SPP1 as a risk factor for patients with acute on chronic liver failure undergoing liver transplantation.

BACKGROUND: Acute on chronic liver failure (ACLF) is characterized by systemic inflammation and significant mortality, calling for accurate assessment due to the diverse prognosis of liver transplantation (LT). METHODS: 8 patients with ACLF and 4 normal controls (NC) underwent peripheral blood mononuclear cells (PBMCs) transcriptomics, whereas 9 patients with ACLF and 3 NC had hepatic CD45+ T cells transcriptomics. Thecandidateindicatorfoundinthetranscriptomicswas confirmedbya retrospective cohort (n = 137) and one prospective cohort (n = 68). RESULTS: Transcriptomics revealed significant differentially expression genes (DEGs) and bioprocesses related to the PBMCs and hepatic CD45+ T cells. Secreted phosphoprotein 1 (SPP1) was identified as a potential indicator for ACLF patients receiving LT, which was supported by evidence from the cross-sectional cohorts. As the condition of ACLF got worse, so did SPP1 levels, which were associated with liver failure and coagulation failure. SPP1 levels prior to LT were considerably greater in non-survivors of ACLF within 90 days than that in survivors. In the derivation cohort and validation cohort, ACLF patients with elevated SPP1 levels had significantly shorter cumulative survival durations than those with low SPP1 levels, P = 0.02 and P < 0.001, respectively. The SPP1-MELD and SPP1-chronic liver failure consortium (CLIF-C) ACLF scores had comparatively larger areas under the receiver operating characteristic curves (AUCs) than MELD (P = 0.0388) and CLIF-C ACLF (P = 0.045). CONCLUSIONS: The circulating SPP1 showed promise as a predictor for ACLF patients receiving LT, which demonstrated the need for tracking the clinical outcome of LT.

Liver-derived extracellular vesicles from patients with hepatitis B virus-related acute-on-chronic liver failure impair hepatic regeneration by inhibiting on FGFR2 signaling via miR-218-5p.

BACKGROUND: Impaired liver regeneration in hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) patients is closely related to prognosis; however, the mechanisms are not yet defined. Liver-derived extracellular vesicles (EVs) may be involved in the dysregulation of liver regeneration. Clarifying the underlying mechanisms will improve the treatments for HBV-ACLF. METHODS: EVs were isolated by ultracentrifugation from liver tissues of HBV-ACLF patients (ACLF_EVs) after liver transplantation, and their function was investigated in acute liver injury (ALI) mice and AML12 cells. Differentially expressed miRNAs (DE-miRNAs) were screened by deep miRNA sequencing. The lipid nanoparticle (LNP) system was applied as a carrier for the targeted delivery of miRNA inhibitors to improve its effect on liver regeneration. RESULTS: ACLF_EVs inhibited hepatocyte proliferation and liver regeneration, with a critical role of miR-218-5p. Mechanistically, ACLF_EVs fused directly with target hepatocytes and transferred miR-218-5p into hepatocytes, acting by suppressing FGFR2 mRNA and inhibiting the activation of ERK1/2 signaling pathway. Reducing the level of miR-218-5p expression in the liver of ACLF mice partially restored liver regeneration ability. CONCLUSION: The current data reveal the mechanism underlying impaired liver regeneration in HBV-ACLF that promotes the discovery of new therapeutic approaches.

Multi-region sequencing with spatial information enables accurate heterogeneity estimation and risk stratification in liver cancer.

