Human umbilical cord-derived cells can often serve as feeder cells to maintain primate embryonic stem cells in a state capable of producing hematopoietic cells

Clinical application of human embryonic stem (ES) cells will require the establishment of methods for their culture, either in the presence or absence of human-derived feeder cells. We have tested the ability of non-immortalized cultured cells derived from human umbilical cord (HUC cells) to support ES cell culture. A primate ES cell line that had been established and maintained with mouse embryonic fibroblasts was cultured on HUC cells for >3 months (HUC-maintained ES cells). These cells retained their expression of alkaline phosphatase, SSEA-4, Oct-3/4, and to a lesser extent Nanog, but did not express Rex-1. Nevertheless, HUC-maintained ES cells could produce ectoderm-, mesoderm- and endoderm-derived cells in teratomata that they formed in immunodeficient mice. We show that HUC-maintained ES cells could give rise to hematopoietic cells, although this ability of HUC cells varied among HUC cell populations derived from different neonates. HUC cells are promising as human material with which to maintain ES cells in a state that retains their ability to produce mature cells, including hematopoietic cells.     

YaeJ is a novel ribosome-associated protein in Escherichia coli that can hydrolyze peptidyl-tRNA on stalled ribosomes

In bacteria, ribosomes often become stalled and are released by a trans-translation process mediated by transfer-messenger RNA (tmRNA). In the absence of tmRNA, however, there is evidence that stalled ribosomes are released from non-stop mRNAs. Here, we show a novel ribosome rescue system mediated by a small basic protein, YaeJ, from Escherichia coli, which is similar in sequence and structure to the catalytic domain 3 of polypeptide chain release factor (RF). In vitro translation experiments using the E. coli-based reconstituted cell-free protein synthesis system revealed that YaeJ can hydrolyze peptidyl–tRNA on ribosomes stalled by both non-stop mRNAs and mRNAs containing rare codon clusters that extend downstream from the P-site and prevent Ala-tmRNA•SmpB from entering the empty A-site. In addition, YaeJ had no effect on translation of a normal mRNA with a stop codon. These results suggested a novel tmRNA-independent rescue system for stalled ribosomes in E. coli. YaeJ was almost exclusively found in the 70S ribosome and polysome fractions after sucrose density gradient sedimentation, but was virtually undetectable in soluble fractions. The C-terminal basic residue-rich extension was also found to be required for ribosome binding. These findings suggest that YaeJ functions as a ribosome-attached rescue device for stalled ribosomes.     

Development of a robust method for establishing B cell lines using Epstein-Barr Virus

B lymphoblastoid cell lines (B-LCLs) are generally established from B lymphocytes by infection with Epstein-Barr virus (EBV). As their genomic structure is stable in culture, B-LCLs are a valuable resource for many types of analysis. The efficiency of establishing B-LCLs from freshly obtained blood samples from healthy individuals is almost 100?%; however, for blood samples stored inappropriately after collection or held in long-term storage as peripheral blood mononuclear cells (PBMCs) in liquid nitrogen, the efficiency of B-LCL establishment can be considerably lower. To date, we have established more than 550 B-LCLs from 685 PBMC samples that have been stored in liquid nitrogen for over 20?yr. The PBMCs were prepared from blood samples donated by individuals belonging to native minority ethnic groups in outlying regions of South America and elsewhere. The establishment of B-LCLs from this material is difficult, and failure results in the waste of valuable and rare samples. We sought to improve our success rate for establishing B-LCLs from these difficult and irreplaceable samples by a detailed examination of each step of the process. The analysis showed that two parameters were particularly critical to the success rate: the density of the PBMCs plated after EBV infection and the EBV titer. These observations shed light on cases where establishment of B-LCLs was hard due to the small number of PBMCs or damage to the cells.     

