Effect Of The Lentivirus-mediated Constitutive Expression Of Foxa2 And Hnf4a Upon The Heptic Differentiation Of Embryonic Stem Cells

Liver cirrhosis, hepatic failure and other end-stage liver diseases caused by factors like viral hepatitis are known as one of the major stubborn illnesses in clinical treatment. And liver transplantation is the only effective therapy at the present time, whose accessibility can be largely reduced by liver source scarcity and high surgery expense. However, a new promise for the treatment of the end-stage liver diseases can be fulfilled by the research on bio-artificial liver, hepatocyte transplantation and liver tissue engineering, if the biggest clinical bottleneck of an ineffective hepatocyte source will finally be eliminated. On the other hand, embryonic stem cells are expected to become a new source of liver cells as they process the characteristics of multi-differentiation, self-renewal and unlimited proliferation. So far, however, the strategies of the directional development and differentiation of embryonic stem cells into hepatocytes remain to be improved.With the new recognition of cell plasticity in recent years, we found more and more evidences showing that transcription factor plays a decisive role in the cell fate determination. Few key transcription factors can even directly promote the differentiation, dedifferentiation, and transdifferentiation of various types of cells. Therefore, it can be hypothesized that there are some specific transcription factors or some transcription-factor combinations capable of facilitating the committed differentiation of ESCs into hepatocytes. For instance, forkhead box A2 (Foxa2) and hepatocyte nuclear factor 4 alpha (Hnf4a) have been known as a significant group of two hepatic transcription factors starting to express during the early and middle stages of liver development. They play a pivotal role in developing the liver and maintaining the hepatic functions. But no report has been published on the influence of their constitutive co-expression upon the committed differentiation of ESCs into hepatocytes. In this article, therefore, the effect and related mechanism of the constitutive expression of Foxa2 and Hnf4a on the differentiation of embryonic stem cells into hepatocytes is explored. Hopefully it will provide a new model for us to obtain hepatocytes from ESCs.The purpose of this research is to introduce a fast and highly effective gene modification to ESCs. As ESCs have been known to be one of the cell groups difficult to be genetic modified, the transient gene modification methods like plasmid transfection and adenovirus are not applicable to ESCs. The third-generation lentivirus vectors might be a good choice for ESC genetic modification for they have the ability to knock the gene into the genome, and they are relatively safe and infective to quiescent cell. Yet problems still remain. For example, the third-generation lentivirus vectors are difficult to be packaged, ESCs are resistant to lentivirus, and gene silencing in the ESCs is prone to happen. Besides, ESC have to be maintained on the mouse embryonic fibroblast (MEF) supplied with a special culture medium containing the leukemia inhibitory factor (LIF). Therefore, during the genetic modification, the selection of positive clones and the preservation of ESC characteristics can be a big problem. Therefore, this paper makes a methodological study on the construction and packaging of the third-generation lentivirus vectors, and the lentivirus-mediated ESC genetic modification at the very beginning.During the research process, the third-generation lentivirus vectors were first constructed which ensured their suitability for fluorescent markers and also their favorability for the genetic modification and the stable expression in ESCs. Second, using the calcium phosphate transfection system and the lentivirus vector 2K7bsd-EFp-EGFP with enhanced green fluorescence, this research developed the method to package lentivirus in large quantity and with high efficiency, and then this method was employed to package the lentivirus particles containing Foxa2 or Hnf4a. Third, using the packaged lentivirus particles 2K7bsd-EFp-EGFP and the mouse embryonic stem cells CCE, this research developed the method of fast genetic modification to ESCs, and then this method was used to package ESCs with the constitutive expression of Foxa2 or Hnf4a. Lastly, utilizing the ESCs with foreign genes and the different inducing environments, this research explored the effect of the constitutive expression of Foxa2, Hnf4a and Foxa2/Hnf4a upon the hepatic differentiation of ESCs.The major findings of this research are listed as follows.1. A series of the