BACKGROUND: Numerous studies have used multi-region sampling approaches to characterize intra-tumor heterogeneity (ITH) in hepatocellular carcinoma (HCC). However, conventional multi-region sampling strategies do not preserve the spatial details of samples, and thus, the potential influences of spatial distribution on patient-wise ITH (represents the overall heterogeneity level of the tumor in a given patient) have long been overlooked. Furthermore, gene-wise transcriptional ITH (represents the expression pattern of genes across different intra-tumor regions) in HCC is also under-explored, highlighting the need for a comprehensive investigation. METHODS: To address the problem of spatial information loss, we propose a simple and easy-to-implement strategy called spatial localization sampling (SLS). We performed multi-region sampling and sequencing on 14 patients with HCC, collecting a total of 75 tumor samples with spatial information and molecular data. Normalized diversity score and integrated heterogeneity score (IHS) were then developed to measure patient-wise and gene-wise ITH, respectively. RESULTS: A significant correlation between spatial and molecular heterogeneity was uncovered, implying that spatial distribution of sampling sites did influence ITH estimation in HCC. We demonstrated that the normalized diversity score had the ability to overcome sampling location bias and provide a more accurate estimation of patient-wise ITH. According to this metric, HCC tumors could be divided into two classes (low-ITH and high-ITH tumors) with significant differences in multiple biological properties. Through IHS analysis, we revealed a highly heterogenous immune microenvironment in HCC and identified some low-ITH checkpoint genes with immunotherapeutic potential. We also constructed a low-heterogeneity risk stratification (LHRS) signature based on the IHS results which could accurately predict the survival outcome of patients with HCC on a single tumor biopsy sample. CONCLUSIONS: This study provides new insights into the complex phenotypes of HCC and may serve as a guide for future studies in this field.

Clinical analysis of deceased donor liver transplantation in the treatment of hepatocellular carcinoma with segmental portal vein tumor thrombus: A long-term real-world study.

Background: Hepatocellular carcinoma (HCC) patients with portal vein tumor thrombus (PVTT) have conventionally been regarded as a contraindication for liver transplantation (LT). However, the outcomes of deceased donor liver transplantation (DDLT) in patients with segmental PVTT remain unknown. The aim of this study is to evaluate the feasibility and effectiveness of DDLT in the treatment of HCC with segmental PVTT. Methods: We retrospectively analyzed 254 patients who underwent DDLT for HCC in our institution from January 2015 to November 2019. To assess the risks of PVTT, various clinicopathological variables were evaluated. Overall (OS) and recurrence-free survival (RFS) analyses based on different PVTT types were performed in HCC patients. Results: Of the 254 patients, a total of 46 patients had PVTT, of whom 35 had lobar PVTT and 11 had segmental PVTT in second-order branches or below. Alpha-fetoprotein (AFP) level, tumor maximal diameter, histological grade, micro-vascular invasion (MVI), RFS, and OS were significantly different between the control and PVTT groups. Lobar PVTT was associated with unfavorable 5-year RFS and OS compared with MVI group (28.6% and 17.1%, respectively). Instead, no significant difference was observed between the segmental PVTT and MVI group in terms of 5-year RFS and OS (RFS: 36.4% vs. 40.4%, p=0.667; OS: 54.5% vs. 45.1%, p=0.395). Further subgroup analysis showed segmental PVTT with AFP levels ≤100 ng/ml presented significantly favorable RFS and OS rates than those with AFP level >100 ng/ml (p=0.050 and 0.035, respectively). Conclusions: In summary, lobar PVTT remains a contraindication to DDLT. HCC patients with segmental PVTT and AFP level ≤100 ng/ml may be acceptable candidates for DDLT.

HNF4α overexpression enhances the therapeutic potential of umbilical cord mesenchymal stem/stromal cells in mice with acute liver failure.

Human umbilical cord mesenchymal stem/stromal cells (hUMSCs) hold promise for treating acute liver failure (ALF). Here, we investigated the therapeutic effect of hUMSCs overexpressing hepatocyte nuclear factor 4α (HNF4α), a transcription factor important for maintaining hepatocyte identity and hepatic functions, in ALF, compared with hUMSCs without overexpression of HNF4α (CON-hUMSCs). The cells were administered into mice via the tail vein for 24 h before exposure to lipopolysaccharide/d-galactosamine (LPS/d-GalN) for 6 h by intraperitoneal injection. HNF4α-hUMSCs ameliorated liver injury in ALF better than CON-hUMSCs. The overexpression of HNF4α enhanced the transcription of interleukin (IL)-10 and promoted M2 macrophage polarization through the IL-10/signal transducer and activator of transcription 3 (STAT3) pathway. HNF4α-hUMSCs could exert a more pronounced therapeutic effect on ALF than CON-hUMSCs, providing a novel therapy for ALF.