IL-6-induced Bcl6 variant 2 supports IL-6-dependent myeloma cell proliferation and survival through STAT3

IL-6 is a growth and survival factor for myeloma cells, although the mechanism by which it induces myeloma cell proliferation through gene expression is largely unknown. Microarray analysis showed that some B-cell lymphoma-associated oncogenes such as Bcl6, which is absent in normal plasma cells, were upregulated by IL-6 in IL-6-dependent myeloma cell lines. We found that Bcl6 variant 2 was upregulated by STAT3. ChIP assay and EMSA showed that STAT3 bound to the upstream region of variant 2 DNA. Expression of p53, a direct target gene of Bcl6, was downregulated in the IL-6-stimulated cells, and this process was impaired by an HDAC inhibitor. Bcl6 was knocked down by introducing small hairpin RNA, resulting in decreased proliferation and increased sensitivity to a DNA damaging agent. Thus, STAT3-inducible Bcl6 variant 2 appears to generate an important IL-6 signal that supports proliferation and survival of IL-6-dependent myeloma cells.     

Is parainfluenza virus a threatening virus for human cancer cell lines?

Immortalized cell lines, such as human cancer cell lines, are an indispensable experimental resource for many types of biological and medical research. However, unless the cell line has been authenticated prior to use, interpretation of experimental results may be problematic. The potential problems this may cause are illustrated by studies in which authentication of cell lines has not been carried out. For example, immortalized cell lines may unknowingly be infected with viruses that alter their characteristics. In fact, parainfluenza virus type 5 (PIV5) poses a threat to the use of immortalized cell lines in biological and medical research; PIV5 infection significantly alters cellular physiology associated with the response to interferon. If PIV5 infection is widespread in immortalized cell lines, then a very large number of published studies might have to be re-evaluated. Fortunately, analyses of a large number of immortalized cell lines indicate that PIV5 infection is not widespread.     

Establishment of mouse embryonic stem cell-derived erythroid progenitor cell lines able to produce functional red blood cells

Background

The supply of transfusable red blood cells (RBCs) is not sufficient in many countries. If erythroid cell lines able to produce transfusable RBCs in vitro were established, they would be valuable resources. However, such cell lines have not been established. To evaluate the feasibility of establishing useful erythroid cell lines, we attempted to establish such cell lines from mouse embryonic stem (ES) cells.

Methodology/Principal Findings

We developed a robust method to obtain differentiated cell lines following the induction of hematopoietic differentiation of mouse ES cells and established five independent hematopoietic cell lines using the method. Three of these lines exhibited characteristics of erythroid cells. Although their precise characteristics varied, each of these lines could differentiate in vitro into more mature erythroid cells, including enucleated RBCs. Following transplantation of these erythroid cells into mice suffering from acute anemia, the cells proliferated transiently, subsequently differentiated into functional RBCs, and significantly ameliorated the acute anemia. In addition, we did not observe formation of any tumors following transplantation of these cells.

Conclusion/Significance

To the best of our knowledge, this is the first report to show the feasibility of establishing erythroid cell lines able to produce mature RBCs. Considering the number of human ES cell lines that have been established so far, the intensive testing of a number of these lines for erythroid potential may allow the establishment of human erythroid cell lines similar to the mouse erythroid cell lines described here. In addition, our results strongly suggest the possibility of establishing useful cell lines committed to specific lineages other than hematopoietic progenitors from human ES cells.     

Validation of multiple single nucleotide variation calls by additional exome analysis with a semiconductor sequencer to supplement data of whole-genome sequencing of a human population

Background

Validation of single nucleotide variations in whole-genome sequencing is critical for studying disease-related variations in large populations. A combination of different types of next-generation sequencers for analyzing individual genomes may be an efficient means of validating multiple single nucleotide variations calls simultaneously.

Results

Here, we analyzed 12 independent Japanese genomes using two next-generation sequencing platforms: the Illumina HiSeq 2500 platform for whole-genome sequencing (average depth 32.4×), and the Ion Proton semiconductor sequencer for whole exome sequencing (average depth 109×). Single nucleotide polymorphism (SNP) calls based on the Illumina Human Omni 2.5-8 SNP chip data were used as the reference. We compared the variant calls for the 12 samples, and found that the concordance between the two next-generation sequencing platforms varied between 83% and 97%.

