The influence of inflammation on the characteristics of adipose-derived mesenchymal stem cells (ADMSCs) and tissue repair capability in a hepatic injury mouse model.

BACKGROUND: Mesenchymal stem cells (MSCs) are adult stem cells with self-renewal and multi-directional differentiation potential and possess the functions of immunomodulation, regulation of cell growth, and repair of damage. Over recent years, MSCs have been found to regulate the secretion of inflammatory factors and to exert regulatory effects on various lymphocytes in inflammatory states, and on the subsequent repair of tissue damage caused by inflammation. In the present study, we analyzed the effects of tissue inflammation on the characteristics of MSCs. METHODS: Human fat derived from the infrapatellar fat pad (IPFP) of knees with differing degrees of inflammation was extracted from specimens derived from total knee arthroplasties. HE and immunohistochemical staining was performed to directly observe the evidence and degree of inflammation in human infrapatellar fat pad tissue in order to classify MSCs cells, by their origin, into highly inflamed and lowly inflamed groups, and to study the effect of tissue inflammation on cell acquisition rates via cellular counting data. Flow cytometry assays were performed to investigate the effect of tissue inflammation on MSC surface marker expression. Trilineage differentiation, including osteogenesis, adipogenesis, and chondrogenesis, was performed to assess the effect of tissue inflammation on the ability of MSCs to undergo directed differentiation. The effect of tissue inflammation on the ability of MSCs to proliferate was investigated via clone formation studies. RNA-sequencing was performed to evaluate the transcriptomes of MSCs derived from different areas of inflammation. The effect of tissue inflammation on tissue repair capacity and safety of MSCs was investigated via a murine model of acute liver injury. RESULTS: The results of cell count data indicate that a high degree of tissue inflammation significantly decreases the acquisition rate of MSCs, and the proportion of CD34+ and CD146+ cells. The results of our trilineage differentiation assay show that a higher degree of inflammation decreases osteogenic differentiation and enhances adipogenic and chondrogenic differentiation of MSCs. However, these differences were not statistically significant. Clone formation assays indicate that the degree of tissue inflammation at the MSC source does not significantly affect the proliferative capacity of MSCs. The transcriptomes of MSCs remain relatively stable in fat pad tissues derived from both highly and lowly inflamed samples. The results of acute liver injury investigations in mice indicate that MSCs of high and low inflammatory tissue origin have no significant difference in their tissue repair capability. CONCLUSIONS: High tissue inflammation at the source of MSCs reduces the acquisition rate of MSCs and the percentage of CD34+ and CD146+ cells acquisition. However, source tissue inflammation may not significantly affect trilineage differentiation potential and proliferative capacity of MSCs. Also, MSCs obtained from differing source degrees of inflammation retain stable and similar transcriptomic profile and are both safe and efficacious for tissue repair/regeneration without detectable differences.

Tumor-Tropic Adipose-Derived Mesenchymal Stromal Cell Mediated Bi2 Se3 Nano-Radiosensitizers Delivery for Targeted Radiotherapy of Non-Small Cell Lung Cancer.

With the successful marriage between nanotechnology and oncology, various high-Z element containing nanoparticles (NPs) are approved as radiosensitizers to overcome radiation resistance for enhanced radiotherapy (RT). Unfortunately, NPs themselves lack specificity to tumors. Due to the inherent tropism nature of malignant cells, mesenchymal stem cells (MSCs) emerge as cell-mediated delivery vehicles for functional NPs to improve their therapeutic index. Herein, radiosensitive bismuth selenide (Bi2 Se3 ) NPs-laden adipose-derived mesenchymal stromal cells (AD-MSCs/Bi2 Se3 ) are engineered for targeted RT of non-small cell lung cancer (NSCLC). The results reveal that the optimized intracellular loading strategy hardly affects cell viability, specific surface markers, or migration capability of AD-MSCs, and Bi2 Se3  NPs can be efficiently transported from AD-MSCs to tumor cells. In vivo biodistribution test shows that the Bi2 Se3 NPs accumulation in tumor is increased 20 times via AD-MSCs-mediated delivery. Therefore, AD-MSCs/Bi2 Se3 administration synchronized with X-ray irradiation controls the tumor progress well in orthotopic A549 tumor bearing mice. Considering that MSCs migrate better to irradiated tumor cells in comparison to nonirradiated ones and MSCs preferentially accumulate within lung tissues after systemic administration into accounts, the tumor-tropic MSCs/NPs system is feasible and promising for targeted RT treatment of NSCLC.

