Short and long term fate of human AMSC subcutaneously injected in mice

AIM:To study the ability of human adipose-derived mesenchymal stem cells(AMSCs)to survive over the short and long term,their biodistribution and their biosafety in vivo in tumor-prone environments.METHODS:We subcutaneously injected human AMSCs from different human donors into immunodeficient SCID mice over both short-(2 and 4 mo)and long-(17 mo)term in young,and aged tumor-prone mice.Presence of human cells was studied by immunohistochemistry and polymerase chain reaction analysis in all organs of injected mice.RESULTS:Subcutaneously injected AMSCs did not form teratomas at any time point.They did not migrate but remained at the site of injection regardless of animal age,and did not fuse with host cells in any organ examined.AMSCs survived in vivo for at least 17 mo after injection,and differentiated into fibroblasts of the subdermic connective tissue and into mature adipocytes of fat tissue,exclusively at the site of injection.CONCLUSION:Our results support the assertion that AMSC may be safe candidates for therapy when injected subcutaneously because of their long term inability to form teratomas.

Cloning and expression of aequorin genes from jellyfish Aequorea and characterization of aequorins activities

Two new aequorin genes,aeqxm and aeqxxm,were isolated from jellyfish Aequorea macrodactyla and Aequorea parva respectively,which are commonly found in the warmer waters on the coastal region of the East China Sea.The DNA sequences of the two genes have no introns and each one contains an ORF of 585 bp in full-length encoding a 195-aa protein.The two genes of aeqxm and aeqxxm share nucleotide homologies of 80.7%and 85.1%with AEVAQ440X respectively,and the corresponding proteins share amino acid homologies of 84.7%and 84.2%with AEVAQ440X.High amino acid homology was found between apoaeqxm and apoaeqxxm.The two genes were cloned into expression vector pTO-T7 respectively,and the expression yields amounted to 40%of the total protein in E.coli BL21.The activities of the two photoproteins were reconstituted by incubating the expressed apoproteins with coelenterazine f.In the presence of Ca ion,both of the regenerated aeqxm and aeqxxm exhibited an emission peak at the wave length of 470 nm.

Portal hypertension-related inflammatory phenotypes: From a vitelline and amniotic point of view

Prehepatic portal hypertension induces a splanchnic low-grade inflammatory response that could switch to high-grade inflammation with the development of severe and life-threatening complications when associated with chronic liver disease. The extraembryonic origin of the portal system maybe determines the regression to an extraembryonic phenotype, i.e., vitellogenic and amniotic, during the evolution of both types of portal hypertension. Thus, prehepatic portal hypertension, or compensated hypertension by portal vein ligation in the rat, is associated with molecular mechanisms related to vitellogenesis, where hepatic steatosis and splanchnic angiogenesis stand out. In turn, extrahepatic cholestasis in the rat induces intrahepatic portal hypertension, or decompensated hypertension, with ascites and hepatorenal syndrome. The splanchnic interstitium, the mesenteric lymphatic system, and the peritoneal mesothelium seem to create an inflammatory pathway that could have a key pathophysiological relevance in the production of ascites. The hypothetical comparison between the ascitic and the amniotic fluid also allows for translational investigation. The induced regression of the splanchnic system to extraembryonic functions by portal hypertension highlights the great relevance of the extraem-bryonic structures even during postnatal life.

Identification of EGF as an Important Regulator for Promoting CYP3A4 Expression in Human Embryonic Stem Cell-Derived Hepatocytes Using TALEN-Based Gene Targeting

Functional human hepatocytes are one of the most significant tools for studying drug absorption, distribution, metabolism and excretion/toxicity （ADME/Tox）, especially for applications in preclinical drug development （Sahi et al., 2010; Godoy et al., 2013）. They provide the closest in vitro model to the human liver and the only model that mimics the drug metabolic profiles found in vivo. However, these cells lose their metabolic function rapidly and dramatically during the in vitro culture process, which largely hinders their wider application in drug development （Sahi et al., 2010; Godoy et al., 2013）. To overcome this obstacle, it is important to regulate the activities of key genes which are responsible for the detoxification metabolic function of human hepatocytes.

