility at mesendodermal genes. Maturation capacity for conversion of PSC-derived hematopoietic precursors to mature blood associates with the amount and pattern of DNA methylation acquired during reprogramming. Our study therefore provides insight into the molecular features that determine the differential capacities seen among human iPSC lines and, through the predictive potential of this information, highlights a way to select optimal iPSCs for clinical applications.

Identification of low-abundance proteins in serum via the isolation of HSP72 complexes.

Heat shock protein 72 (HSP72) is an intracellular molecular chaperone that is overexpressed in tumor cells, and has also been detected in extracellular regions such as the blood. HSP72 forms complexes with peptides and proteins that are released from tumors. Accordingly, certain HSP72-binding proteins/peptides present in the blood of cancer patients may be derived from tumor cells. In this study, to effectively identify low-abundance proteins/peptides in the blood as tumor markers, we established a method for isolating HSP72-binding proteins/peptides from serum. Nine HSP72-specific monoclonal antibodies were conjugated to N-hydroxysulfosuccinimide-activated Sepharose beads (NHq) and used to isolate HSP72 complexes from serum samples. Precipitated proteins were then identified by LC-MS/MS analysis. Notably, this approach enabled the isolation of low-abundance proteins from serum without albumin removal. Moreover, by subjecting the serum samples of ten patients with multiple myeloma (MM) to NHq analysis, we identified 299 proteins present in MM HSP72 complexes, including 65 intracellular proteins. Among the intracellular proteins detected, 21 were present in all serum samples tested, while 11 were detected in both the conditioned media from cultured multiple myeloma cells and serum from MM patients. These results suggest that the NHq method can be applied to discover candidate tumor markers.

Comparative proteomics analysis of gastric cancer stem cells.

Cancer stem cells (CSCs) are responsible for cancer progression, metastasis, and recurrence. To date, the specific markers of CSCs remain undiscovered. The aim of this study was to identify novel biomarkers of gastric CSCs for clinical diagnosis using proteomics technology. CSC-like SP cells, OCUM-12/SP cells, OCUM-2MD3/SP cells, and their parent OCUM-12 cells and OCUM-2MD3 cells were used in this study. Protein lysates from each cell line were analyzed using QSTAR Elite Liquid Chromatography with Tandem Mass Spectrometry, coupled with isobaric tags for relative and absolute quantitation technology. Candidate proteins detected by proteomics technology were validated by immunohistochemical analysis of 300 gastric cancers. Based on the results of LC-MS/MS, eight proteins, including RBBP6, GLG1, VPS13A, DCTPP1, HSPA9, HSPA4, ALDOA, and KRT18, were up-regulated in both OCUM-12/SP cells and OCUM-2MD3/SP cells when compared to their corresponding parent cells. RT-PCR analysis indicated that the expression level of RBBP6, HSPA4, DCTPP1, HSPA9, VPS13A, ALDOA, GLG1, and CK18 was high in OCUM-12/SP and OCUM-2MD3/SP, in compared with the control of parent OCUM-12 and OCUM-2MD3. These proteins were significantly associated with advanced invasion depth, lymph node metastasis, distant metastasis, or advanced clinical stage. RBBP6, DCTPP1, HSPA4, and ALDOA expression in particular were significantly associated with a poor prognosis in the 300 gastric cancer patients. RBBP6 was determined to be an independent prognostic factor. The motility-stimulating ability of OCUM-12/SP cells and OCUM-2MD3/SP cells was inhibited by RBBP6 siRNA. These findings might suggest that the eight proteins, RBBP6, GLG1, VPS13A, DCTPP1, HSPA9, HSPA4, ALDOA, and KRT18, utilizing comparative proteomics analysis, were perceived to be potential CSC markers of gastric cancer. Of the eight candidate proteins, RBBP6 was suggested to be a promising prognostic biomarker and a therapeutic target for gastric cancer.

Establishment of neutralizing rat monoclonal antibodies for fibroblast growth factor-2.

Fibroblast growth factor-2 (FGF-2) plays a critical role in endothelial survival, proliferation, and angiogenesis and is localized on the cell membrane by binding to heparan sulfate proteoglycans. Here we established a neutralizing monoclonal antibody, 1B9B9, against FGF-2 using the rat medial iliac lymph node method. 1B9B9 blocked the binding of FGF-2 to its receptor, inhibiting FGF-2-induced proliferation and corresponding downstream signaling in endothelial cells. Treatment of human umbilical vein endothelial cells with 1B9B9 reduced the basal phosphorylation levels of Akt and MAPK. Furthermore, continued treatment with 1B9B9 induced cell death by apoptosis. Compared with FGF-2 knockdown, 1B9B9 significantly reduced cell survival. In addition, the combination of FGF-2 siRNA and 1B9B9 showed a synergistic effect. The data indicate that 1B9B9 established by the rat iliac lymph node method is a fully compatible neutralizing antibody.

