Liver in a dish

There exists a worldwide shortage of donor livers for transplant. This may not pose a problem in the future, as Takebe et al. have recently grown functional “liver buds” from stem cells in a dish.Since the discovery of human induced pluripotent stem cells (hiPSCs), the promise of generating organs from patients'' iPSCs has received considerable attention as an alternative to donor organ transplantation. Over the past few years, much progress has been made in the differentiation of various somatic cell types from human pluripotent stem cells (hPSCs). However, only a limited number of reports have described the generation of three-dimensional organoids from human stem cells in vitro, including the optic cup¹, the pituitary epithelium², and from adult stem cells — the gut epithelium³. These experimental systems share several common features: 1) they all begin with ES cells or adult stem cells, 2) the cells grow as floating aggregates, and 3) all three organoids (optic cup, pituitary epithelium, and gut crypt) are epithelial structures⁴. In addition, one particularly unexpected finding has emerged from each of these experiments, namely that a high level of self-organization seems to play a substantial role in establishing local tissue architecture and assembly of the resulting organoid.Despite these remarkable examples of organogenesis in vitro, the likelihood of growing a complex vascularized organ in dish, such as liver, has seemed less plausible. Takebe et al.⁵ have made the implausible possible by focusing on the first steps of organogenesis, namely the cellular interactions that occur during liver bud development. The earliest stage of liver organogenesis involves the outgrowth of a group of endodermal and mesenchymal cells from the posterior foregut that soon thereafter become vascularized to form a liver bud. During these morphogenetic changes, a key element to the formation of a liver bud is the orchestration of signals between three types of cells: liver, mesenchymal and endothelial progenitors. Takebe et al. posited that they might be able to recapitulate liver bud formation in vitro by mixing hepatic endoderm cells together with endothelial and mesenchymal cells. To test this idea, they prepared hepatic endoderm cells (hiPSC-HEs) from hiPSCs by directed differentiation, and then co-cultured them with human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (MSCs). Two days later, the cells had self-assembled into a 5-mm-long, three-dimensional tissue that was reminiscent of “liver bud” structures in vivo. To further mature these hiPSC-derived “liver buds” (hiPSC-LBs), they transplanted them into immune-compromised mice where the hiPSC-LBs connected with the host vasculature within 48 h and formed functional vascular networks similar in density and morphology to those of human adult livers. Transplanted hiPSC-LBs started functioning about 10 days later, producing human albumin and metabolizing drugs in a similar fashion to human liver. Perhaps most remarkably, Takebe et al. demonstrated that these hiPSC-LBs could rescue liver function when transplanted to mice with liver failure.The differences between Takebe and his colleagues'' study and other studies designed to reproduce organogenesis in vitro are that they started with several different cell types; the cells were grown initially in a two-dimensional petri dish; and the result was a solid liver organoid that can be vascularized and function after transplantation. For many, the most striking finding is the high level of self-organization in this experimental differentiation system. By analogy, it is equivalent to delivering all of the materials necessary to build a house to a construction site and returning several days later to find a fully assembled home. Clearly the principles of self-organization and self-assembly are playing much more profound roles during differentiation than we previously thought and it is likely what has been reported by Takebe et al. represents only the tip of the iceberg. One takeaway from the way that Takebe and his colleagues'' tackled the problem of in vitro organogenesis may be their focus on the earliest processes in organ development, as it is likely to identify the right combination of cell types for organogenesis to proceed. Nonetheless, this study has raised several new questions. How does self-organization and self-assembly occur in vitro? What is the molecular logic of this process? How can we manipulate a self-organizing system so that we might guide it in the direction we want it to go? And ultimately, could we use a similar strategy to produce other complex solid organs in vitro, e.g., lung, kidney, and pancreas?As summarized by Takebe et al., this study demonstrates a “proof-of-concept” that “organ-bud transplantation provides a promising new approach to study regenerative medicine”. However, a significant amount of work will be required before these findings can be translated into a therapy. First, these little liver buds do not form a complete adult liver. They are missing a number of critical cell types, chief among them biliary epithelial cells and thus bile ducts. How to produce a fully functional liver remains a challenge. Second, in order to translate these findings into human therapies, a key step will be to scale this process so that one can produce a liver bud large enough for transplantation into humans. Of course, there is always the question about safety when it comes to stem cell-based therapies. Undifferentiated stem cells left in transplants tend to form tumors and the use of oncogenes for iPS reprogramming needs to be resolved before iPS cells can be considered for human therapy. Despite the reality that clinical therapies based on this report remain a distant promise, it is inspirational to consider how quickly the field is moving and exciting to speculate about what might come next. If one considers that a drug has been identified to specifically eliminate pluripotent but not differentiated hPSCs⁶ and that a recent report showed that pluripotent stem cells could be induced from mouse somatic cells by using only small molecules⁷, we may have good reason to believe that one day in the not too distant future we could grow patient-customized organs for transplantation (Figure 1).Open in a separate windowFigure 1This figure outlines the strategy of generating organs from patients'' iPSCs as an alternative to transplantation. Patient-derived pluripotent stem cells (iPSCs) can be differentiated in vitro to desired cell types. As demonstrated by Takebe et al.⁵, different cell types can be co-cultured in dish to recapitulate the earliest process of organogenesis and form three-dimensional organ buds. These in vitro produced organ buds could be used for transplantation in the future.     