Cyclophilins A and B oppositely regulate renal tubular epithelial cell phenotype

Restoration of kidney tubular epithelium following sublethal injury sequentially involves partial epithelial–mesenchymal transition(pEMT),proliferation,and further redifferentiation into specialized tubule epithelial cells(TECs).Because the immunosuppressant cyclosporine-A produces pEMT in TECs and inhibits the peptidyl-prolyl isomerase(PPIase)activity of cyclophilin(Cyp)proteins,we hypothesized that cyclophilins could regulate TEC phenotype.Here we demonstrate that in cultured TECs,CypA silencing triggers loss of epithelial features and enhances transforming growth factorβ(TGFβ)-induced EMT in association with upregulation of epithelial repressors Slug and Snail.This pro-epithelial action of CypA relies on its PPIase activity.By contrast,CypB emerges as an epithelial repressor,because CypB silencing promotes epithelial differentiation,prevents TGFβ-induced EMT,and induces tubular structures in 3D cultures.In addition,in the kidneys of CypB knockout mice subjected to unilateral ureteral obstruction,inflammatory and pro-fibrotic events were attenuated.CypB silencing/knockout leads to Slug,but not Snail,downregulation.CypB support of Slug expression depends on its endoplasmic reticulum location,where it interacts with calreticulin,a calcium-buffering chaperone related to Slug expression.As CypB silencing reduces ionomycin-induced calcium release and Slug upregulation,we suggest that Slug expression may rely on CypB modulation of calreticulin-dependent calcium signaling.In conclusion,this work uncovers new roles for CypA and CypB in modulating TEC plasticity and identifies CypB as a druggable target potentially relevant in promoting kidney repair.

Biocompatibility and internalization of molecularly imprinted nanoparticles

Molecularly imprinted polymers （MIP） are receiving increasing attention thanks to their robustness, stability, and inexpensive manufacture compared with their bio-analogues such as antibodies. The molecular imprinting process can be defined as the generation of molecular recognition sites in a synthetic polymer. The template-derived sites created within a polymeric matrix allow MIPs （often referred as plastic antibodies） to selectively recognize and bind to the target molecule. Therefore, MIPs can be used in sensors and in separation and diagnostics. Owing to their size and functional properties, MIP nanoparticles （NPs） can potentially be used in biomedicine, but comprehensive analysis of their interaction with cells and in vitro toxicological tests must be performed first. Herein, we report the synthesis of bare and core-shell imprinted NPs using an innovative solid-phase approach and the toxicological evaluation of such NPs in different cell lines （HaCaT, MEFs, HT1080, and macrophages）. We also evaluated the influence of the protein corona on particle stability, the internalization of NPs in cells, and the influence of various surface coatings. Studies on the metabolic effects of imprinted NPs on fibroblasts showed that bare MIPs do not alter cell metabolism, whereas some issues arise when specific particle coatings are used. Furthermore, in vitro cytokine release studies revealed that macrophages were not activated in the presence of the MIPs evaluated in this study. The results suggest that MIP NPs are biocompatible, paving the way for their in vivo application.

Rapid generation of gene-targeted EPS-derived mouse models through tetraploid complementation

One major strategy to generate genetically modified mouse models is gene targeting in mouse embryonic stem(ES)cells,which is used to produce gene-targeted mice for wide applications in biomedicine.However,a major bottleneck in this approach is that the robustness of germiine transmission of gene-targeted ES cells can be significantly reduced by their genetic and epigenetic instability after long-term culturing,which impairs the efficiency and robustness of mouse model generation.Recently,we have established a new type of pluripotent cells termed extended pluripotent stem(EPS)cells,which have superior developmental potency and robust germline competence compared to conventional mouse ES cells.In this study,we demonstrate that mouse EPS cells well maintain developmental potency and genetic stability after long-term passage.Based on gene targeting in mouse EPS cells,we established a new approach to directly and rapidly generate gene-targeted mouse models through tetraploid complementation,Haibo Li and Chaoran Zhao contributed equally to this work.Electronic supplementary material The online version of this article(https://doi.org/10.1007/s13238-018-0556-1)contains supplementary material,which is available to authorized users.which could be accomplished in approximately 2 months.Importantly,using this approach,we successfully constructed mouse models in which the human interleukin 3(IL3)or interleukin 6(IL6)gene was knocked into its corresponding locus in the mouse genome.Our study demonstrates the feasibility of using mouse EPS cells to rapidly generate mouse models by gene targeting,which have great application potential in biomedical research.