Nan You

The differentiation of MSCs into functional hepatocyte-like cells in a liver biomatrix scaffold and their transplantation into liver-fibrotic mice.

Hepatocytes derived from mesenchymal stem cells (MSCs) hold great potential for cell-based therapies for liver diseases. The cell-based therapies are critically dependent on the hepatic differentiation of the MSCs with a high efficiency and on a considerable scale. Recent results have shown that decellularized organs provide a three-dimensional extracellular matrix for the lineage restriction of stem cell maturation. In this study, we compared the cell proliferation and hepatic differentiation of murine MSCs in a biomatrix scaffold from rat liver and in the presence and absence growth factors (GF) with a two-dimensional substrate. In the absence or presence of GF, the dynamic cultured scaffold (DCS) stimulated the MSCs to express endodermal and hepatocyte-specific genes and proteins associated with improved functions, and the cells exhibited the ultrastructural characteristics of mature hepatocytes. When transplanted into CCl(4)-injured mice, the cells pretreated with a combination of the DCS and GF exhibited increased survival, liver function, engraftment into the host liver and further hepatic differentiation. The paracrine effect of the transplanted cells on hepatic stellate cells and native hepatocytes played a key role in the treatment of the liver pathology. These studies define an effective method that facilitates the hepatic differentiation of MSCs exhibiting extensive functions and support further research into the use of a decellularized liver matrix as a bioscaffold for liver tissue engineering.

MicroRNAs involved in neoplastic transformation of liver cancer stem cells

BackgroundThe existence of cancer stem cells in hepatocellular carcinoma (HCC) has been verified by characterizing side population (SP) cells based on efflux of Hoechst 33342 dye from stem cells. Recent advances in microRNA (miRNA) biology have revealed that miRNAs play an important role in embryonic development and tumorigenesis. However, it is still unclear which miRNAs participate in the neoplastic transformation of liver cancer stem cells (LCSCs) during hepatocarcinogenesis.MethodsTo identify the unique set of miRNAs differentially regulated in LCSCs, we applied SP sorting to primary cultures of F344 rat HCC cancer cells treated with diethylnitrosamine (DEN) and normal syngenic fetal liver cells, and the stem-like characteristics of SP cells were verified through detecting expression of CD90.1, AFP and CK-7. Global miRNA expression profiles of two groups of SP cells were screened through microarray platform.ResultsA total of 68 miRNAs, including miR-10b, miR-21, miR-470*, miR-34c-3p, and let-7i*, were identified as overexpressed in SP of HCC cells compared to fetal liver cells. Ten miRNAs were underexpressed, including miR-200a* and miR-148b*. These miRNAs were validated using stem-loop real-time reverse transcriptase polymerase chain reaction (RT-PCR).ConclusionsOur results suggest that LCSCs may have a distinct miRNA expression fingerprint during hepatocarcinogenesis. Dissecting these relationships will provide a new understanding of the function of miRNA in the process of neoplastic transformation of LCSCs.

The Significance of Notch1 Compared with Notch3 in High Metastasis and Poor Overall Survival in Hepatocellular Carcinoma

