Immortalization And Functional Studies Of Marmoset Hepatocytes

Objective: Liver is a pivotal organ in regulating many physiological processes, suchasglycogen storage, lipid metabolism, protein secretion and drug detoxification. Hepatocytes are the major cell type in the liver and thus play a key role in maintaining the xenobiotics metabolism,detoxificationand homoeostasis. Therefore, hepatocytes have been considered as an important cellular model for studying liver metabolism, pathogenesis, cell transplantation and drug screening. Rodent hepatocytes have been widely applied in drug screening, however most drugs failed in clinical trials due to genetic, anatomic and physiological differences between human and rodents. Therefore, primate hepatocytesis gradually emerging as a better model systemfor basic and clinical researches given their unique traits and similarity to human being genetically, anatomically and physiologically. Common marmosets are new world primates and have been used as animal models for human diseases such as Parkinson's and other diseases. In particular, Common marmoset shows sensitivity to liver diseases and thus has been utilized successfully as HCV chimeras infection models in vivo. However, there is no suitable primate hepatocyte cell model available. In this study, we aim to establish a common marmoset hepatocyte cell line with the capability of proliferation, metabolism and repopulationin vitroandin vivo and thus provide a suitable hepatocyte cell model for elucidating the pathogenic mechanism of liver disease, drug screening and therapeutics.Methods: 1.Isolation and passage of Common marmoset primary hepatocytes: We isolated primary hepatocytes from 2-week-old liver derived from aborted common marmoset anddigested with collagenase IV. Subsequently, cells were seeded onto 6-well plate with hepatocyte maintenance medium consisting of Williams' E(Sigma) supplementedwith 20ng/ml HGF,20ng/ml EGF,1x Insulin-transferrin-sodium(GIBCO), 10%10099 FBS(GIBCO), 10?M dexamethasone, 10% P/S, 10% Glu, 10ng/ml nicotinamide, 75 mg bovine serum albumin and 10ng/ml nicotinamide. 2.Methodological optimization for immortalizing common marmoset hepatocytes: To obtain immortalizedcommon marmosethepatocytes,we transfected primaryhepatocyteswith SV40LT- or h TERT–expressing lentivirusesseparately or together and screened for positive clones with GFP and puromycin resistance. 3.Establishment of immortalized common marmoset hepatocyte cell lines: To verify the marmoset hepatocyte cell clones, we testedthe capacity of different clones to proliferate and the morphology after SV40 LT lentivirus transfection. 4. Examination of chromosome stability and tumorigenicity in marmoset hepatocyte cell clones: To confirm the intactness of chromosomes, we analyzed the karyotype and chromosome number with Giemsa's and DAPI staining. Also, we inoculated hepatocyte cells into immune-deficient nude mice subcutaneously to examine the tumorigenicity. 5. Gene expression profile analysis of hepatocyte cell clones: To verify the expression of hepatic genes in marmoset hepatocyte cell clones, we measured the liver-related gene expression at m RNA and protein levels with semi-quantitative RT-PCR and confocal microscopy. 6. Analysis of ultrastructure and basic physiological function of marmoset hepatocyte cell clones:Electron microscopy was used to analyze the ultrastructure including mitochondrion, hepatic glycogen and microvilli. Glycogen storage and lipid staining and ICG excretion were used to investigate in vitro function of marmoset hepatocyte clones. 7. Validation of primary metabolic and biliary excretion ability of marmoset hepatocyte cell clones:Semi-quantitative RT-PCR was used to measure levels of primary liver metabolism-related CYP gene expression prior to and after rifampin and 3-methylcholanthrene drug induction. Furthermore, the GFP-labelled CDFDA uptake and localization experiment was used to validate the biliary excretion functionin vitro. 8.In vivo functional studies of marmoset hepatocyte cell clones in FAH-/- mice: To test the repopulation and repair capability of the marmoset hepatocyte cell clone MHCL-1, we inoculated1x107 hepatocytes into the spleen of Fumarylacetoacetatehydrolase-deficient(FAH-/-) mice, a mouse model showing deficiency in tyrosine metabolism. The survival rate, liver function and body-weight were monitored to evaluate the effect. 