Comparison of Teratoma Formation between Embryonic Stem Cells and Parthenogenetic Embryonic Stem Cells by Molecular Imaging

With their properties of self-renewal and differentiation, embryonic stem (ES) cells hold great promises for regenerative therapy. However, teratoma formation and ethical concerns of ES cells may restrict their potential clinical applications. Currently, parthenogenetic embryonic stem (pES) cells have attracted the interest of researchers for its self-renewing and pluripotent differentiation while eliciting less ethic concerns. In this study, we established a model with ES and pES cells both stably transfected with a double-fusion reporter gene containing renilla luciferase (Rluc) and red fluorescent protein (RFP) to analyze the mechanisms of teratoma formation. Transgenic Vegfr2-luc mouse, which expresses firefly luciferase (Fluc) under the promoter of vascular endothelial growth factor receptor 2 (Vegfr2-luc), was used to trace the growth of new blood vessel recruited by transplanted cells. Bioluminescence imaging (BLI) of Rluc/Fluc provides an effective tool in estimating the growth and angiogenesis of teratoma in vivo. We found that the tumorigenesis and angiogenesis capacity of ES cells were higher than those of pES cells, in which VEGF/VEGFR2 signal pathway plays an important role. In conclusion, pES cells have the decreased potential of teratoma formation but meanwhile have similar differentiating capacity compared with ES cells. These data demonstrate that pES cells provide an alternative source for ES cells with the risk reduction of teratoma formation and without ethical controversy

Alginic Acid-Coated Chitosan Nanoparticles Loaded with Legumain DNA Vaccine: Effect against Breast Cancer in Mice

<div><p>Legumain-based DNA vaccines have potential to protect against breast cancer. However, the lack of a safe and efficient oral delivery system restricts its clinical application. Here, we constructed alginic acid-coated chitosan nanoparticles (A.C.NPs) as an oral delivery carrier for a legumain DNA vaccine. First, we tested its characteristic in acidic environments <i>in vitro</i>. DNA agarose electrophoresis data show that A.C.NPs protected DNA better from degradation in acidic solution (pH 1.5) than did chitosan nanoparticles (C.NPs). Furthermore, size distribution analysis showed that A.C.NPs tended to aggregate and form micrometer scale complexes in pH<2.7, while dispersing into nanoparticles with an increase in pH. Mice were intragastrically administrated A.C.NPs carrying EGFP plasmids and EGFP expression was detected in the intestinal Peyer’s patches. Full-length legumain plasmids were loaded into different delivery carriers, including C.NPs, attenuated <i>Salmonella typhimurium</i> and A.C.NPs. A.C.NPs loaded with empty plasmids served as a control. Oral vaccination was performed in the murine orthotopic 4T1 breast cancer model. Our data indicate that tumor volume was significantly smaller in groups using A.C.NPs or attenuated <i>Salmonella typhimurium</i> as carriers. Furthermore, splenocytes co-cultured them with 4T1 cells pre-stimulated with CoCl₂, which influenced the translocation of legumain from cytoplasm to plasma membrane, showed a 4.7 and 2.3 folds increase in active cytotoxic T lymphocytes (CD3⁺/CD8⁺/CD25⁺) when treated with A.C.NPs carriers compared with PBS C.NPs. Our study suggests that C.NPs coated with alginic acid may be a safe and efficient tool for oral delivery of a DNA vaccine. Moreover, a legumain DNA vaccine delivered orally with A.C.NPs can effectively improve autoimmune response and protect against breast cancer in mice.</p> </div

A.C.NPs loaded with DNA pass through the acidic gastric barrier and are taken up by macrophages and dendritic cells in the intestinal Peyer’s patches.

<p>Naked EGFP DNA plasmids, C.NPs-EGFP, and A.C.NPs-EGFP were separately given to BALB/c mice (n = 5) via intragastric gavage at a daily dose equivalent to 30 µg plasmid DNA per mouse for three consecutive days. Peyer’s patches were isolated and analyzed by flow cytometry. PE-conjugated F4/80 and APC-conjugated CD11c antibodies were used to stain (A) the macrophages and (B) dendritic cells, respectively. Histograms of the percentage of EGFP-positive (C) macrophages and (D) dendritic cells. (E) Histograms of the ratio of F4/80- or CD11c- positive cells to total EGFP-positive cells. Data are presented as mean ± SD of three independent experiments (**P<0.01; n = 5).</p

Diagram shows the protective effect of A.C.NPs on DNA against enzymatic and acidic degradation.

<p>(A) Schematic of A.C.NPs-legumain preparation. (B) Schematic representation of A.C.NPs-legumain passing through the acidic gastrointestinal track and taken up by antigen-presenting cells in the intestinal Peyer’s patches.</p

Illustration of A.C.NPs-legumain synthesis.

<p>Illustration of A.C.NPs-legumain synthesis.</p

Characteristics of C.NPs-legumain and A.C.NPs- legumain in an acidic environment.

<p>(A) Nanoparticles were treated in different acidity levels (pH 1.8∼12) for 2 hours. C.NPs-legumain and A.C.NPs-legumain particle diameter and zeta potential measurements at 37°C. (B) Representative images of A.C.NPs at pH 1.5 (left, scale bar = 1µm) and pH 7.0 (right, scale bar = 100 nm). (C) FTIR spectra of A.C.NPs-legumain at pH 1.5 and pH 7.0.</p