Background The prognosis for patients with hepatocellular carcinoma (HCC) is poor, and the mechanisms underlying the development of HCC remain unclear. Notch1 and Notch3 may be involved in malignant transformation, although their roles remain unknown. Materials and Methods HCC tissues were stained with anti-Notch1 or -Notch3 antibody. The migration and invasion capacities of the cells were measured with transwell cell culture chambers. RT-PCR was used to measure the expression of Notch1 and Notch3 mRNA. Additionally, western blot analysis was used to assess the protein expression of Notch1, Notch3, CD44v6, E-cadherin, matrix metalloproteinase-2 (MMP-2), MMP-9, and urokinase-type plasminogen activator (uPA). RNA interference was used to down-regulate the expression of Notch1 and Notch3. Cell viability was assessed using MTT. Results Based on immunohistochemistry, high Notch1 expression was correlated with tumor size, tumor grade, metastasis, venous invasion and AJCC TNM stage. High Notch3 expression was only strongly correlated with metastasis, venous invasion and satellite lesions. Kaplan-Meier curves demonstrated that patients with high Notch1 or Notch3 expression were at a significantly increased risk for shortened survival time. In vitro, the down-regulation of Notch1 decreased the migration and invasion capacities of HCC cells by regulating CD44v6, E-cadherin, MMP-2, MMP-9, and uPA via the COX-2 and ERK1/2 pathways. Down-regulation of Notch3 only decreased the invasion capacity of HCC cells by regulating MMP-2 and MMP-9 via the ERK1/2 pathway. Conclusions Based on the migration and invasion of HCC, we hypothesize that targeting Notch1 may be more useful than Notch3 for designing novel preventive and therapeutic strategies for HCC in the near future.

Prognostic significance of metadherin overexpression in hepatitis B virus-related hepatocellular carcinoma.

Metadherin (MTDH), which is an HIV-1 or TNF-α-inducible transcript, has a crucial role in several types of cancer by regulating multiple cellular signaling processes. However, to date, the role of MTDH in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) is still unclear. In the present study, we detected MTDH expression in normal liver, chronic hepatitis B and HBV-related HCC tissues. The data showed that MTDH expression levels were elevated in the hepatitis B tissues and especially in the HBV-related HCC tissues compared to normal liver tissues. There was a trend for gradually increased MTDH expression from normal liver tissue to hepatitis B and HBV-related HCC tissues. Furthermore, a statistical analysis revealed that MTDH expression significantly correlated with the American Joint Committee on Cancer (AJCC, 7th edition) stage (P=0.020), T classification (P=0.007), N classification (P=0.044), vascular invasion (P=0.006) and histological differentiation (P=0.020) in the HBV-related HCC patients. In addition, patients with high MTDH levels had shorter survival times compared to those with low MTDH expression (P=0.001). Taken together, these results suggest that MTDH could be a potential prognostic marker for overall survival and tumor progression and a chemotherapeutic target in HBV-related HCC patients.

Differences in the Properties and Mirna Expression Profiles between Side Populations from Hepatic Cancer Cells and Normal Liver Cells

Aims Because hepatic cancer stem cells (HCSCs) are believed to derive from the conversion of hepatic normal stem cells (HNSCs), the identification of the differences that distinguish HCSCs from HNSCs is important. Methods The HCC model was established in F344 rats by DEN induction. Using FACS analysis, side population cells from HCC (SP-HCCs) were isolated from the epithelial-like cells of HCC tissues, and the side population cells from normal liver (SP-NLCs) were isolated from syngeneic normal liver cells. The expression of stem cell markers was detected in both freshly isolated and amplified subpopulations. After induction with HGF, the differentiation of each subpopulation was analyzed by detection of early and late liver markers. In vivo, the biological characteristics of SP-HCCs and SP-NLCs were analyzed by repairing injured livers or forming tumors in nude mice. In addition, the expression of miRNAs was examined in both populations by miRNA array and QRT-PCR. Results SP-NLCs and SP-HCCs were 4.30±0.011% and 2.100±0.010% of the whole population, respectively. Both SP-NLCs and SP-HCCs displayed greater expression of stem cell markers (CD133 and EpCAM) than NSP-NLCs and NSP-HCCs, respectively (P<0.01), both after fresh isolation and amplification. Upon HGF induction, SP-NLCs generated many ALB positive cells and few CK-7 positive cells, but NSP-NLCs could generate only ALB positive cells. In contrast, SP-HCCs gave rise to only AFP positive cells. As few as 5×105 SP-NLCs were capable of repairing liver injury, while the same number of NSP-NLCs could not repair the liver. Furthermore, only 1×104 SP-HCCs were necessary to initiate a tumor, while NSP-HCCs could not form a tumor. Compared to SP-NLCs, 68 up-regulated and 10 down-regulated miRNAs were present in SP-HCCs (P<0.01). Conclusion Based on the decisive roles of some miRNAs in the genesis of HCSCs, miRNAs may contribute to the different characteristics that distinguish SP-HCCs from SP-NLCs.