Conclusions

Our results show the versatility and usefulness of the combination of exome sequencing with whole-genome sequencing in studies of human population genetics and demonstrate that combining data from multiple sequencing platforms is an efficient approach to validate and supplement SNP calls.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-673) contains supplementary material, which is available to authorized users.     

Lipocalin 2-mediated growth suppression is evident in human erythroid and monocyte/macrophage lineage cells

Lipocalin 2 (LCN2), a secreted protein of the lipocalin family, induces apoptosis in some types of cells and inhibits bacterial growth by sequestration of the iron-laden bacterial siderophore. We have recently reported that LCN2 inhibits the production of red blood cells in the mouse. Here we analyzed the role of LCN2 in human hematopoiesis. Expression of LCN2 was observed not only in mature cells such as those of the granulocyte/macrophage and erythroid lineages but also in hematopoietic stem/progenitor cells. We also examined expression of two candidate receptors for LCN2, brain type organic cation transporter (BOCT) and megalin, in various cell types. BOCT showed relatively high levels of expression in erythroid and hematopoietic stem/progenitor cells but lower levels in granulocyte/macrophage and T lymphoid cells. Megalin was expressed at high levels in T lymphoid and erythroid cells but at lower levels in granulocyte/macrophage lineage cells. LCN2 suppressed the growth of erythroid and monocyte/macrophage lineages in vitro, but did not have this effect on cells of other lineages. In addition, immature hematopoietic stem/progenitor cells were not sensitive to LCN2. These results demonstrate a lineage-specific role for LCN2 in human hematopoiesis that is reminiscent of its effects upon mouse hematopoiesis and strongly suggest an important in vivo function of LCN2 in the regulation of human hematopoiesis.     

Ras-mediated signaling pathway regulates the expression of a low-molecular-weight heat-shock protein in fission yeast.

In fission yeast, Schizosaccharomyces pombe, deficiency of ras1 gene causes an abnormal cell shape and abolishes mating ability. However, target genes of this signaling pathway are largely unknown because of the lack of an appropriate analysis system. To overcome this problem, we have started a novel project to categorize entire genes based on their expression levels under different growth conditions. Using this strategy, we screened genes whose expression levels were affected in the presence or absence of the ras1 gene product. For this purpose, we utilized high-density arrays of clones covering the entire genome of the fission yeast, and probed with labelled cDNA derived from various strains and growth conditions. Here, we demonstrate the detection of a low-molecular-weight heat-shock protein gene, hsp16, whose expression is very likely to be regulated by a ras-mediated signaling pathway, but not by the heat-shock response.     

Highly specific marker genes for detecting minimal gastric cancer cells in cytology negative peritoneal washings

Peritoneal wash cytology plays a pivotal role in the decision for gastric cancer treatment because advanced gastric cancer often turns out incurable with peritoneal metastasis. Molecular detection of minimal cancer cells from peritoneal washings may overcome the sensitivity boundary of conventional cytology and contribute to the prediction of the disease outcome. To select marker candidates out of ten thousands of genes, we performed microarray analyses in 12 gastric cell lines and 8 peritoneal washings of early stage cases. With 40 candidates selected by the above expression profiling, RT-PCR in 16 representative peritoneal wash samples was performed to identify genes specific to cytology positive samples. The finally selected five genes, CK20, FABP1, MUC2, TFF1, and TFF2, were then evaluated for their utility as a marker for minimal residual disease in 99 peritoneal wash samples. Nested RT-PCR using the five genes showed positive results highly specific to incurable cases (91-100%). With a high specificity, the combination of these five genes succeeded in identifying 6 out of 20 (30%) additional patients with all types of early recurrence that could not be predicted by the conventional method. The six newly identified recurrences included four non-peritoneal ones, showing that RT-PCR using the five genes without a real-time quantitative PCR technique contributes to the detection of minimal residual disease.