CRISPR editing of the GLI1 first intron abrogates GLI1 expression and differentially alters lineage commitment.

GLI1 is one of three GLI family transcription factors that mediate Sonic Hedgehog signaling, which plays a role in development and cell differentiation. GLI1 forms a positive feedback loop with GLI2 and likely with itself. To determine the impact of GLI1 and its intronic regulatory locus on this transcriptional loop and human stem cell differentiation, we deleted the region containing six GLI binding sites in the human GLI1 intron using CRISPR/Cas9 editing to produce H1 human embryonic stem cell (hESC) GLI1-edited clones. Editing out this intronic region, without removing the entire GLI1 gene, allowed us to study the effects of this highly complex region, which binds transcription factors in a variety of cells. The roles of GLI1 in human ESC differentiation were investigated by comparing RNA sequencing, quantitative-real time PCR (q-rtPCR), and functional assays. Editing this region resulted in GLI1 transcriptional knockdown, delayed neural commitment, and inhibition of endodermal and mesodermal differentiation during spontaneous and directed differentiation experiments. We found a delay in the onset of early osteogenic markers, a reduction in the hematopoietic potential to form granulocyte units, and a decrease in cancer-related gene expression. Furthermore, inhibition of GLI1 via antagonist GANT-61 had similar in vitro effects. These results indicate that the GLI1 intronic region is critical for the feedback loop and that GLI1 has lineage-specific effects on hESC differentiation. Our work is the first study to document the extent of GLI1 abrogation on early stages of human development and to show that GLI1 transcription can be altered in a therapeutically useful way.

A Stem Cell-Derived Platform for Studying Single Synaptic Vesicles in Dopaminergic Synapses.

The exocytotic release of dopamine is one of the most characteristic but also one of the least appreciated processes in dopaminergic neurotransmission. Fluorescence imaging has yielded rich information about the properties of synaptic vesicles and the release of neurotransmitters in excitatory and inhibitory neurons. In contrast, imaging-based studies for in-depth understanding of synaptic vesicle behavior in dopamine neurons are lagging largely because of a lack of suitable preparations. Midbrain culture has been one of the most valuable preparations for the subcellular investigation of dopaminergic transmission; however, the paucity and fragility of cultured dopaminergic neurons limits their use for live cell imaging. Recent developments in stem cell technology have led to the successful production of dopamine neurons from embryonic or induced pluripotent stem cells. Although the dopaminergic identity of these stem cell-derived neurons has been characterized in different ways, vesicle-mediated dopamine release from their axonal terminals has been barely assessed. We report a more efficient procedure to reliably generate dopamine neurons from embryonic stem cells, and it yields more dopamine neurons with more dopaminergic axon projections than midbrain culture does. Using a collection of functional measurements, we show that stem cell-derived dopamine neurons are indistinguishable from those in midbrain culture. Taking advantage of this new preparation, we simultaneously tracked the turnover of hundreds of synaptic vesicles individually using pH-sensitive quantum dots. By doing so, we revealed distinct fusion kinetics of the dopamine-secreting vesicles, which is consistent within both preparations.

Two new sesquiterpenoids from endophytic fungus J3 isolated from Mangrove Plant Ceriops tagal.

Two new sesquiterpenoids, named 2α-hydroxyxylaranol B (1) and 4ß-hydroxyxylaranol B (2), together with a known diterpenoid 3,4-seco-sonderianol (3) were isolated from the fermentation of endophytic fungus J3 of Ceriops tagal. Their structures were elucidated based on spectroscopic methods including 1D and 2D NMR (HMQC, (1)H-(1)H COSY and HMBC). All compounds were evaluated for their cytotoxic activities by MTT method, and compound 3 exhibited cytotoxic activities against K562, SGC-7901, and BEL-7402 cell lines.

Roles of Th17 cells in pulmonary granulomas induced by Schistosoma japonicum in C57BL/6 mice.