A survey of optimal strategy for signature-based drug repositioning and an application to liver cancer.

Pharmacologic perturbation projects, such as Connectivity Map (CMap) and Library of Integrated Network-based Cellular Signatures (LINCS), have produced many perturbed expression data, providing enormous opportunities for computational therapeutic discovery. However, there is no consensus on which methodologies and parameters are the most optimal to conduct such analysis. Aiming to fill this gap, new benchmarking standards were developed to quantitatively evaluate drug retrieval performance. Investigations of potential factors influencing drug retrieval were conducted based on these standards. As a result, we determined an optimal approach for LINCS data-based therapeutic discovery. With this approach, homoharringtonine (HHT) was identified to be a candidate agent with potential therapeutic and preventive effects on liver cancer. The antitumor and antifibrotic activity of HHT was validated experimentally using subcutaneous xenograft tumor model and carbon tetrachloride (CCL4)-induced liver fibrosis model, demonstrating the reliability of the prediction results. In summary, our findings will not only impact the future applications of LINCS data but also offer new opportunities for therapeutic intervention of liver cancer.

DJ-1 Deficiency in Hepatocytes Improves Liver Ischemia-Reperfusion Injury by Enhancing Mitophagy.

BACKGROUND & AIMS: DJ-1 is universally expressed in various tissues and organs and is involved in the physiological processes in various liver diseases. However, the role of DJ-1 in liver ischemia-reperfusion (I/R) injury is largely unknown. METHODS: In this study, we first examined the DJ-1 expression changes in the liver tissues of mice and clinical donor after hepatic I/R by both quantitative polymerase chain reaction and Western blotting assays. Then we investigated the role of DJ-1 in I/R injury by using a murine liver I/R model. RESULTS: We demonstrated that DJ-1 down-regulation in both human and mouse liver tissues in response to I/R injury and Dj-1 deficiency in hepatocytes but not in myeloid cells could significantly ameliorate I/R induced liver injury and inflammatory responses. This hepatoprotective effect was dependent on enhanced autophagy in Dj-1 knockout mice, because inhibition of autophagy by 3-methyladenine and chloroquine could reverse the protective effect on hepatic I/R injury in Dj-1 knockout mice. CONCLUSIONS: Dj-1 deficiency in hepatocytes significantly enhanced mitochondrial accumulation and protein stability of PARKIN, which in turn promotes the onset of mitophagy resulting in elevated clearance of damaged mitochondria during I/R injury.

Co-encapsulation of HNF4α overexpressing UMSCs and human primary hepatocytes ameliorates mouse acute liver failure.

