Reprogramming Of Primary Mouse Hepatocytes Into Insulin-Producing Cells By Transfection With Multicistronic Vectors

[Background]Diabetes mellitus (DM) is a set of clinical syndrome characterized by chronic elevated blood glucose (blood sugar) levels and has a variety of presentation. With the rapid development of economy, changes of lifestyle and aging of the population, the prevalence of diabetes rises rapidly in China. At present, the prevalence of diabetes is about9.7%, about90million in total. Furthermore, the prevalence of impaired glucose regulation (IGR) is close to15%. Diabetes has become the third biggest threat to human health of chronic noncommunicable diseases except malignant tumor and cardiovascular disease, which seriously affects the patients’quality of life and life span and has become a heavy burden of the whole country. How to control diabetes and its complications effectively is a worldwide problem in current medical field which is urgent to be solved.According to the existing data, the core in the pathogenesis of diabetes is considered as relative or absolute insufficiency of insulin caused by dysfunction, reduction or even deficiency of islet β cells, attributing to the damage of β cell. Diet controlling, physical exercise, oral medication and insulin injection are conventional therapies of DM. However, all the therapies mentioned can neither cure the disease, nor prevent its related complications. Therefore, it is real need to explore a new treatment to cure diabetes and its complications.Scientists developed pancreas transplantation, simultaneous pancreas-kidney transplantation and islet transplantation to replace the non-functioning islet cell since last century and they had been proved effective in certain degree. Once, it was believed that’Edmonton Protocol’was a promising treatment which can cure diabetes at the beginning of21th century. Nevertheless, wide clinical application of islet transplantation has been blocked by both scarcity of donor cells and requirement for life-long immunosuppressive therapy. At present, transformation or trans-differentiation from abundant autologous non-islet cells into insulin-producing cells (IPCs) via a safe and effective induction is recognized as a promising strategy for curing diabetes. Both domestic and foreign researchers have tried to use embryonic stem cells, adult stem cells for transformation or trans-differentiation. However, when used to regenerate IPCs, the embryonic stem cells confront with the tumorigenicity and ethical problems, and defects also exist in adult stem cells including the complicated process of isolation and purification and a relatively low differentiation rate. Recently, it is documented by some groups that hepatocytes can be trans-differentiated into IPCs which secrete insulin in a glucose-dependent manner by cell reprogramming technology, and result in amelioration of hyperglycemia in streptozotocin-treated mice. Advantages of this protocoll are as follow:firstly, on the phase of embryogenesis, the same as pancreatic isletβ cells, hepatocytes also derived from endoderm, conversion between hepatocytes and pancreatic islet β cells may therefore require fewer epigenetic changes; secondly, liver has a powerful capacity of repair and regeneration, and can serve as the source of cells for trans-differentiation. Nevertheless, most of the previous methods were based on viral vectors such as adenovirus, adeno-associated virus or lentivirus, whose process were complicated, without mentionging the risk of tumorigenicity and immune response. A non-viral approach for islet regeneration is needed. Plasmid pcDNA3.1(+) can be designed to a multicistronic vector, and co-express some forgien genes with a high efficiency. Compared with other non-viral technique, many desirable features such as less damage to target cells, easy to operate and stable can be found in this method. Recently, it is shown that among more than1100transcription factors associated with the development of pancreas, the specific combination of Pancreatic and duodenal homeobox factor1(Pdx-1), Neurogenin3(Ngn3) and Musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) was a most efficient precept in converting non-pancreatic β cells into IPCs. It was also comfirmed by our previous study that fetal liver cell line of mice BNL CL.2can be induced to display some features of pancreatic β cells by co-expressing Ngn3+Pdx-1+MafA via multicistronic vectors.In order to futher explore the feasibility of trans-differentiation from hepatocytes into IPCs for autologous transplantation to solve problems including scarcity of donor cells and immune rejection, we hypothesize that primary hepatocytes can be trans-differentiated into IPCs by co-expressing Ngn3+Pdx-1+MafA via multicistronic vectors. In this study, we first constructed the plasmids, and then utilized Lipofectamine2000to introduce the3factors into mice primary hepatocytes. The existence of IPCs was confirmed by genetic and protein analysis.[Objective]1. To construct plasmid vectors to co-express Ngn3, Pdx-1and MafA basing on= pcDNA3.1(+).2. To transfect the vectors into mice primary hepatocytes, and to comform the trans-differentiation from hepatocytes into IPCs.[Methods]1. Plasmid pcDNA3.1(+) was serve as backbone to construct vectors as follow: pcDNA3.1(+)-MafA ORF+ngn33’UTR+pdx-13’UTR, pcDNA3.1(+)-ngn3ORF+pdx-13’UTR, pcDNA3.1(+)-MafA ORF+ngn33’UTR, pcDNA3.1(+)-MafA ORF, pcDNA3.1(+)-ngn3ORF and pcDNA3.1(+)-pdx-1ORF.2. Primary hepatocytes were acquired by reverse in situ collagenase perfusion method and were cultured.3. There were7groups in our study, Lipofectamine2000was used to introduce the vectors into the primary hepatocytes.4. Detections of Real-time PCR, Western blotting and Enzyme-linked Immunosorbent Assay were conducted after transfection.[Results]1. Sequencing results shows that the target genes are cloned correctly which means we have constructed all the6vectors successfully.2. The primary hepatocytes were transfected by Lipofectamine2000and subsequently cultured. The transfection efficiency was30-40%.3. Reverse transcription PCR analysis indicates that gene expressed in the corresponding groups. Detetions of Real-time PCR, Western blotting show that hepatocytes transfected vector pcDNA3.1(+)-MafA ORF+ngn33’UTR+pdx-13’UTR were more similar to islet than the rest groups.4. It was proved by analysis of ELISA that insulin biosynthesis and secretion in multicistronic group was higher than other groups. [Conclusion]1. Basing on the pcDNA3.1(+), we constructed the plasmid carrying Pdx-1, Ngn3and MafA and another5vectors which were used in control groups successfully.2. It was more effectively to induce the trans-differentiation from primary hepatocytes into IPCs by transfecting the vector pcDNA3.1(+)-MafA ORF+ngn33’UTR+pdx-13’UTR with a non-viral procedure.