third-generation lentiviral expression vectors 2K7bsd-EFp-EGFP, 2K7neo-EFp-Foxa2, 2K7bsd-EFp-Hnf4a, 2K7bsd-ALBep-EGFP were successfully constructed which ensured their suitability for fluorescent markers and also their favorability for the gene modification to and the stable expression in ESCs, by using elements including the third-generation lentivirus vectors 2K7bsd and 2K7neo, the human elongation factor 1 alpha promoter (EFp), the mouse albumin enhancer promoter (ALBep), the woodchuck hepatitis virus posttranscriptional response element (WPRE), cDNA ecoding the open reading frame (ORF) of human Foxa2 and human Hnf4a.2. Based on the calcium phosphate transfection method, 293FT cells, packaging plasmids (pLP1, pLP2, pLP/VSVG) and lentiviral expression vector 2K7bsd-EFp-EGFP were used to generate lentivirus, this research also optimized the density and quality of plasmids, the proportion of expression vector particles and packaging plasmids, the pH of transfection fluid, the status of 293FT cells, the transfection time, and the titer of lentivirus concentration at its harvest time. Then it developed a method of packaging lentiviral expression vectors with high efficiency and in large quantities. Finally, employing this lentivirus packaging method, a large amount of lentivirus particles 2K7bsd-EFp-EGFP, 2K7neo-EFp-Foxa2 and 2K7bsd-EFp-Hnf4a, were produced and packaged.3. This research developed a method of mass-producing, packaging and freezing mouse embryonic fibroblasts. The MEFs were also tested and proved supportive of ESCs. Its packaging was inexpensive and easy, thus good for the maintenance and culture of mouse ESCs.4. Using the lentivirus particles 2K7bsd-EFp-EGFP prepared beforehand, the lentivirus mediated mouse ESCs genetic modification was optimized from the aspects of the lentiviral transfection time and mode, the ESC culture medium, the drug screening time after transfection, the addition of feeder layer cells, the selection of clones. Thus, a fast and efficient method for mouse ESCs genetic modification was developed. Using the mouse ESCs genetic modification method and the lentivirus particles prepared beforehand, we carried out the genetic modification of ESCs, and successfully constructed the cell lines of CCE-EGFP, CCE-Foxa2, CCE-Hnf4a, and CCE-Foxa2/ Hnf4a, which also passed the tests of PCR and enzyme digestion, Western Blot and immunofluorescent detection.5. According to the results from the experiment of culturing CCE-Foxa2, CCE-Hnf4a, CCE-Foxa2/Hnf4a and those from the experiment of teratoma formation the ESCs transfected with foreign genes could still sustain their normal proliferation and morphological characteristics as they had. Transplanting the three types of cells into nude mice could all form a teratoma, where the cell tissues of the three embryonic layers could be observed with no significance difference between them.6. This research studied the effect of the Foxa2 and Hnf4a constitutive expression upon the hepatic differentiation of ESCs. After testing the four cell groups CCE, CCE-Foxa2, CCE-Hnf4a, CCE-Foxa2/ Hnf4a in three different inducing culture media, and after morphological observation, realtime fluorescent quantitative PCR analysis and immunofluorescent detection, it was found that there are no significant differentiation between the four cell groups in their differentiation of ESCs into hepatocytes if ESCs were given autonomy and not interfered by any inducer in vitro. However, if inducers like DMSO interfered, Foxa2 could significantly facilitate the differentiation of ESCs into hepatocytes,while Hnf4a failed to demonstrate any significant effect. Under the same condition, the constitutive co-expression of Foxa2 and Hnf4a could achieve some favorable effect upon the differentiation but not so significant an effect as that of Foxa2 working alone.In conclusion, to construct the required third-generation lentiviral expression vectors, this research firstly devised a fast and efficient mass-producing and packaging method based on the calcium phosphate transfection method. Then, by this packaging method, such lentivirus particles were prepared. Next, the fast lentivirus-mediated ESCs genetic modification was developed, and the ESCs lines which are stably expressive of target genes were also packaged. Lastly, these cells were used for the research in the effect of the constitutive expression of Foxa2 and Hnf4a upon the committed differentiation of ESCs into hepatocytes. Therefore, this dissertation has provided a reliable method for further research on the ESC gene modification, and has also demonstrated an innovative way of investigating the effect of other transcription factors upon the differentiation of ESCs into hepatocytes.