Xiaopeng Yu

Establishment and Characterization of Immortalized Porcine Hepatocytes for the Study of Hepatocyte Xenotransplantation

BACKGROUND In light of the critical shortage of donor livers, xenogeneic sources offer the best alternative to human hepatocytes for the treatment of acute liver failure. This study investigated whether a combination of simian virus 40 large T antigen (SV40 LT) and human telomerase catalytic subunit (hTERT) genes could immortalize primary porcine hepatocytes that could reverse acute liver failure (ALF) in rats. METHODS We cotransfected SV40 LT and hTERT genes into primary porcine hepatocytes to examine the features of the transfected cell lines. We characterized the potentially therapeutic effect of immortalized porcine hepatocytes in a rat model of ALF induced by 90% hepatectomy. RESULTS An immortalized porcine hepatocyte cell line, HepLi, was expanded by >250 passages. HepLi cells maintained the defining characteristics of primary porcine hepatocytes, including porcine albumin secretion, urea production, and diazepam metabolism. Intrasplenic transplantation of HepLi cells significantly improved liver function, and significantly prolonging the survival of rats with ALF. CONCLUSIONS Cotransfection of SV40 LT and hTERT immortalized primary porcine hepatocytes without tumorigenicity in vitro. The Immortalized porcine hepatocytes served as a potential cell resource for xenotransplantation.

Evaluation of a novel choanoid fluidized bed bioreactor for future bioartificial livers

AIM To construct and evaluate the functionality of a choanoid-fluidized bed bioreactor (CFBB) based on microencapsulated immortalized human hepatocytes. METHODS Encapsulated hepatocytes were placed in the constructed CFBB and circulated through Dulbeccos Modified Eagles Medium (DMEM) for 12 h, and then through exchanged plasma for 6 h, and compared with encapsulated cells cultivated under static conditions in a spinner flask. Levels of alanine aminotransferase (ALT) and albumin were used to evaluate the CFBB during media circulation, whereas levels of ALT, total bilirubin (TBil), and albumin were used to evaluate it during plasma circulation. Mass transfer and hepatocyte injury were evaluated by comparing the results from the two experimental conditions. In addition, the viability and microstructure of encapsulated cells were observed in the different environments. RESULTS The bioartificial liver model based on a CFBB was verified by in vitro experiments. The viability of encapsulated cells accounting for 84.6% ± 3.7% in CFBB plasma perfusion was higher than the 74.8% ± 3.1% in the static culture group (P < 0.05) after 6 h. ALT release from cells was 29 ± 3.5 U/L vs 40.6 ± 3.2 U/L at 12 h (P < 0.01) in the CFBB medium circulation and static medium culture groups, respectively. Albumin secretion from cells was 234.2 ± 27.8 μg/1 × 10(7) cells vs 167.8 ± 29.3 μg/1 × 10(7) cells at 6 h (P < 0.01), 274.4 ± 34.6 μg/1 × 10(7) cells vs 208.4 ± 49.3 μg/1 × 10(7) cells (P < 0.05) at 12 h, in the two medium circulation/culture groups, respectively. Furthermore, ALT and TBil levels were 172.3 ± 24.1 U/L vs 236.3 ± 21.5 U/L (P < 0.05), 240.1 ± 23.9 μmol/L vs 241.9 ± 31.4 μmol/L (P > 0.05) at 6 h in the CFBB plasma perfusion and static plasma culture groups, respectively. There was no significant difference in albumin concentration between the two experimental plasma groups at any time point. The microstructure of the encapsulated hepatocytes remained healthier in the CFBB group compared with the static culture group after 6 h of plasma perfusion. CONCLUSION The CFBB can function as a bioartificial liver based on a bioreactor. The efficacy of this novel bioreactor is promising for the study of liver failure.

Establishment and characterization of an immortalized human hepatic stellate cell line for applications in co-culturing with immortalized human hepatocytes.

Background and objective. The liver-specific functions of hepatocytes are improved by co-culturing hepatocytes with primary hepatic stellate cells (HSC). However, primary HSC have a short lifespan in vitro, which is considered a major limitation for their use in various applications. This study aimed to establish immortalized human HSC using the simian virus 40 large T antigen (SV40LT) for applications in co-culturing with hepatocytes and HSC in vitro. Methods. Primary human HSC were transfected with a recombinant retrovirus containing SV40LT. The immortalized human HSC were characterized by analyzing their gene expression and functional characteristics. The liver-specific functions of hepatocytes were evaluated in a co-culture system incorporating immortalized human hepatocytes with HSC-Li cells. Results. The immortalized HSC line, HSC-Li, was obtained after infection with a recombinant retrovirus containing SV40LT. The HSC-Li cells were longitudinally spindle-like and had numerous fat droplets in their cytoplasm as shown using electron microscopy. Hepatocyte growth factor (HGF), VEGF Receptor 1(Flt-1), collagen type Iα1 and Iα2 mRNA expression levels were observed in the HSC-Li cells by RT-PCR. Immunofluorescence staining showed that the HSC-Li cells were positive for α-smooth muscle actin (α-SMA), platelet-derived growth factor receptor-beta (PDGFR-β), vimentin, and SV40LT protein expression. The HSC-Li cells produced both HGF and transforming growth factor-beta1 (TGF-β1) in a time-dependent manner. Real-time PCR showed that albumin, CYP3A5, CYP2E1, and UGT2B7 mRNA expression generally increased in the co-culture system. The enzymatic activity of CYP1A2 under the co-culture conditions also generally increased as compared to the monoculture of immortalized human hepatocytes. Conclusions. We successfully established the immortalized human HSC cell line HSC-Li. It has the specific phenotypic and functional characteristics of primary human HSC, which would be a useful tool to develop anti-fibrotic therapies. Co-culturing with the HSC-Li cells improved the liver-specific functions of hepatocytes, which may be valuable and applicable for bioartificial liver systems.