A 3D screening approach identifies the compound epitajixanthone hydrate as a new inhibitor of cancer cell growth and invasion.

With unique advantages, the small-molecule anticancer drugs have recently gained growing attention. Particular strategies, exemplified by high-throughput screening, fragment-based drug discovery, virtual screening and knowledge-based design, have been developed to identify active compounds. However, such screens generally rely on sophisticated and expensive instrumentations. Herein, we developed a simple spheroids 3D culture system to enable direct screening of small molecules with reliable results. Using this system, we screened 27 fungal natural products and three fungal crude extracts for their inhibitory effects on cancer cell growth, and invasion. We identified that the compound M23 (epitajixanthone hydrate, a derivative of prenylxanthone) and the crude extracts (MPT-191) from the fungi Taxus chinensis showed potential anticancer activity. The effect of epitajixanthone hydrate on cancer cell growth and invasion were further confirmed by the assays of cells viability, trans-well migration and invasion, colony formation and cells reattachment. Overall, Epitajixanthone hydrate was identified as an effective inhibitor of cancer cell growth and invasion by our simple and fast screening platform.

Prenylated Indole Diterpene Alkaloids from a Mine-Soil-Derived Tolypocladium sp.

Ten new prenylated indole diterpene alkaloids, tolypocladin A-J (1-10), including four chlorinated metabolites, have been isolated from a culture of a mine-soil-derived fungus, Tolypocladium sp. XL115. The structures and absolute configurations of 1-10 were determined by spectroscopic analysis, ECD calculations, and comparison with known compounds. Compounds 1 and 8 displayed significant antimicrobial activities. In addition, compound 1 also showed weak cytotoxic activity against all tested human cancer cell lines and suppressed the growth and viability of the patient-derived HCC cells T1224.

Three new α-pyrone derivatives from the plant endophytic fungus Penicillium ochrochloronthe and their antibacterial, antifungal, and cytotoxic activities.

Three new 3,4,6-trisubstituted α-pyrone derivatives, namely 6-(2'R-hydroxy-3'E,5'E-diene-1'-heptyl)-4-hydroxy-3-methyl-2H-pyran-2-one (1), 6-(2'S-hydroxy-5'E-ene-1'-heptyl)-4-hydroxy-3-methyl-2H-pyran-2-one (2), and 6-(2'S-hydroxy-1'-heptyl)-4 -hydroxy-3-methyl-2H-pyran-2-one (3), together with one known compound trichodermic acid (4), were isolated from the solid-substrate fermentation culture of Penicillium ochrochloronthe associated the roots of Taxus media. Compounds 1-4 displayed the antimicrobial activity selectively against tested fungal and bacterial strains with minimum inhibitory concentration (MIC) values ranging from 12.5 to 100 µg/ml. Furthermore, we found that only compound 4 exhibited moderate cytotoxicity against five human cancer cells (A549, LN229, MGC, LOVO, and MDA231) with IC50 values of 51.45, 23.43, 39.16, 46.97, and 42.85 µg/ml, respectively.

Pellet coculture of osteoarthritic chondrocytes and infrapatellar fat pad-derived mesenchymal stem cells with chitosan/hyaluronic acid nanoparticles promotes chondrogenic differentiation.

