Feihu Hu

Exosome Uptake through Clathrin-mediated Endocytosis and Macropinocytosis and Mediating miR-21 Delivery

Background: Exosomes can transfer information between cells and facilitate tumor development. Results: PC12 cell-derived exosomes enter into BMSCs through clathrin-mediated endocytosis and macropinocytosis, and decrease the expression of TGFβRII and TPM1 through miR-21. Conclusion: The results dissect the pathway of exosome internalization and demonstrate their regulation ability. Significance: These findings enhanced our knowledge of the internalization and function of tumor exosomes. Exosomes are nanoscale membrane vesicles secreted from many types of cells. Carrying functional molecules, exosomes transfer information between cells and mediate many physiological and pathological processes. In this report, utilizing selective inhibitors, molecular tools, and specific endocytosis markers, the cellular uptake of PC12 cell-derived exosomes was imaged by high-throughput microscopy and statistically analyzed. It was found that the uptake was through clathrin-mediated endocytosis and macropinocytosis. Furthermore, PC12 cell-derived exosomes can enter and deliver microRNAs (miRNAs) into bone marrow-derived mesenchymal stromal cells (BMSCs), and decrease the expression level of transforming growth factor β receptor II (TGFβRII) and tropomyosin-1 (TPM1) through miR-21. These results show the pathway of exosome internalization and demonstrate that tumor cell-derived exosomes regulate target gene expression in normal cells.

Dynamics of exosome internalization and trafficking

Cells release exosomes into extracellular medium. Although the important roles of exosomes in many physiological and pathological processes are being revealed, the mechanism of exosome–cell interaction remains unclear. In this article, employing real‐time fluorescence microscopy, the motion of exosomes on the plasma membrane or in the cytoplasm of recipient PC12 cells was observed directly. In addition, several motion modes of exosomes were revealed by single particle tracking (SPT). The changes between motion modes were also detected, presenting the dynamic courses of exosome attachment onto plasma membrane and exosome uptake. Octadecyl rhodamine B chloride (R18) was found to be useful to distinguish endocytosis from fusion during exosome uptake. Colocalization with organelle markers showed exosomes were sorted to acidic vesicles after internalization. The results provide new sight into the exosome–cell interaction mode and the intercellular trafficking of exosomes. This study will help to understand the roles of exosomes at cell level. J. Cell. Physiol. 228: 1487–1495, 2013.

Effects of Epidermal Growth Factor and Basic Fibroblast Growth Factor on the Proliferation and Osteogenic and Neural Differentiation of Adipose-Derived Stem Cells

Stem cells used for clinical tissue regeneration therapy should have the capacity of self-renewal, high proliferation, and differentiation and be able to be transplanted in large numbers. Although high concentrations of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) may induce the differentiation of stem cells, these factors have been widely used to enhance the propagation of stem cells, including adipose-derived mesenchymal stem cells (ASCs). However, the effects of low concentrations of EGF and bFGF on stem cells need to be evaluated carefully. This study illustrates that low concentrations of EGF (5 ng/mL) and bFGF (10 ng/mL) increase the proliferative ability of ASCs and induce the typical spindle-shaped cell morphology. EGF and bFGF added to medium promoted neural lineage differentiation and impaired the mesodermal differentiation ability of ASCs. This study demonstrates that even low concentrations of EGF and bFGF may limit the differentiation ability of stem cells during stem cell expansion in vitro. EGF and bFGF supplementation should be carefully considered in stem cells for clinical applications.

Comprehensive annotation of microRNA expression profiles

BackgroundMicroRNAs (miRNAs) regulate many biological processes by post-translational gene silencing. Analysis of miRNA expression profiles is a reliable method for investigating particular biological processes due to the stability of miRNA and the development of advanced sequencing methods. However, this approach is limited by the broad specificity of miRNAs, which may target several mRNAs.ResultIn this study, we developed a method for comprehensive annotation of miRNA array or deep sequencing data for investigation of cellular biological effects. Using this method, the specific pathways and biological processes involved in Alzheimer’s disease were predicted with high correlation in four independent samples. Furthermore, this method was validated for evaluation of cadmium telluride (CdTe) nanomaterial cytotoxicity. As a result, apoptosis pathways were selected as the top pathways associated with CdTe nanoparticle exposure, which is consistent with previous studies.ConclusionsOur findings contribute to the validation of miRNA microarray or deep sequencing results for early diagnosis of disease and evaluation of the biological safety of new materials and drugs.

Solution-based synthesis of SnO2 nanoparticle/CdS nanowire heterostructures

Heterostructures of SnO2 nanoparticles (NP)/CdS nanowires (NW) were successfully fabricated by a two-step chemical solution method. In the first step, CdS nanowires were synthesized by a simple solvothermal route. In the second step, SnO2 nanoparticles were grown on the surface of the CdS nanowires in a chemical solution of SnCl4·5H2O and anhydrous ethanol at 200 °C. Field-emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) were adopted to characterize the as-prepared products. The possible formation mechanism of the SnO2 NP/CdS NW heterostructures has been phenomenologically discussed. The effects of reaction conditions, such as deposition reaction time, reaction temperature, and solvent were also studied.

