Expression of c-kit receptor in human cholangiocarcinoma and in vivo treatment with imatinib mesilate in chimeric mice

AIM： To investigate the c-kit expression in biliary tract cancer cell lines and histological sections from patients with extrahepatic cholangiocarcinoma （CC） and to evaluate the efficacy of in vitro and in vitro treatment with imatinib mesilate. METHODS： The protein expression of c-kit in the human biliary tract cancer cell lines Mz-ChA-2 and EGI-1 and histological sections from 19 patients with extrahepatic CC was assessed by immunoblotting, immunocytochemistry, and immunohistochemistry. The anti-proliferative effect of imatinib mesilate on biliary tract cancer cell lines Mz-ChA-2 and EGI-1 was studied in vitro by automated cell counting. In addition, immunodeficient NMRI mice （Taconic^TM） were subcutaneously injected with 5 × 10^6 cells of cell lines MzChA-2 and EGI-1. After having reached a tumour volume of 200 mm^3, daily treatment was started intraperitoneally with imatinib mesilate at a dose of 50 mgikg or normal saline （NS）. Tumor volume was calculated with a Vernier caliper. After 14 d, mice were sacrificed with tumors excised and tumor mass determined.RESULTS： Immunoblotting revealed presence of c-kit in Mz-ChA-2 and absence in EGI-1 cells. Immunocytochemistry with c-kit antibodies displayed a cytoplasmatic and membraneous localization of receptor protein in Mz-ChA-2 cells and absence of c-kit in EGI-1 cells, c-kit was expressed in 7 of 19 （37%） extrahepatic humanCC tissue samples, 2 showed a moderate and 5 a rather weak immunostaining. Imatinib mesilate at a low concentration of 5 μmoliL caused a significant growth inhibition in the c-kit positive cell line Mz-ChA-2 （31%）, but not in the c-kit negative cell line EGI-1 （0%） （P〈0.05）. Imatinib mesilate at an intermediate concentration of 10 μmoliL inhibited cellular growth of both cell lines （51% vs 57%）. Imatinib mesilate at a higher concentration of 20 μmoliL seemed to have a general toxic effect on both cell lines. The IC50 values were 9.7 μmoliL and 11 μmoliL, respectively. After 14 d of in vitro treatment with imatinib mesilate, using the chimeric mouse model, c-kit positive Mz-ChA-2 tumors had a significantly reduced volume and mass as compared to NS treatment （P〈 0.05）. In contrast to that, treatment of mice bearing c-kit negative EGI-1 tumors did not result in any change of tumor volume and mass as compared to NS treatment. CONCLUSION： c-kit expression is detectable at a moderate to low protein level in biliary tract cancer. Imatinib mesilate exerts marked effects on tumor growth in vitro andin vitro dependent on the level of c-kit expression.

Efficient production of chimeric mice from embryonic stem cells injected into 4-to 8-cell and blastocyst embryos

Background： Production of chimeric mice is a useful tool for the elucidation of gene function. After successful isolation of embryonic stem （ES） cell lines, there are many methods for producing chimeras, including co-culture with the embryos, microinjection of the ES ceils into pre-implantation embryos, and use of tetraploid embryos to generate the full ES-derived transgenic mice. Here, we aimed to generate the transgenic ES cell line, compare the production efficiency of chimeric mice and its proportion to yield the male chimeric mice by microinjected ES cells into 4- to 8-cell and blastocysts embryos with the application of Piezo-Micromanipulator （PMM）, and trace the fate of the injected ES cells. Results： We successfully generated a transgenic ES cell line and proved that this cell line still maintained pluripotency. Although we achieved a satisfactory chimeric mice rate, there was no significant difference in the production of chimeric mice using the two different methods, but the proportion of the male chimeric mice in the 4- to 8-cell group was higher than in the blastocyst group. We also found that there was no tendency for ES cells to aggregate into the inner cell mass using in vitro culture of the chimeric embryos, indicating that they aggregated randomly. Conclusions： These results showed that the PMM method is a convenient way to generate chimeric mice and microinjection of ES cells into 4- to 8-cell embryos can increase the chance of yielding male chimeras compared to the blastocyst injection. These results provide useful data in transgenic research mediated by ES cells.

Liver chimeric mice with tupaia hepatocyte transplantation as an animal model for hepatitis B virus infection and antiviral therapy

The human liver chimeric mouse is a milestone animal model for hepatitis B virus(HBV)infection.Such mice with primary human hepatocyte(PHH)transplantation are adequate to support chronic HBV infection for several weeks and to evaluate antiviral drugs.However,the drawbacks of PHHs include lack of available donors,poor expansion in vitro and ethical issues that limit the application of human liver chimeric mice,necessitating the search for alternatives.Here,we transplanted primary tupaia hepatocytes(PTHs)into the livers of immunodeficient mice and achieved high liver chimerism within six weeks.These tupaia liver chimeric mice are adequate to support chronic infection of the four common HBV genotypes A,B,C and D for 36 weeks,as well as evaluate of antiviral drugs,including hepatitis B immune globulin(HBIG),monoclonal antibody and nucleoside analogues(NAs),for preventative therapy and treatment post infection.In conclusion,the tupaia liver chimeric mouse model provides a convenient,efficient and stable animal model for chronic HBV infection and long-term drug evaluation.

Establishment of Embryonic Stem Cell Lines Derived from Outbred Mouse Embryos and Production of Chimeras

The aim of the present investigation was to determine if embryonic stem (ES) cells could be isolated from outbred mouse embryos (KM) and if chimeras could be producedly using outbred ES cells. Three ES cell lines,designated KE1, KE2, and KE5,were isolated from 5 Kunming albino blastocysts. Normal diploid composition of these cell lines was above 70%. By using C57BL/6J and 615 blastocysts as host embryos, one chimera was obtained in living pups. It was shown for the first time that chimeras can be produced by using outhred ES cells. This work implies that to establish ES cell lines from other animal embryos is possible. More interestingly, white color dots from ES cells on the coat of this chimeric mouse enlarged a lot after half a year, indicated that ES cells were inhibited by cells from outbred mouse or the cells of outbred mouse grew vigiously.

Pluripotent stem cells secrete Activin A to improve their epiblast competency after injection into recipient embryos

It is not fully clear why there is a higher contribution of pluripotent stem cells （PSCs） to the chimera produced by injection of PSCs into 4-cell or 8-cell stage embryos compared with blastocyst injection. Here, we show that not only embryonic stem cells （ESCs） but also induced pluripotent stem cells （iPSCs） can generate F0 nearly 100% donor cell-derived mice by 4-cell stage embryo injection, and the approach has a ＂dose effect＂. Through an analysis of the PSC-secreted proteins, Activin A was found to impede epiblast （EPI） lineage development while promoting trophectoderm （TE） differentiation, resulting in replacement of the EPI lineage of host embryos with PSCs. Interestingly, the injection of ESCs into blastocysts cultured with Activin A （cultured from 4-cell stage to early blastocyst at E3.5） could increase the contribution of ESCs to the chimera. The results indicated that PSCs secrete protein Activin A to improvetheir EPI competency after injection into recipient embryos through influencing the development of mouse early embryos. This result is useful for optimizing the chimera production system and for a deep understand- ing of PSCs effects on early embryo development.