Improved synchronous light scattering method for measuring baker’s yeast biomass using thickened suspensions

Measuring yeast biomass is important in the processes of microbial fermentations. It has been demonstrated that synchronous light scattering (SLS) signals could be applied for the quantification of model bioparticles such as Saccharomyces cerevisiae. In this study, an improved synchronous light scattering method was developed for yeast biomass estimation. The settlement of yeast cells during SLS signals measuring process was studied, and hydrolysis anionic polyacrylamide was added into yeast suspensions to increase the stability of the cells in liquid environment. By simultaneously scanning both the excitation and emission monochromators of a common spectrofluorometer with same starting excitation and emission wavelength (namely, ?λ = 0), the SLS intensity was found to be proportional to the yeast concentration in the range from 0 to 4.9 × 10⁶ cell/mL (R ² = 0.9907), the detection limit is 8.1 × 10³ cell/mL. The developed method exhibited good stability and sensitivity in the recovery test and growth curve drawing process, demonstrating the potential of the method in practical application of biomass estimation.     

Grape seed proanthocyanidin extracts prevent high glucose-induced endothelia dysfunction via PKC and NF-κB inhibition

In our study, it has been detected in vivo and in vitro that GSPE reversed high glucose-induced the increase of ICAM-1 and VCAM-1. It is shown that by western blotting detection, GSPE significantly inhibited the activation of NF-κB induced by high glucose while there was significant decrease of the expression of PKC with GSPE intervention. By adding the NF-κB blocker PDTC and the PKC inhibitor peptide 19–31(10^?6 M), no significant difference was found in the levels of VCAM-1 and ICAM-1 among GSPE group, the PKC inhibitor peptide 19–31-added GSPE group and the PDTC-added GSPE group. So the conclusion could be drawn that PKC inhibition must be involved in GSPE decreasing the level of ICAM-1 and VCAM-1.We proved for the first time that GSPE prevented high glucose-induced the increase of ICAM-1 and VCAM-1 by PKC and NF-κB inhibition. These findings show a novel mechanism of the action GSPE preventing endothelial dysfunction, which may have clinical application values.     

Development of rRNA-targeted probes for detection of Prorocentrum micans (Dinophyceae) using whole cell in situ hybridization