Reduced graphene oxide/gold nanoparticle aerogel for catalytic reduction of 4-nitrophenol

Currently, it is of great significance and a challenge to develop facile synthetic routes to obtain a novel plasmonic heterogeneous catalyst with high activity and long lifetime for the reduction of a refractory organic compound like 4-nitrophenol (4-NP). To this end, a three-dimensional (3D) porous framework named reduced graphene oxide/gold nanoparticle aerogel (rGO/Au NPA) was constructed by individual GO sheets and HAuCl4 under the reduction of trisodium citrate dihydrate (Na3Cit) via a one-step hydrothermal method. The abundant Au NPs having a diameter of 7–160 nm can be easily in situ incorporated into graphene sheets to form a 3D hierarchical monolith by the reduction of Na3Cit, which was well-disclosed by field-emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM). Such 3D rGO/Au NPA with interconnected porous structure displays a good thermal stability and large Brunauer–Emmett–Teller specific surface area of 37.8325 m2 g−1. More importantly, the fabricated rGO/Au NPA can act as a heterogeneous catalyst, exhibiting an outstanding catalytic activity and good reusability towards the reduction of 4-NP due to the synergetic effect between Au NPs and graphene sheets. Additionally, the mechanism of enhanced catalytic efficiency for the 3D rGO/Au NPA catalyst has also been proposed.

Comparative analysis of microRNA expression in human mesenchymal stem cells from umbilical cord and cord blood

Human mesenchymal stem cells (MSCs) derived from both umbilical cord (UC) and cord blood (CB) share similar characteristics, and their differences are largely unknown. Besides the significant difference in cell morphology, differentiation ability and development processes of the two different origin MSCs, a different expression pattern of microRNAs between the two kinds of MSCs was also obtained. By comprehensively annotating the differently expressed global microRNAs (miRNAs), a series of biological pathways were predicted. We found that miRNAs significantly repressed insulin signaling in UCMSCs, while neural related processes were more repressed in CBMSCs. Particularly, TGF-β and Notch signaling were differently activated in both MSCs, unveiling their distinct angiogenesis potentials. Taken together, this study illustrates that MSCs from UC and CB display distinct properties, which indicates different potentials for clinical usage.

MicroRNA profiling analysis revealed different cellular senescence mechanisms in human mesenchymal stem cells derived from different origin

Mesenchymal stem cells (MSCs) from human umbilical cord (UC) and cord blood (CB) share many common properties and exhibit promising clinical potential. Cellular senescence, which induces the loss of stem cells characters and disrupts their therapeutic functions, has been demonstrated to be under the regulation of microRNAs (miRNAs). In this study, we compared the miRNA profiles in early and late passage UCMSCs and CBMSCs based on deep sequencing. 224 and 170 miRNAs were significantly altered in UCMSCs and CBMSCs respectively. A functional annotation of the predicted miRNA targets revealed a series of common senescence pathways. However, Functional enrichment analysis revealed different bioprocesses involved in cellular senescence of UC- and CB-MSCs. The common miRNAs shared by the two kinds of MSCs also exert different function in terms of GO enrichment analysis. Our results supported MSCs derived from different origin may undergo senescence through different path.

Exosomes Transfer Among Different Species Cells and Mediating miRNAs Delivery

Exosomes, the natural vehicles of intercellular communication, transfer proteins, mRNAs, and microRNAs (miRNAs) and mediate many physiological and pathological processes. It is not clear that whether exosomal miRNAs could regulate gene expression across species, though some studies suggest interactions of exosomal miRNAs between cells. In this report, we have isolated exosomes from rat PC12 cells and assessed their internalization by human cancer Hela cells. The internalized exosomes were located in Hela lysosomes. Human PTEN expression was significantly deregulated due to miR‐21 delivered by rat cell exosomes. Our results prove that exosomes could incorporate between cells of different species and could regulate the protein expressions in the recipient cells by delivering the enclosed miRNAs. Thus our study foreshadows a futuristic treatment approach of utilizing miRNA enclosed exosome vehicles sans species concerns in combating various diseases/ regulating abnormal proteins. J. Cell. Biochem. 118: 4267–4274, 2017.

MiR-218 Induces Neuronal Differentiation of ASCs in a Temporally Sequential Manner with Fibroblast Growth Factor by Regulation of the Wnt Signaling Pathway

Differentiation of neural lineages from mesenchymal stem cells has raised the hope of generating functional cells as seed cells for nerve tissue engineering. As important gene regulators, microRNAs (miRNAs) have been speculated to play a vital role in accelerating stem cell differentiation and repairing neuron damage. However, miRNA roles in directing differentiation of stem cells in current protocols are underexplored and the mechanisms of miRNAs as regulators of neuronal differentiation remain ambiguous. In this study, we have determined that miR-218 serves as crucial constituent regulator in neuronal differentiation of adipose stem cells (ASCs) through Wnt signaling pathway based on comprehensive annotation of miRNA sequencing data. Moreover, we have also discovered that miR-218 and Fibroblast Growth Factor-2 (FGF2) modulate neuronal differentiation in a sequential manner. These findings provide additional understanding of the mechanisms regulating stem cell neuronal differentiation as well as a new method for neural lineage differentiation of ASCs.