BACKGROUND: Cell source plays a key role in cell-based cartilage repair and regeneration. Recent efforts in cell coculture have attempted to combine the advantages and negate the drawbacks of the constituent cell types. The aim of this study was to evaluate the chondrogenic outcome of articular chondrocytes (ACs) and infrapatellar fat pad (IPFP)-derived mesenchymal stem cells (MSCs) in direct coculture. METHODS: ACs and IPFP MSCs from the same patients with knee osteoarthritis (OA) were cocultured in monolayer and in pellets. The monocultures of each cell type were also used as controls. Morphological and histologic analysis, immunofluorescence staining, reverse transcription-polymerase chain reaction, and enzyme-linked immunosorbent assay were performed to characterize the chondrogenic differentiation of cocultures. Furthermore, the effects of chitosan/hyaluronic acid (CS/HA) nanoparticle exposure on the chondrogenesis of cocultures were examined. RESULTS: In both monolayer and pellet coculture, the hypertrophy of MSCs and the inflammatory activities of ACs were inhibited, although the chondrogenic production in coculture was not promoted compared with that in monoculture. In addition, the exposure of CS/HA nanoparticles to pellet coculture improved the production of type II collagen and aggrecan. CONCLUSIONS: We demonstrate for the first time that pellet coculture of ACs and IPFP MSCs with CS/HA nanoparticles could promote chondrogenic outcome while preventing the inflammatory status of ACs and the hypertrophic differentiation of MSCs. These findings suggest that the combination of ACs, IPFP MSCs, and CS/HA might be useful in cartilage repair in knee OA.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus.

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells (hPSCs) is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

MicroRNAs as key mediators of hepatic detoxification.

MicroRNAs (miRNAs) are a class of short noncoding RNAs that modulate gene expression at both transcriptional and post-transcriptional levels. Many studies have extensively revealed the significance of miRNAs in mediating liver development and diseases. However, their role in hepatic detoxification processes has been explored only recently. In this review, we summarized the up-to-date knowledge about miRNA dependent regulation of enzymes involved in all three phases of the drugs and xenobiotics detoxification process. We also discussed the role of miRNA in regulating some upstream nuclear receptors involving gene expression of enzymes for detoxification process in liver. The toxicological significance of miRNAs in liver diseases and future research perspectives are finally presented.

Efficient Recombinase-Mediated Cassette Exchange in hPSCs to Study the Hepatocyte Lineage Reveals AAVS1 Locus-Mediated Transgene Inhibition.

Tools for rapid and efficient transgenesis in "safe harbor" loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes.

MicroRNAs: the fine modulators of liver development and function.

MicroRNAs are a class of small non-coding RNAs involved in the transcriptional and post-transcriptional regulation of gene expression. The function of miRNAs in liver disease including hepatocellular carcinoma (HCC), hepatitis, and alcoholic liver disease, have been widely studied and extensively reviewed. Increasing evidence demonstrates that miRNAs also play a critical role in normal liver development and in the fine-tuning of fundamental biological liver processes. In this review, we highlight the most recent findings on the role of miRNAs in liver specification and differentiation, liver cell development, as well as in the many metabolic functions of the liver, including glucose, lipid, iron and drug metabolism. These findings demonstrate an important role of miRNAs in normal liver development and function. Further researches will be needed to fully understand how miRNAs regulate liver generation and metabolic function, which should then lead to greater insights in liver biology and perhaps open up the possibility to correct errors that cause liver diseases or metabolic disorders.

Hepatic differentiation of human embryonic stem cells on microcarriers.

Translation of stem cell research to industrial and clinical settings mostly requires large quantities of cells, especially those involving large organs such as the liver. A scalable reactor system is desirable to ensure a reliable supply of sufficient quantities of differentiated cells. To increase the culture efficiency in bioreactor system, high surface to volume ratio needs to be achieved. We employed a microcarrier culture system for the expansion of undifferentiated human embryonic stem cells (hESCs) as well as for directed differentiation of these cells to hepatocyte-like cells. Cells in single cell suspension were attached to the bead surface in even distribution and were expanded to 1×10(6)cells/ml within 2 days of hESC culture with maintenance of the level of pluripotency markers. Directed differentiation into hepatocyte-like cells on microcarriers, both in static culture and stirred bioreactors, induced similar levels of hepatocyte-like cell differentiation as observed with cells cultured in conventional tissue culture plates. The cells expressed both immature and mature hepatocyte-lineage genes and proteins such as asialoglycoprotein receptor-1 (ASGPR-1) and albumin. Differentiated cells exhibited functional characteristics such as secretion of albumin and urea, and CYP3A4 activity could be detected. Microcarriers thus offer the potential for large-scale expansion and differentiation of hESCs induced hepatocyte-like cells in a more controllable bioreactor environment.

Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5' terminal nucleotide.