BACKGROUND: Acute liver failure (ALF) is a complicated condition that is characterized by global hepatocyte death and often requires immediate liver transplantation. However, this therapy is limited by shortage of donor organs. Mesenchymal stem cells (MSCs) and hepatocytes are two attractive sources of cell-based therapies to treat ALF. The combined transplantation of hepatocytes and MSCs is considered to be more effective for the treatment of ALF than single-cell transplantation. We have previously demonstrated that HNF4α-overexpressing human umbilical cord MSCs (HNF4α-UMSCs) promoted the expression of hepatic-specific genes. In addition, microencapsulation allows exchange of nutrients, forming a protective barrier to the transplanted cells. Moreover, encapsulation of hepatocytes improves the viability and synthetic ability of hepatocytes and circumvents immune rejection. This study aimed to investigate the therapeutic effect of microencapsulation of hepatocytes and HNF4α-UMSCs in ALF mice. METHODS: Human hepatocytes and UMSCs were obtained separately from liver and umbilical cord, followed by co-encapsulation and transplantation into mice by intraperitoneal injection. LPS/D-gal was used to induce ALF by intraperitoneal injection 24 h after transplantation. In addition, Raw 264.7 cells (a macrophage cell line) were used to elucidate the effect of HNF4α-UMSCs-hepatocyte microcapsules on polarization of macrophages. The protein chip was used to define the important paracrine factors in the conditioned mediums (CMs) of UMSCs and HNF4α-UMSCs and investigate the possible mechanism of HNF4α-UMSCs for the treatment of ALF in mice. RESULTS: HNF4α-UMSCs can enhance the function of primary hepatocytes in alginate-poly-L-lysine-alginate (APA) microcapsules. The co-encapsulation of both HNF4α-UMSCs and hepatocytes achieved better therapeutic effects in ALF mice by promoting M2 macrophage polarization and reducing inflammatory response mainly mediated by the paracrine factor HB-EGF secreted by HNF4α-UMSCs. CONCLUSIONS: The present study confirms that the co-encapsulation of HNF4α-UMSC and hepatocytes could exert therapeutic effect on ALF mainly by HB-EGF secreted by HNF4α-UMSCs and provides a novel strategy for the treatment of ALF.

Blocking GSDMD processing in innate immune cells but not in hepatocytes protects hepatic ischemia-reperfusion injury.

Pyroptosis, a proinflammatory form of programmed cell death, plays important roles in the pathogenesis of many diseases. Inflammasome activation, which has been shown in hepatic ischemia-reperfusion injury (IRI), is demonstrated to be closely associated with pyroptosis, indicating that pyroptosis may occur and perform functions in hepatic IRI. However, there is no direct evidence showing the function of pyroptosis in hepatic IRI. In this study, by detecting the pyroptosis markers, we showed that pyroptosis may be induced during hepatic IRI. Furthermore, by adopting caspase-1 inhibitors, we showed that inhibition of pyroptosis could significantly ameliorate liver injury and suppress inflammatory response during hepatic IRI. Interestingly, caspase-1 inhibitors have no protective effects on in vitro hepatocytes under hypoxic reoxygenation condition. To investigate pyroptosis induced in which specific cell types may affect hepatic IRI, we generated hepatocyte-specific Gsdmd-knockout (Hep-Gsdmd-/-) and myeloid-specific Gsdmd-knockout (LysmCre+Gsdmdf/f) mice. Functional experiments showed that compared to control mice (Gsdmdf/f), there were alleviated liver injury and inflammation in LysmCre+Gsdmdf/f mice, but not in AlbCre+Gsdmdf/f mice. In parallel in vitro studies, cytokine expression and production decreased in bone-marrow-derived macrophages and Kupffer cells from LysmCre+Gsdmdf/f mice compared to their controls. Our findings demonstrated that pyroptosis in innate immune cells aggravates hepatic IRI and implied that hepatic IRI could be protected by blocking pyroptosis, which may become a potential therapeutic target in the clinic.

Cholangiocarcinoma: anatomical location-dependent clinical, prognostic, and genetic disparities.

BACKGROUND: Anatomical location is considered in diagnostic and therapeutic approaches of cholangiocarcinoma (CCA). However, disparities and its extents in proportion of surgical candidates, prognostic factors, prognostic genetic networks, susceptibility for lymph node dissection, and disease stage at diagnosis remain to be confirmed. METHODS: A total of 11,710 patients with cholangiocarcinoma from Surveillance, Epidemiology, and End Results Cancer Registries (SEER) and 45 CCA patients with paired tumor and normal specimens from The Cancer Genome Atlas were studied. Kaplan-Meier estimation, Cox proportional hazards regression, Pearson's correlation, comparison between anatomical location (distal, intrahepatic, and perihilar)-dependent CCAs, differential expressive gene stratification, potential interactive gene identification, and confirmation on pathways of the prognostic networks were carried out. RESULTS: Survival outcomes were most favorable in the distal type, followed by perihilar and intrahepatic types, but postsurgical prognosis was slightly higher in intrahepatic type compared to perihilar type. Distant historic stage at diagnosis was noticed in intrahepatic type. Significant prognostic factors and their hazards ratios were dependent to the anatomical location. In addition, lymph node dissection provided significant survival benefits in perihilar type only. Furthermore, prognosis-predictive genes, as well as potential processes and pathways, were significantly among the anatomical location-dependent types that the genes barely overlapped. CONCLUSIONS: There are disparities in almost all aspects among distal, intrahepatic, and perihilar CCAs. Anatomical location needs to be considered in treatment, prognostic estimation, identifying targets, and developing therapeutic approaches for CCA.