Hsc70 contributes to cancer cell survival by preventing Rab1A degradation under stress conditions.

Heat shock cognate protein 70 (Hsc70) acts as a molecular chaperone for the maintenance of intracellular proteins, which allows cancer cells to survive under proteotoxic stress. We attempted to use Hsc70 to identify key molecules in cancer cell survival. Here, we performed mass-spectrometry-based proteomics analysis utilizing affinity purification with anti-Hsc70 antibodies; as a result, 83 differentially expressed proteins were identified under stress conditions. This result implies that there was a change in the proteins with which Hsc70 interacted in response to stress. Among the proteins identified under both serum-depleted and 5-fluorouracil-treated conditions, Rab1A was identified as an essential molecule for cancer cell survival. Hsc70 interacted with Rab1A in a chaperone-dependent manner. In addition, Hsc70 knockdown decreased the level of Rab1A and increased the level of its ubiquitination under stress conditions, suggesting that Hsc70 prevented the degradation of Rab1A denatured by stress exposure. We also found that Rab1A knockdown induced cell death by inhibition of autophagosome formation. Rab1A may therefore contribute to overcoming proteotoxic insults, which allows cancer cells to survive under stress conditions. Analysis of Hsc70 interactors provided insight into changes of intracellular status. We expect further study of the Hsc70 interactome to provide a more comprehensive understanding of cancer cell physiology.

Peace, a MYB-like transcription factor, regulates petal pigmentation in flowering peach 'Genpei' bearing variegated and fully pigmented flowers.

Flowering peach Prunus persica cv. Genpei bears pink and variegated flowers on a single tree. The structural genes involved in anthocyanin biosynthesis were expressed strongly in pink petals but only very weakly or not at all in variegated petals. A cDNA clone encoding a MYB-like gene, isolated from pink petals was strongly expressed only in pink petals. Introduction of this gene, via biolistics gave magenta spots in the white areas of variegated petals, therefore this gene was named as Peace (peach anthocyanin colour enhancement). Differences in Peace expression determine the pattern of flower colouration in flowering peach. The R2R3 DNA-binding domain of Peace is similar to those of other plant MYBs regulating anthocyanin biosynthesis. Key amino acids for tertiary structure and the motif for interaction with bHLH proteins were conserved in Peace. Phylogenetic analysis indicates that Peace is closely related to AtMYB123 (TT2), which regulates proanthocyanidin biosynthesis in Arabidopsis, and to anthocyanin regulators in monocots rather than to regulators in dicots. This is the first report that a TT2-like R2R3 MYB has been shown to regulate anthocyanin biosynthesis.

Establishment of a 5-fluorouracil-resistant triple-negative breast cancer cell line.

Triple-negative breast cancers (TNBCs) are defined as tumors that lack expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. Clinically, TNBC patients are treated with cytotoxic drugs including 5-fluorouracil (5-FU). However, TNBCs develop resistance to such drugs after a series of treatments. To elucidate the mechanisms of drug resistance, establishment of drug-resistant cancer cell lines should be one of the most useful model systems. However, 5-FU-resistant TNBC cell lines have not been previously reported. In this study, we established a 5-FU-resistant cell line, MDA-MB-231/5-FU, from the human TNBC cell line MDA-MB-231, by repeated exposure to stepwise increases in the concentration of 5-FU. The IC50 value of 5-FU for MDA-MB-231/5-FU was 5.5-fold that for the parental cells. The MDA-MB-231/5-FU cell line acquired resistance to not only 5-FU, but also vinorelbine, paclitaxel and gemcitabine. Additionally, we performed iTRAQ-based quantitative proteomics in MDA-MB-231/5-FU cells and the parental cells in order to characterize MDA-MB-231/5-FU. The proteins upregulated in the newly established cells were mainly classified into the categories of 'DNA recombination', 'cell cycle', 'complex assembly', 'cytoskeleton organization', 'transport' and 'negative regulation of cell death'. These proteins may be related to mechanisms of drug resistance in TNBCs. Our established MDA-MB-231/5-FU cell line should be a useful tool for identifying new mechanisms of drug resistance and new drug targets in TNBCs.