Prorocentrum is a common dinoflagellate genus along the Chinese seacoast, which frequently causes harmful algal blooms. Efforts to understand and prevent blooms caused by these harmful species require the development of methods for rapid and precise identification and quantification so that an adequate early warning of harmful algal blooms may be given. Here, we report the development and application of rRNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH) to aid in the detection of Prorocentrum micans. The hypervariable D1–D2 regions of a large subunit rDNA of a strain isolated from East China Sea identified as P. micans were first sequenced to design species-specific probes. Analysis of sequences identified as P. micans and deposited in GenBank revealed significant base differences among them and phylogenetic analyses revealed multiple clades within the taxon P. micans. Thus, it is likely that more than one taxonomic and genetically distinct entity has been identified as P. micans, if not misidentified. A series of probes were identified to one of these clades and tested for their specificity. Second, whole cell in situ hybridization procedures were established and the optimal probes were screened among the candidate probes. Next, cross-reactivity was performed to test the specificity of the probes and the detection reliability under various culture conditions, including different nutrient levels, temperatures, and light intensities. Finally, an improved protocol for natural samples was applied to the field material. The designed rRNA-targeted probe was specific, showing no cross-reactivity with other microalgae. The optimized detection protocol could be completed within 1.5 h. All target cells were speculated to be identified during all stages of their whole growth cycle under different culture conditions because the difference in fluorescence intensities throughout the experiment was not significant (p?>?0.05). The cell densities determined by FISH and light microscopy (LM) were comparable, without any significant difference (p?>?0.05) between them. In general, the established FISH probe was promising for specific, rapid, precise detection of a selected set of P. micans in natural samples and served as a good detection model for other Prorocentrum in the future.     

Synergistic antitumor activity of reversine combined with aspirin in cervical carcinoma in vitro and in vivo

A recent report showed that reversine treatment could induce murine myoblasts dedifferentiation into multipotent progenitor cells and inhibit proliferation of some tumors, and other reports showed that apoptosis of lung adenocarcinoma cells could be induced by aspirin. The aim of the present study was to evaluate the synergistic antitumor effects of reversine and aspirin on cervical cancer. The inhibition rate of reversine and aspirin on cervical cancer cell lines’ (HeLa and U14) was determined by MTT method, cell cycle of HeLa and U14 cells was analyzed by FACS, mitochondrial membrane potential of HeLa and U14 was detected using a JC-1 kit. HeLa and U14 colony formation was analyzed by soft agar colony formation assay. The expression of caspase-3, Bcl-2/Bax, cyclin D1 and p21 was detected by qRT-PCR and Western Blotting. Moreover, tumor weight and tumor volume was assessed using a murine model of cervical cancer with U14 cells subcutaneously (s.c.) administered into the neck, separately or combined with drug administration via the intraperitoneal (i.p.) route. The inhibition rate of cells in the combination group (10 μmol/L reversine, 10 mmol/L aspirin) increased significantly in comparison to that when the drugs were used alone (P < 0.05); moreover, this combination could synergistically inhibit the proliferation of five cervical cancer cell lines (HeLa, U14, Siha, Caski and C33A). In the therapeutic mouse model, tumor weight and tumor volume of cervical cancer bearing mice was more reduced when compared with the control agents (P < 0.05) in tumor-bearing mice. The combination of reversine and aspirin exerts synergistic growth inhibition and apoptosis induction on cervical cancers cells.     

Potential antitumor mechanisms of phenothiazine drugs

In this study, three kinds of phenothiazine drugs were analyzed to explore their potential antitumor mechanisms. First, target proteins that could interact with chlorpromazine, fluphenazine and trifluoperazine were predicted. Then, the target proteins of the three drugs were intersected. Cell signaling pathway enrichment and related disease enrichment were conducted for the intersected proteins to extract the enrichment categories associated with tumors. By regulation network analysis of the protein interactions, the mechanisms of action of these target proteins in tumor tissue were clarified, thus confirming the potential antitumor mechanisms of the phenothiazine drugs. The final results of cell signaling pathway enrichment and related disease enrichment showed that the categories with the highest score were all found in tumors. Target proteins belonging to the tumor category included signaling pathway members such as Wnt, MAPK and retinoic acid receptor. Moreover, another target protein, MAPK8, could indirectly act on target proteins CDK2, IGF1R, GSK3B, RARA, FGFR2 and MAPK10, thereby affecting tumor cell division and proliferation. Therefore, phenothiazine drugs may have potential antitumor effects, and tumor-associated target proteins play important roles in the process of cell signaling transduction cascades.     