Hepatocyte nuclear factor 4A improves hepatic differentiation of immortalized adult human hepatocytes and improves liver function and survival.

Immortalized human hepatocytes (IHH) could provide an unlimited supply of hepatocytes, but insufficient differentiation and phenotypic instability restrict their clinical application. This study aimed to determine the role of hepatocyte nuclear factor 4A (HNF4A) in hepatic differentiation of IHH, and whether encapsulation of IHH overexpressing HNF4A could improve liver function and survival in rats with acute liver failure (ALF). Primary human hepatocytes were transduced with lentivirus-mediated catalytic subunit of human telomerase reverse transcriptase (hTERT) to establish IHH. Cells were analyzed for telomerase activity, proliferative capacity, hepatocyte markers, and tumorigenicity (c-myc) expression. Hepatocyte markers, hepatocellular functions, and morphology were studied in the HNF4A-overexpressing IHH. Hepatocyte markers and karyotype analysis were completed in the primary hepatocytes using shRNA knockdown of HNF4A. Nuclear translocation of ß-catenin was assessed. Rat models of ALF were treated with encapsulated IHH or HNF4A-overexpressing IHH. A HNF4A-positive IHH line was established, which was non-tumorigenic and conserved properties of primary hepatocytes. HNF4A overexpression significantly enhanced mRNA levels of genes related to hepatic differentiation in IHH. Urea levels were increased by the overexpression of HNF4A, as measured 24h after ammonium chloride addition, similar to that of primary hepatocytes. Chromosomal abnormalities were observed in primary hepatocytes transfected with HNF4A shRNA. HNF4α overexpression could significantly promote ß-catenin activation. Transplantation of HNF4A overexpressing IHH resulted in better liver function and survival of rats with ALF compared with IHH. HNF4A improved hepatic differentiation of IHH. Transplantation of HNF4A-overexpressing IHH could improve the liver function and survival in a rat model of ALF.

Overexpression of hepatocyte nuclear factor 4α in human mesenchymal stem cells suppresses hepatocellular carcinoma development through Wnt/ß-catenin signaling pathway downregulation.

Mesenchymal stem cells (MSCs) hold promise as cellular vehicles for the delivery of therapeutic gene products because they can be isolated, expanded, and genetically modified in vitro and possess tumor-oriented homing capacity in vivo. (1) Hepatocyte nuclear factor 4α (HNF4α) is a dominant transcriptional regulator of hepatocyte differentiation and hepatocellular carcinogenesis (HCC). (2,3) We have previously demonstrated that overexpression of HNF4α activates various hepatic-specific genes and enhances MSC differentiation. (4) However, the extent that overexpression of HNF4α in MSCs influences HCC progression has yet to be examined. Here we sought to investigate what effect MSCs overexpressing HNF4α (MSC-HNF4α) have on human hepatoma cells in vitro and in vivo. Conditioned medium collected from in vitro MSC-HNF4α cultures significantly inhibited hepatoma cell growth and metastasis compared with controls. Additionally, nude mice administered MSC-HNF4α exhibited significantly smaller tumors compared with controls in vivo. Immunoblot analysis of HCC cells treated with MSC-HNF4α displayed downregulated ß-catenin, cyclinD1, c-Myc, MMP2 and MMP9. Taken together, our results demonstrate that MSC-HNF4α inhibits HCC progression by reducing hepatoma cell growth and metastasis through downregulation of the Wnt/ß-catenin signaling pathway.