In schistosomiasis, limited information is available about the role of interleukin-17 (IL-17) in lung, despite the fact that this cytokine plays a crucial role during pro-inflammatory immune responses. In our study, we observed CD4(+)T cells changed after the infection. Furthermore, ELISA and FACS results revealed that Schistosomajaponicum infection could induce a large amount of IL-17 in mouse pulmonary lymphocytes. IL-17-producing cells, including Th17 cells, CD8(+)T (Tc) cells, γδT cells and natural killer T cells, was also associated with the development of lung inflammatory diseases. FACS results indicated that Th17 cell was the main source of IL-17 in the infected pulmonary lymphocytes after phorbol-12-myristate-13-acetate (PMA) and Ionomycin stimulation. Moreover, FACS results revealed that the percentage of Th17 cells continued to increase as over the course of S. japonicum infection. Additionally, cytokines co-expression results demonstrated that Th17 cells could express more IL-4 and IL-5 than IFN-γ. Reducing IL-17 activity by using anti-IL-17 ameliorated the damage and decreased infiltration of inflammatory cells in infected C57BL/6 mouse lungs. Collectively, these results suggest Th17 cells is the major IL-17-producing cells population and IL-17 contributes to pulmonary granulomatous inflammatory during the S. japonicum infection.

Ursolic acid promotes cancer cell death by inducing Atg5-dependent autophagy.

Ursolic acid (UA) has been reported to possess anticancer activities. Although some of the anticancer activities of UA have been explained by its apoptosis-inducing properties, the mechanisms underlying its anticancer actions are largely unknown. We have found that UA-activated autophagy induced cytotoxicity and reduced tumor growth of cervical cancer cells TC-1 in a concentration-dependent manner. UA did not induce apoptosis of TC-1 cells in vitro as determined by annexin V/propidium iodide staining, DNA fragmentation, and Western blot analysis of the apoptosis-related proteins. We found that UA increased punctate staining of light chain 3 (LC3), which is an autophagy marker. LC3II, the processed form of LC3I which is formed during the formation of double membranes, was induced by UA treatment. These results were further confirmed by transmission electron microscopy. Wortmannin, an inhibitor of autophagy, and a small interfering RNA (siRNA) for autophagy-related genes (Atg5) reduced LC3II and simultaneously increased the survival of TC-1 cells treated with UA. We also found that LC3II was significantly reduced and that survival was increased in Atg5-/- mouse embryonic fibroblast (MEF) cells compared to Atg5+/+ MEF cells under UA treatment. However, silencing BECN1 by siRNA affected neither the expression of LC3II nor the survival of TC-1 cells under UA treatment. These results suggest that autophagy is a major mechanism by which UA kills TC-1 cells. It is Atg5 rather than BECN1 that plays a crucial role in UA-induced autophagic cell death in TC-1 cells. The activation of autophagy by UA may become a potential cancer therapeutic strategy complementing the apoptosis-based therapies. Furthermore, regulation of Atg5 may improve the efficacy of UA in cancer treatment.

Inhibition of the Rho signaling pathway improves neurite outgrowth and neuronal differentiation of mouse neural stem cells.

Neurons in the adult mammalian CNS do not spontaneously regenerate axons after injury due to CNS myelin and other inhibitory factors. Previous studies have showed that inhibition of the Rho-ROCK pathway promotes axonal outgrowth in primary neurons or in spinal cord injury models. Furthermore, RhoA inhibitor C3 transferase has a potential effect to induce neural differentiation in primary cultured neurons and cell lines. As stem cells and stem cell-derived neural progenitor cells have emerged as a regenerative medicine for stroke, Parkinson's disease and other neurological disorders, strategies that can promote axonal outgrowth and neuronal differentiation appear to have promising benefits in the cell-based therapy. Currently, how changes in the Rho-ROCK pathway may affect the neurite outgrowth and neuronal differentiation of stem cells has been poorly understood. The present investigation examined the effects of RhoA inhibition on neurite outgrowth and neuronal differentiation of neural stem cells (NSCs) isolated from the subventricular zone (SVZ) of the mouse. Our results show that inhibition of RhoA leads to neurite outgrowth of NSCs not only on normal culture substrate, poly-D-lysine (PDL), but also on myelin substrate. Moreover, inhibition of RhoA improves neuronal differentiation of NSCs and up-regulates biomarkers of neuronal gene expression. These results support that the Rho signaling pathway plays an important role in neurite development and neuronal differentiation of NSCs.