Estimation of Endocarpon pusillum Hedwig carbon budget in the Tengger Desert based on its photosynthetic rate

This study investigated the photosynthetic rate of the lichen Endocarpon pusillum at the Chinese Academy of Sciences Shapotou Desert Research Station and estimated its annual contribution to the carbon budget in the ecosystem. The software SigmaPlot 10.0 with “Macro-Area below curves” was used to calculate the carbon fixation capacity of the lichen. The total carbon budget (ΣC) of the lichen was obtained by subtracting the respiratory carbon loss (ΣDR) from the photosynthetic carbon gain (ΣNP). Because water from precipitation plays an important role in photosynthesis in this ecosystem, the annual carbon budget of E. pusillum at the station was estimated based on the three-year average precipitation data from 2009 to 2011. Our results indicate that the lichen fixes 14.6 g C m^?2 annually. The results suggest that artificial inoculation of the crust lichen in the Tengger Desert could not only help reduce the sand and dust storms but also offer a significant carbon sink, fixing a total of 438000 t of carbon over the 30000 km² of the Tengger Desert. The carbon sink could potentially help mitigate the atmospheric greenhouse effect. Our study suggests that the carpet-like lichen E. pusillum is an excellent candidate for “Bio-carpet Engineering” of arid and semi-arid regions.     

A New Functional Site W115 in CdtA Is Critical for Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin

Aggregatibacter actinomycetemcomitans, a specific pathogen of localized aggressive periodontitis, produces a cytolethal distending toxin (CDT) that arrests eukaryotic cells irreversibly in G0/G1 or G2/M phase of the cell cycle. Although structural studies show that the aromatic patch region of CdtA plays an important role in its biological activity, the functional sites of CdtA have not been firmly established. In this study, site-specific mutagenesis strategy was employed for cdtA point mutations construction so as to examine the contributions of individual amino acids to receptor binding and the biological activity of holotoxin. The binding ability was reduced in CdtA^Y181ABC holotoxin and the biological function of CDT was not weaken in CdtA^Y105ABC, CdtA^Y125ABC, CdtA^F109ABC and CdtA^S106NBC holotoxin suggesting that these sites were not critical to CDT. But the binding activity and cell cycle arrest ability of holotoxin complexes were inhibited in CdtA^W115GBC. And this site did not affect the holotoxin assembly by size exclusion chromatography. Therefore, W115 might be a critical site of CdtA binding ability. These findings suggest that the functional sites of CdtA are not only in the aromatic patch region. W115, the new functional site is critical for receptor binding and cell cycle arrest, which provides potential targets for pharmacological disruption of CDT activity.     

Decreased Serum Hepcidin Concentration Correlates with Brain Iron Deposition in Patients with HBV-Related Cirrhosis

Purpose

Excessive brain iron accumulation contributes to cognitive impairments in hepatitis B virus (HBV)-related cirrhotic patients. The underlying mechanism remains unclear. Hepcidin, a liver-produced, 25-aminoacid peptide, is the major regulator of systemic iron metabolism. Abnormal hepcidin level is a key factor in some body iron accumulation or deficiency disorders, especially in those associated with liver diseases. Our study was aimed to explore the relationship between brain iron content in patients with HBV-related cirrhosis and serum hepcidin level.

Methods

Seventy HBV-related cirrhotic patients and forty age- sex-matched healthy controls were enrolled. Brain iron content was quantified by susceptibility weighted phase imaging technique. Serum hepcidin as well as serum iron, serum transferrin, ferritin, soluble transferrin receptor, total iron binding capacity, and transferrin saturation were tested in thirty cirrhotic patients and nineteen healthy controls. Pearson correlation analysis was performed to investigate correlation between brain iron concentrations and serum hepcidin, or other iron parameters.

Results

Cirrhotic patients had increased brain iron accumulation compared to controls in the left red nuclear, the bilateral substantia nigra, the bilateral thalamus, the right caudate, and the right putamen. Cirrhotic patients had significantly decreased serum hepcidin concentration, as well as lower serum transferring level, lower total iron binding capacity and higher transferrin saturation, compared to controls. Serum hepcidin level negatively correlated with the iron content in the right caudate, while serum ferritin level positively correlated with the iron content in the bilateral putamen in cirrhotic patients.

Conclusions

Decreased serum hepcidin level correlated with excessive iron accumulation in the basal ganglia in HBV-related cirrhotic patients. Our results indicated that systemic iron overload underlined regional brain iron repletion. Serum hepcidin may be a clinical biomarker for brain iron deposition in cirrhotic patients, which may have therapeutic potential.