[Cryopreservation of microencapsulated human hepatocytes].

OBJECTIVE: To establish a stable method of isolation, culture and cryopreservation of adult primary hepatocytes to provide potential hepatocyte resources for therapeutic usage in hepatocyte transplantation and bioartificial liver support systems for the treatment of acute and chronic liver diseases,and for experimental usage as an in vitro model of the liver. METHODS: Adult hepatocytes from 20 human donors undergoing partial hepatectomy were isolated using a two-step extracoporeal collagenase perfusion technique.Seven preincubation time points (2h,6h,12h,24h,36h,48h and 72h) were selected for optimization.After pre-incubation at 4 degrees C for 12-24h in HepatoZYME-SFM (the optimal condition),hepatocytes were microencapsulated using alginate-poly-L-lysine-alginate microcapsules,transferred to a complete medium containing 10% dimethyl sulphoxide and immediately placed into an isopropanol progressive freezing container for overnight freezing at -80 degrees C followed by immersion in liquid nitrogen the next day.During the post-thawing culture period,the cells were tested for albumin secretion,urea synthesis,cell cycling,transcription and protein synthesis (measuring mRNA and protein levels),and the morphological structure and pathology,for comparison with the features from before microencapsulated cryopreservation (PMC). RESULTS: The viability and plating efficiency of the hepatocytes isolated using the two-step extracorporeal collagenase perfusion technique were 75.0+/-4.6% and 72.0+/-6.0%,respectively.The pre-incubation times of 12h and 24h (viability:61.4+/-4.8% and 62.0+/-5.6%; plating efficiency:3.2+/-5.8% and 62.6+/-3.6%,respectively) showed significantly higher albumin secretion than all other time points tested (F =40.3,all P less than 0.05).Compared with the immediate cryopreservation (immediately frozen control) hepatocytes,the PMC hepatocytes showed significantly better transcription and protein synthesis and higher albumin secretion and urea levels.The PMC group did not show a significantly different level of albumin production from the directly cultured hepatocytes (culture day 2:ll9.2ng/ml vs.131.36ng/ml,P =0.051; day 3:110ng/ml vs.120.4ng/ml,P=0.063; day 4:98.2ng/ml vs.109.8ng/ml,P more than 0.05).However,over culturing days 2,3 and 4,comparison of the PMC hepatocytes to the immediate cryopreservation hepatoeytes showed the former to have significantly higher secretion of albumin (119.2ng/ml vs.101.2ng/ml,110.0ng/ml vs.87.6ng/ml and 98.2ng/ml vs.73.8ng/ml; all P less than 0.05) and urea level (7.83 mug/ml vs.6.79 mug/ml,6.83 mug/ml vs.5.89 mug/ml and 5.85 mug/ml vs.4.83 mug/ml; all P less than 0.05).The post-thawed PMC hepatoeytes showed preservation of the morphological structure,while the immediate cryopreservation hepatocytes did not. CONCLUSION: The two-step extracorporeal collagenase perfusion technique after partial hepatectomy is a novel,simple,and reliable method for hepatocyte isolation.Pre-incubation at 4 degrees C for 12-24h before the microencapsulation cryopreservation allows for efficient recovery of functional and morphological integrity after thawing and provides viable hepatoeytes that may be useful for clinical applications in pharmacotoxicology,bioartificial liver therapy and cell therapy in humans.

Induction of highly functional hepatocytes from human umbilical cord mesenchymal stem cells by HNF4α transduction.