Argonaute (AGO) proteins recruit small RNAs to form the core of RNAi effector complexes. Arabidopsis encodes ten AGO proteins and a large network of small RNAs. How these small RNAs are sorted into specific AGO complexes remains largely unknown. We have cataloged small RNAs resident in four AGO complexes. We found that AGO2 and AGO4 preferentially recruit small RNAs with a 5' terminal adenosine, whereas AGO1 harbors microRNAs (miRNAs) that favor a 5' terminal uridine. AGO5 predominantly binds small RNAs that initiate with cytosine. Changing the 5' terminal nucleotide of an miRNA predictably redirected it into a different AGO complex and alters its biological activity. These results reveal a role for small RNA sequences in assorting among AGO complexes. This suggests that specialization of AGO complexes might involve remodeling the 5' end-binding pocket to accept certain small RNA sequences, perhaps explaining the evolutionary drive for miRNAs to initiate with uridine.

Glucose inhibits the expression of triose phosphate/phosphate translocator gene in wheat via hexokinase-dependent mechanism.

Although triose phosphate/phosphate translocator is known to play an important role in regulating the distribution of assimilates in wheat chloroplasts, the mechanism of triose phosphate/phosphate translocator gene control has not yet been clearly elucidated. We first showed that glucose inhibited the expression of triose phosphate/phosphate translocator gene in wheat by reverse transcription-polymerase chain reaction and Western blotting. The triose phosphate/phosphate translocator expression was seriously impaired by 5 mmol/L glucose, and it responded slowly, more than 48 h, to level as low as 1 mmol/L glucose. Both glucose and 2-deoxyglucose inhibited the expression of triose phosphate/phosphate translocator gene, but 2-deoxyglucose-6-P, product of phosphorylated 2-deoxyglucose, cannot be further metabolized, therefore the further metabolism of phosphorylated glucose by hexokinase is not a prerequisite for triggering glucose-regulated expression of triose phosphate/phosphate translocator gene. Glucose had little inhibitory effect on the expression of triose phosphate/phosphate translocator gene when hexokinase activity was reduced or eliminated by transforming wheat protoplasts with a hexokinase antisense construct or treating protoplasts with glucosamine, an inhibitor of hexokinase. Therefore, it appears essential for hexokinase to retain phosphorylation activity for glucose to regulate the expression of triose phosphate/phosphate translocator gene. The treatment of protoplasts with glucose-6-phosphate resulting in no inhibition of triose phosphate/phosphate translocator expression demonstrated that phosphorylation via hexokinase is necessary for glucose inhibiting triose phosphate/phosphate translocator expression. All the data suggest that triose phosphate/phosphate translocator is regulated by glucose via a hexokinase-dependent pathway.

NADK2, an Arabidopsis chloroplastic NAD kinase, plays a vital role in both chlorophyll synthesis and chloroplast protection.

As one of terminal electron acceptors in photosynthetic electron transport chain, NADP receives electron and H(+) to synthesize NADPH, an important reducing energy in chlorophyll synthesis and Calvin cycle. NAD kinase (NADK), the catalyzing enzyme for the de novo synthesis of NADP from substrates NAD and ATP, may play an important role in the synthesis of NADPH. NADK activity has been observed in different sub-cellular fractions of mitochondria, chloroplast, and cytoplasm. Recently, two distinct NADK isoforms (NADK1 and NADK2) have been identified in Arabidopsis. However, the physiological roles of NADKs remain unclear. In present study, we investigated the physiological role of Arabidiposis NADK2. Sub-cellular localization of the NADK2-GFP fusion protein indicated that the NADK2 protein was localized in the chloroplast. The NADK2 knock out mutant (nadk2) showed obvious growth inhibition and smaller rosette leaves with a pale yellow color. Parallel to the reduced chlorophyll content, the expression levels of two POR genes, encoding key enzymes in chlorophyll synthesis, were down regulated in the nadk2 plants. The nadk2 plants also displayed hypersensitivity to environmental stresses provoking oxidative stress, such as UVB, drought, heat shock and salinity. These results suggest that NADK2 may be a chloroplast NAD kinase and play a vital role in chlorophyll synthesis and chloroplast protection against oxidative damage.