Control of in vitro neural differentiation of mesenchymal stem cells in 3D macroporous, cellulosic hydrogels.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent cells that can be induced to differentiate into multiple cell lineages, including neural cells. They are a good cell source for neural tissue-engineering applications. Cultivation of human (h)MSCs in 3D scaffolds is an effective means for the development of novel neural tissue-engineered constructs, and may serve as a promising strategy in the treatment of nerve injury. AIM: This study presents the in vitro growth and neural differentiation of hMSCs in 3D macroporous, cellulosic hydrogels. RESULTS: The number of hMSCs cultivated in the 3D scaffolds increased by more than 14-fold after 7 days. After 2 days induction, most of the hMSCs in the 3D scaffolds were positive for nestin, a marker of neural stem cells. After 7 days induction, most of the hMSCs in the 3D scaffolds showed glial fibrillary acidic protein, tubulin or neurofilament M-positive reaction and a few hMSCs were positive for nestin. After 14 days induction, hMSCs in the 3D scaffolds could completely differentiate into neurons and glial cells. The neural differentiation of hMSCs in the 3D scaffolds was further demonstrated by real-time PCR. CONCLUSION: These results show that the 3D macroporous cellulosic hydrogel could be an appropriate substrate for neural differentiation of hMSCs and its possible applications in neural tissue engineering are discussed.

Recombinant human NGF-loaded microspheres promote survival of basal forebrain cholinergic neurons and improve memory impairments of spatial learning in the rat model of Alzheimer's disease with fimbria-fornix lesion.

Neurotrophic factors are used for the experimental treatment of neurological disorders, such as Alzheimer's disease. However, delivery of the neurotrophic factors into the brain remains a big challenge. Recombinant human nerve growth factor (NGF)-loaded microspheres were fabricated and characterized in vitro and in vivo in our previous study. The present study was to assess the therapeutic benefit of rhNGF-loaded microspheres in treating the rat model of Alzheimer's disease with fimbria-fornix lesion. Recombinant human NGF-loaded microspheres were implanted into the basal forebrain of the rats with fimbria-fornix lesion. Four weeks after implantation in the basal forebrain, immunohistochemical analysis showed that rhNGF-loaded microspheres had a significant effect on the survival of axotomized cholinergic neurons in the medial septum (MS) and vertical diagonal branch (VDB) (p<0.05). Y-maze tests showed rhNGF-loaded microspheres can significantly improve the ability of spatial learning and memory of the rats with fimbria-fornix lesion (p<0.05). These results indicate that rhNGF-loaded microspheres are an effective means for the treatment of Alzheimer's disease.

[A novel anti-DNA antibody modified coronary stent for site-specific plasmid DNA delivery].

OBJECTIVE: To explore the feasibility of using an endovascular metal stent as a highly efficient and site-specific gene delivery system. METHODS: Stents were formulated with a collagen coating. Anti-DNA monoclonal antibodies were covalently bound to the collagen surface by a cross linking reagent of N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). Binding capacity and stability of antibody and plasmid DNA on stents were quantified by radioactive labeling. The gene transduction efficiency was evaluated in cell culture and in rabbits. RESULTS: The amount of antibodies binding on collagen matrix through SPDP reaction was 15 times higher than that of through physical absorption (P < 0.005). The binding stability of plasmid was significantly better than the control groups (P < 0.01). There was no harmful effect on cell growth with the anti-DNA antibody modified stents. The stents retrieved from cell culture after 72 hours of incubation in A10 cells showed numerous transducted cells only infiltrating the surface coating indicating a highly localized and efficient gene delivery pattern. Results of in vivo gene transfer by this modified stent revealed (2.8 +/- 0.7)% of total cells transduction and the higher transduction location was neointimal layer (about 7%). No distal spread of vector was detectable in the anti-DNA antibody modified stent implantation animals. CONCLUSIONS: Anti-DNA antibody modified stents represent a novel highly efficient and site-specific gene delivery system which can deliver various kinds of plasmid vectors. The release of plasmid DNA tethered on the stents could be controlled in some conditions. This novel system provided a novel platform for cardiovascular site-specific gene therapy.