AIM: To investigate the differentiation potential of human umbilical mesenchymal stem cells (HuMSCs) and the key factors that facilitate hepatic differentiation. METHODS: HuMSCs were induced to become hepatocyte-like cells according to a previously published protocol. The differentiation status of the hepatocyte-like cells was examined by observing the morphological changes under an inverted microscope and by immunofluorescence analysis. Hepatocyte nuclear factor 4 alpha (HNF4α) overexpression was achieved by plasmid transfection of the hepatocyte-like cells. The expression of proteins and genes of interest was then examined by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) or real-time RT-PCR methods. RESULTS: Our results demonstrated that HuMSCs can easily be induced into hepatocyte-like cells using a published differentiation protocol. The overexpression of HNF4α in the induced HuMSCs significantly enhanced the expression levels of hepatic-specific proteins and genes. HNF4α overexpression may be associated with liver-enriched transcription factor networks and the Wnt/ß-Catenin pathway. CONCLUSION: The overexpression of HNF4α improves the hepatic differentiation of HuMSCs and is a simple way to improve cellular sources for clinical applications.

Role of BMSCs in liver regeneration and metastasis after hepatectomy.

Hepatocellular carcinoma (HCC), which develops from liver cirrhosis, is highly prevalent worldwide and is a malignancy that leads to liver failure and systemic metastasis. While surgery is the preferred treatment for HCC, intervention and liver transplantation are also treatment options for end-stage liver disease. However, the success of partial hepatectomy and intervention is hindered by the decompensation of liver function. Conversely, liver transplantation is difficult to carry out due to its high cost and the lack of donor organs. Fortunately, research into bone-marrow stromal cells (BMSCs) has opened a new door in this field. BMSCs are a type of stem cell with powerful proliferative and differential potential that represent an attractive tool for the establishment of successful stem cell-based therapy for liver diseases. A number of different stromal cells contribute to the therapeutic effects exerted by BMSCs because BMSCs can differentiate into functional hepatic cells and can produce a series of growth factors and cytokines capable of suppressing inflammatory responses, reducing hepatocyte apoptosis, reversing liver fibrosis and enhancing hepatocyte functionality. Additionally, it has been shown that BMSCs can increase the apoptosis rate of cancer cells and inhibit tumor metastasis in some microenvironments. This review focuses on BMSCs and their possible applications in liver regeneration and metastasis after hepatectomy.

In vitro analysis of cryopreserved alginate-poly-L-lysine-alginate-microencapsulated human hepatocytes.

BACKGROUND: The availability of well-characterized human hepatocytes that can be frozen and thawed will be critical for cell therapy. We addressed whether human hepatocytes can recover after microencapsulated cryopreservation and investigated whether these cryopreserved microencapsulated hepatocytes can be used for clinical applications. METHODS: Adult hepatocytes of 18 separate donors were isolated with a two-step extracorporeal collagenase perfusion technique. After pre-incubation at 4 degrees C for 12-24 h in HepatoZYME-SFM, hepatocytes were microencapsulated using alginate-poly-L-lysine-alginate microcapsules. The microencapsulated hepatocytes were transferred to a complete medium containing 10% dimethyl sulphoxide. They were immediately placed into an isopropanol progressive freezing container at -80 degrees C overnight and immersed in liquid nitrogen the next day. During the post-thawing culture period, albumin secretion, urea synthesis, cell cycle, mRNA and protein levels, as well as the morphology and pathology structure of pre-incubation before microencapsulated cryopreservation (PMC) groups were analysed. RESULTS: Compared with the immediate cryopreservation (IC) groups, we found significant improvement in the mRNA and protein levels in the attached cells, and higher secretion of albumin and urea levels after thawing. In the attached cultured human cryopreserved/thawed hepatocytes from the PMC group, albumin production was not significantly different from those of the direct culture groups on days 2, 3 and 4. The preserved morphology in the PMC group compared with the IC group was obvious. CONCLUSIONS: The results of the present study suggested recovery of the functional and morphological integrity of human hepatocytes after pre-incubation at 4 degrees C for 12-24 h before microencapsulated cryopreservation. These studies offer the possibility for clinical applications in pharmacotoxicology, bioartificial liver and cell therapy in humans.