Transporting carriers for intracellular targeting delivery via non-endocytic uptake pathways

Abstract To develop novel therapies for clinical treatments, it increasingly depends on sophisticated delivery systems that facilitate the drugs entry into targeting cells. Profound understanding of cellular uptake routes for transporting carriers promotes the optimization of performance in drug delivery systems. Although endocytic pathway is the most important part of cellular uptake routes for many delivery systems, it suffers the trouble of enzymatic degradation of transporting carriers trapped in endosomes/lysosomes. Therefore, it is desirable to develop alternative transporting methods for delivery systems via non-endocytic pathways to achieve more effective intracellular delivery. In this review, we summarize the literature exploring transporting carriers that mediate intracellular delivery via non-endocytic pathways to present the current research status in this field. Cell-penetrating peptides, pH (low) insertion peptides, and nanoparticles are categorized to exhibit their ability to directly transport various cargos into cytoplasm via non-endocytic uptake in different cell lines. It is hoped that this review can spur the interesting on development of drug delivery systems via non-endocytic uptake pathway.

Adipogenic placenta-derived mesenchymal stem cells are not lineage restricted by withdrawing extrinsic factors: developing a novel visual angle in stem cell biology.

Current evidence implies that differentiated bone marrow mesenchymal stem cells (BMMSCs) can act as progenitor cells and transdifferentiate across lineage boundaries. However, whether this unrestricted lineage has specificities depending on the stem cell type is unknown. Placental-derived mesenchymal stem cells (PDMSCs), an easily accessible and less invasive source, are extremely useful materials in current stem cell therapies. No studies have comprehensively analyzed the transition in morphology, surface antigens, metabolism and multilineage potency of differentiated PDMSCs after their dedifferentiation. In this study, we showed that after withdrawing extrinsic factors, adipogenic PDMSCs reverted to a primitive cell population and retained stem cell characteristics. The mitochondrial network during differentiation and dedifferentiation may serve as a marker of absent or acquired pluripotency in various stem cell models. The new population proliferated faster than unmanipulated PDMSCs and could be differentiated into adipocytes, osteocytes and hepatocytes. The cell adhesion molecules (CAMs) signaling pathway and extracellular matrix (ECM) components modulate cell behavior and enable the cells to proliferate or differentiate during the differentiation, dedifferentiation and redifferentiation processes in our study. These observations indicate that the dedifferentiated PDMSCs are distinguishable from the original PDMSCs and may serve as a novel source in stem cell biology and cell-based therapeutic strategies. Furthermore, whether PDMSCs differentiated into other lineages can be dedifferentiated to a primitive cell population needs to be investigated.

Efficient generation of functional hepatocyte-like cells from mouse liver progenitor cells via indirect co-culture with immortalized human hepatic stellate cells

BACKGROUND Differentiation of liver progenitor cells (LPCs) to functional hepatocytes holds great potential to develop new strategies for hepatocyte transplantation and the screening of drug-induced cytotoxicity. However, reports on the efficient and convenient hepatic differentiation of LPCs to hepatocytes are few. The present study aims to investigate the possibility of generating functional hepatocytes from LPCs in an indirect co-culture system. METHODS Mouse LPCs were co-cultured in Transwell plates with an immortalized human hepatic stellate cell line (HSC-Li) we previously established. The morphology, expression of hepatic markers, and functions of mouse LPC-derived cells were monitored and compared with those of conventionally cultured LPCs. RESULTS Co-culturing with HSC-Li cells induced differentiation of mouse LPCs into functional hepatocyte-like cells. The differentiated cells were morphologically transformed into hepatocyte-like cells 3 days after co-culture initiation. In addition, the differentiated cells expressed liver-specific genes and possessed hepatic functions, including glycogen storage, low-density lipoprotein uptake, albumin secretion, urea synthesis, and cytochrome P450 1A2 enzymatic activity. CONCLUSIONS Our method, which employs indirect co-culture with HSC-Li cells, can efficiently induce the differentiation of LPCs into functional hepatocytes. This finding suggests that this co-culture system can be a useful method for the efficient generation of functional hepatocytes from LPCs.

Development of a novel artificial liver support system

Background/introduction: Artificial liver support treatment is a promising alternative to liver transplantation. An ideal artificial liver support system (ALSS) should be a combination of a nonbiological liver (NBL) device and a bioreactor based bioartificial liver (BAL). Material and methods: A novel ALSS which can not only fulfill toxin-removal functions of NBL but also provide biotransformation and synthetic functions of BAL is constructed. The unique dual-chamber reservoir can improve the efficiency of material exchange. The funnel-shaped fluidized bed bioreactor can provide an ideal physiological environment for hepatocytes. Quick bubble handling function improves the security during treatment. The software design provides error correction function. Our control center is an industrial personal computer and most components are integrated via the RS485 buses. The whole control system consists of three parts: a pump drive module, a sensor network and a human-machine communication interface. To verify our design, we test the system on miniature pigs. Results: The system runs normally in all treatment modes and meets the clinical requirements. Functions of all components are verified. Conclusions: The system provides a reliable research platform for artificial liver support treatment.