9. Investigation of hepatic stem cell properties of marmoset hepatocyte cellclones:Immunoassaying of stem cell-related genes such as AFP and SOX9 and 3D gel culture were used to investigate the potential hepatic stem cell characters of marmoset hepatocyte clones. 10. Other studies on primary marmoset hepatocytes: Crispr-Cas9 system was used to knock-out the tumor suppressor gene TP53 in primary marmoset hepatocytes to examine whether TP53 knock-out can interfere with cell proliferation and tumorigenicity. Immune-deficient nude mice were used for examining the tumorigenicity of TP53 knock-out cells.Result: 1. Marmoset primary hepatocytes with normal epithelial morphology and limited proliferation capacitywere obtained. 2. The SV40 LT lentivirus over-expression system performed better than others in immortalizing marmoset primary hepatocytes. 3.MHCL-1 and MHCL-2 clones could proliferate more than 100 passages, demonstrating the indefinite proliferation capacity. The hepatocyte clones were involved in EMT during the immortalization as demonstrated by the expression of E-cadherin and a-SMA(EMT markers), but the morphology was maintained during passage. 4.Giemsa's and DAPI staining confirmed theintactness of chromosomes in the hepatocyte cell clones with no rearrangement or deletion and the number of chromosomes is 46, suggesting the hepatocyte cell clones are normal. In addition, the tumorigenicity experiments in nude mice showed that no tumor was formed in 5hepatocyte-transplanted mice, further supporting the hepatocyte cell clones are normal hepatocytes. 5.Semi-quantitative RT-PCR and confocal microscopic results showed the expression of typical hepatic gene including ALB, AAT and HNF4 A, though levels of epithelial cell-related genessuch as CK19,CK7 and CK18 were low, strengthening the conclusion that the immortalized hepatocyte clones are normal hepatocytes. 6.ICG uptake andsecretion, lipid or glycogen storage results demonstrated that the immortalized hepatocyte cell clones preserve the basic physiological function. 7.Examination on levels of CYP3A4, CYP1A1 and CYP1A2 before andafterrafampicin and 3-methcholanthrene induction indicated that the hepatocyte cell clones show near normal levels of CYP3A4, CYP1A1 and CYP1A2 expression and is responsive to drug induction as shown by real-time PCR results. 8.In vivo functional studies demonstrated that the marmoset hepatocyte cell clone MHCL-1 could repopulate the liver in the FAH mouse as demonstrated by theimproved liver function,prolongedlifespan and increased body-weight 2 months post-inoculation, compared with untreated FAH mice. Consistently, FAH and GFP immunostaining results further confirmed the repair and repopulation capability of MHCL-1. 9. The semi-quantitative RT-PCR and confocal microscopic results showed the expression of stem cell gene such as AFP and SOX9. And also the collagen-based 3D gel culture induced the formation ofcholangiocytes. These results suggested that the hepatocyte cell clones are likely liver stem cells. 10. DNA sequencing and Westernblot results confirmed theknock-out of TP53 in marmoset primary hepatocytes and further cell transplantation experiments in nude mice indicated the presence of the teratoma-like structures, suggesting that the knock-out of TP53 can affect the hepatocyte differentiation. Conclusions: 1.Twonormal common marmosethepatocyte cell clones are established, showing the unlimited proliferation capacity, normal hepatic gene expression, and a correct number of chromosomes as well as the absence of tumorigenicity. 2. The common marmoset hepatocyte cell clonesdemonstrate typical liver physiology and the expression of CYP genes as well as the responsiveness to drug induction, and thus can be used for drug screening, pharmacology and toxicity test. 4.The common marmoset hepatocyte cell clones show the repopulationcapacityin vivo and can be used for cell transplantation studies. 5.The common marmoset hepatocyte cell clones show the expression of stem cell markers and can be used for further stem cell studies.