A simple isolation and cryopreservation method for adult human hepatocytes.

OBJECTIVE: The objective of this study was to establish a stable method of isolation, culture and cryopreservation of adult primary hepatocytes to provide potential hepatocyte resources for the treatment of acute and chronic liver diseases by hepatocyte transplantation and bioartificial liver support systems, and for the use of hepatocytes as an in vitro model of the liver. METHODS: Adult hepatocytes of 20 separate donors were isolated with a two-step extracoporeal collagenase perfusion technique. Seven preincubation time points (2h, 6h, 12h, 24h, 36h, 48h and 72h) were selected, then the hepatocytes were transferred to HepatoZYME-SFM medium containing 10% FBS and 10% DMSO, and were immediately put into an isopropanol progressive freezing container at -80 degrees C overnight and immersed in liquid nitrogen the next day. During the postthawing culture period, viability, plating efficiency, albumin secretion and urea synthesis were analyzed. RESULTS: The viability and plating efficiency of hepatocytes after partial hepatectomy using two-step extracorporeal collagenase perfusion technique were 75.0+/-4.6% and 72.0+/-6.0% respectively. Preincubation at 4? for 12 hours or 24 hours proved to be the optimal time at which the albumin secretion was higher than at other time points (p<0.05). Compared to the immediate cryopreservation groups (IC), we also found significant improvement in viability (61.4+/-4.8%/62.0+/-5.6% vs. 53.4+/-4.2%, p<0.05), plating efficiency (63.2+/-5.8%/62.6+/-3.6% vs. 55.2+/-4.6%, p<0.05), albumin secretion and urea synthesis (p<0.05) at these time points. CONCLUSIONS: The two-step extracorporeal collagenase perfusion technique after partial hepatectomy provides a novel, simple, and reliable method for hepatocyte isolation. The results of the present study suggest that recovery of human hepatocytes after isolation preincubation at 4 degrees C for 12 hours to 24 hours prior to cryopreservation can obtain hepatocytes ideal for use in pharmacotoxicology, bioartificial liver and cell therapy research purposes.

Establishment of a three-dimensional co-culture system by porcine hepatocytes and bone marrow mesenchymal stem cells in vitro.

AIM: The application of porcine hepatocytes in liver support systems has been hampered by the short-term survival. Co-cultivation of hepatocytes with non-parenchymal cells may be beneficial for optimizing cell functions via heterotypic interactions. In this study, we present a new cultivation system of porcine hepatocytes and mesenchymal stem cells (MSCs) in a randomly distributed co-culture manner. METHODS: Mononuclear cells were isolated from bone marrow aspirate of swines (n = 3) by density gradient centrifugation. MSCs were characterized by flow cytometry with CD29, CD44, CD45 and CD90, respectively. Then freshly isolated hepatocytes were simultaneously inoculated with MSCs in a hepatocyte dominant manner. The morphological and functional changes of heterotypic interactions were characterized. RESULTS: Ninety percent MSCs of passage 3 were positive for CD29, CD44 and CD90, but negative for CD45. A rapid attachment and self-organization of three-dimensional hepatocyte aggregates were encouraged. The cell ultrastructure indicating heterotypic junctions remained similar to that of hepatocytes in vivo. Fluorescence microscopy further verified that MSCs served as a feeder layer for hepatocyte aggregates. Hepatocyte performance levels such as albumin secretion, urea synthesis and CYP3A1 induction were all significantly enhanced in co-culture group compared with hepatocyte homo-culture (P < 0.05). The best hepatic function levels were achieved on day 2 and moderately decreased in the following co-culture days. Moreover, the cell cycle of hepatocytes manifested the same trend in parallel to the enhancement of hepatocyte functionality. CONCLUSIONS: A three-dimensional co-culture system by porcine hepatocytes and bone marrow MSCs was for the first time established in vitro. Enhanced liver-specific functions make such a co-culture system a promising tool for tissue engineering, cell biology, and bioartificial liver devices.