| Subject Resource:This work was supported by grants from the Major Project of Chinese National Programs for Fundamental Research and Development (973Program)(2010CB945502).Background and Objectives, significanceIPS cells refer to the somatic cells reprogramming to pluripotent stem cells. These stem cells and ES cells are very similar in morphology, epigenetics, gene expression profiles and specific differentiation potential, but they are better than the ES cells in the clinical application of immune rejection, limited sources and ethical issues. IPS cells will develop into a complete individual if a certain number of them are transplanted into the reproductive system of mice, which has been proved by some experiments. IPS cells are one of the most promising seed cells in the field of cell therapy and tissue or organ regeneration.IPS cells were first produced in2006by Takahashi and Yamanaka. They imported genes related to the versatility into mouse fibroblasts by retroviruses (Oct4, Sox2, c-Myc and Klf4), then the cells were screened by expression of the factor Fbx15. Eventually, the pluripotent stem cells with the certain characteristics of embryonic stem cells were obtained and named "induced pluripotent stem cells". In June2007, the same group obtained Nanog+-iPS cell lines which were closer to embryonic stem cells by using Nanog instead of Fbx15.The iPS technology is developed rapidly after2008, it has been further improved and perfected. In view of the c-Myc oncogene, importing c-Myc significantly increased incidence of tumors, which could hinder its clinical application. In order to avoid the risk of cancer caused by retroviral vector, Shinya Mi laboratory and the Konrad Hawke Stringer laboratory of Harvard University successfully prepared the iPS cells of the non-viral integrated mice in2009. In addition, scholars in Harvard University increased efficiency more than100times in somatic cell reprogramming to iPS cells by adding specific compounds (such as baerpuluo acid). These results suggested the possibility of restructuring through the pure chemical substances, which provided a more secure, practical technology platform of application iPS cells for cell therapy in the future.With the advent of the iPS cell technology, the pathological process of some genetic diseases or degenerative diseases can be accurately observed in culture dishes. Recently, Park et al. have successfully used iPS technology to establish10stem cell lines which were derived from human genetic or degenerative diseases, including ADA-SCI D, HD, PD, JDM and so on. Meanwhile, there were a large number of experiments and research on iPS cells differentiating into cells with specific functions in vitro, they can be used to study the treatment of retinopathy, the cochlear nerve, cardiovascular disease, brain aspects and diabetes. However, till now there are few reports on whether iPS cells could participate in liver regeneration or repair, as well as how to work. Chinese scholars have specifically induced iPS cells in vitro, under simulated physiological conditions these cells were differentiated into functional hepatocytes following the normal developmental path directly. However, in order to avoid the higher cost, the lower induction and the differences between in vivo and in vitro, foreign scholars injected the iPS cells to the embryos of pregnant FAH mice whose performance was liver failure and renal tubular necrosis due to the lack of enzyme. It was found that serum indexes of hepatic function and the livers of the FAH mice injected with the iPS cells were normal after the birth; the mice without injection were died of the hepatic failure, structural disorder of liver tissue and extensive necrosis of hepatocytes in the6th week after birth. This experiment successfully demonstrated that in vivo the iPS cells derived from hepatocytes can be differentiated into functional hepatocytes. However, the transplanted cells were mature, functional hepatocytes derived from the hepatic issue which came from the complete individual developed from the iPS cell but not the iPS cells themselves or EBs. Moreover, the cost of this experiment was higher than the former.In view of above, mouse model of acute liver injury was established by the combined use of D-(+)-Galactosamine hydrochloride (D-GaIN), lipopolysaccharide (LPS) and partial hepatectomy, which were more serious than single use. Then EBs derived from iPS cells were transplanted into those mice. The serum indexes of hepatic function (ALT, AST, ALB), the distribution of iPS cells in vivo (GFP)and the differentiation of iPS cells in vivo(ALB and CK18) were detected to test whether EBs derived from iPS cell would be induced into functional hepatocytes and whether these hepatocytes would involve in liver regeneration or not.Methods1. Established experimental model of chemical liver injury in mice.A total of50male BALB/c mice were equally and randomly assigned to2groups. D-GaIN/LPS/1/3PH group:All mice were pretreated with2intraperitoneal injections of D-GaIN (500mg/kg) and LPS (50ug/kg) within an interval of1hour.1/3PH group:All mice were pretreated with2intraperitoneal injections of saline (500mg/kg) and saline (50μg/kg) within an interval of1hour,2. One-third partial hepatectomy in mice.24hours later, all mice were performed with1/3partial hepatectomy. All mice were anaesthetized with aether, and were operated with a vertical incision under the xiphoid0.5～1.0cm, deligating the left lateral lobe of liver along the root of ligation and then resection. Put the side of the ligation into the abdominal. Suture the abdominal cavity and skin, disinfect with Iodine.3. Calculation of liver weight/body weight (LW/BW) and liver regeneration rate.There are five lobes of liver in BALB/c mice and the weight of left lateral lobe accounts for35%-41%total liver weight. Thus, the left lateral lobectomy of liver is equal to one-third partial hepaectomy theoretically. 4. Observation of the hepatic morphology and the distribution of the mouse iPSC-derived EBs.On the third, seven and fourteen day after one-third partial hepatectomy, all mice were given euthanasia. Removed their livers and wigh. Fixed the livers conventionally and then embed in paraffine, made into4μm olefin slice up. The liver cell injury was observed at3,7and14days with hematoxylin-eosin staining. Trace the fate of the EBs in vivo by the liver frozen sections at day40after transplantation.5. Immunofluorescence of BrdU, CK19and C-kit.Injection of BrdU:All mice were pretreated with2intraperitoneal injections of BrdU (75mg/kg) within an interval of8hours.24hours later, euthanasia were given. Follow the instruction of the immunohistochemical specification. Restain these slices by hematoxylin before looking at it through the microscope.(BrdU:redyed by eosin and hematoxylin).6. Double staining immunofluorescence of GFP and CK18/ALB.Liver frozen sections were recovered antigen with citrate buffer and blocked in PBST before primary antibody incubations in PBST overnight at4℃. After washing in PBS, sections were incubated with PE-conjugated streptavidin for90minutes at37℃. After further washing, biotinylated secondary antibodies was added for overnight at4℃before extensive washing in PBS and mounting in Prolong Gold with4,6-diamidino-2-phenylindole (DAPI). The antibodies used were as follows:anti-Oct4, anti-SSEA4, anti-EGFP, anti-albumin, and anti-CK18.Results1. The liver weight/body weight (LW/BW) and liver regeneration rate indicated that the liver gross structure basically restored to normal, whose quality had close to preoperative level at postoperative day9. The liver weight had almost recovered to normal at day14and then developed according to the normal growth rhythm, the residual liver instead increased. Instead of to a new part of liver, the excisional part turned to a light yellow hyperplastic tissue. However, the liver weight in mice of D-GaIN/LPS/1/3PH group still less than normal at day14. 2. Hematoxylin-eosin staining demonstrated that significant degeneration changes in hepatic cells in the D-GaIN/LPS-treated group, whose hepatic sinusoid and central venous presented hyperemia, Kupffer’s cell increased. And yet, only mild inflammatory reaction was observed in liver in the non-treated group after partial hepatectomy.3. BrdU staining showed that hepatocyte proliferation at day7was significantly detected in the non-treated group, but few in the D-GaIN/LPS-treated group. CK19and C-kit immunohistochemistry demonstrated that visible oval cells proliferation, which appeared in portal area and differentiated into hepatic cells to recover the injured liver, were seen in mice of D-GaIN/LPS-treated group. However, there were fewer C-kit and CK18positive cells in the livers of1/3PH group until14days.4. The liver function parameter, such as AST、ALT and ALB, returned to normal at40days after EBs transplantation. We can see the focal shape distribution of GFP positive cells in the livers of EBs-transplanted group. Moreover, the hepatic frozen section double staining immunofluorescence demonstrated that there were some GFP and CK18/ALB double positive cells around the membrane of hepatocytes.Conclusion1. The result proved that we had successfully established astrocyte the mouse D-GaIN/LPS/1/3PH model.2. D-GaIN and LPS have a inhibitory effect for mature hepatocytes after partial hepatectomy.3. The result proved that after hepatectomy, given of D-GaIN and LPS, the residual liver cells that exhibited incomplete differentiation and impaired proliferation fail to restore liver function, turned to stimulate the endogenous or exogenous stem cells proliferation, differentiation and recovered the injuried liver, which can provide a selective growth advantage for transplanted cells.4. We found that, the EBs derived from mouse iPSC can differentiated into hepatocyte-like cells40days after transplantation, showing spotty distribution in liver frozen section and expressing CK18and albumin. However, Some limitations of these EBs should be noticed. In our research, we found teratoma was formed in three of EBs-injected mice. Therefore, the safety of the mouse iPSC-derived EBs still needed to be improved. |
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