Notch is the key factor in the process of fetal liver stem/progenitor cells differentiation into hepatocytes.

Cell transplantation is efficient method to therapy end‐stage liver disease (ESLD). How to punctually induce stem cell differentiation into hepatocyte is still a challenge. Notch plays important roles in embryonic development and cell differentiation. However, during the differentiation process from fetal liver stem/progenitor cells (FLSPCs) to mature hepatocytes, the contribution of Notch, especially which Notch receptor is primarily responsible, is unknown. First, specific Notch receptor responsible for FLSPCs differentiation was identified. On both tissue level and cell level, we found that Notch3 was the only receptor greater expressed in liver tissue at embryonic day (ED) 14 and FLSPCs, compared with the adult liver and BRL cells, respectively. Second, morphological phenotypic and functional aspects were analyzed to evaluate whether Notch inhibition by GSIs (γ‐secretase inhibitors, inhibitor of Notch) promotes the differentiation of FLSPCs into hepatocytes. Results showed that N‐[N‐(3, 5‐Difluorophenacetyl)‐L‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT) as GSIs was able to induce FLSPCs differentiation into hepatocytes. The differentiated FLSPCs showed similar morphology to mature hepatocytes, expressed hepatic markers indicative of a mature developmental stage, and displayed similar functionality to mature hepatocytes. The differentiation efficiency by GSIs was similar to that by hepatocyte growth factor (HGF) induction. More specifically, as the differentiation of FLSPCs progressed towards hepatocytes, the expression of Notch3 was gradually down‐regulated, consistent with the down‐regulation of other stem cell markers. These findings imply that Notch3 may not only be a regulator of FLSPCs differentiation into hepatocytes, but also be a potential marker of FLSPCs.

Tg737 inhibition results in malignant transformation in fetal liver stem/progenitor cells by promoting cell-cycle progression and differentiation arrest.

Cancer stem/progenitor cells (CSPCs) may originate from the malignant transformation of normal stem cells. However, the mechanism by which normal stem cells undergo such transformation is not understood. Our previous studies provided evidence that Tg737 may play an important role in carcinogenesis of liver stem cells. In this study, we investigated the role of Tg737 in the malignant transformation of fetal liver stem/progenitor cells (FLSPCs). We inhibited Tg737 in FLSPCs using short hairpin RNA (shRNA). The microscopic observations of freshly purified Tg737 normal FLSPCs (nFLSPCs) and Tg737‐silent FLSPCs (sFLSPCs), which showed high expression levels of stem cell markers, revealed no significant morphological changes in sFLSPCs. Following RNAi of Tg737, the mRNA and protein levels of sFLSPCs decreased by 81.81% and 80.10% as shown by PCR, Western blot and immunocytochemistry analyses. Excluding apoptosis‐related effects, we found that silencing of Tg737 resulted in enhanced cell proliferation through promoting cell‐cycle progression via upregulation of cyclin D1 and cyclin B expression (P < 0.05). Silencing of Tg737 also resulted in significant arrest of cell differentiation (P < 0.05), stable expression of both albumin (ALB) and alpha fetoprotein (AFP) (P > 0.05) and quiescent ultrastructure. Assessment of cell malignant traits by transwell migration assays and by growth of xenograft tumors in athymic mice showed that reduced expression of Tg737 greatly promoted cell invasion and hepatocarcinogenesis of FLSPCs (P < 0.05). This work shows that inactivation of Tg737 may play an important role in malignant transformation of FLSPCs.

Loss of heterozygosity of the tumor suppressor gene Tg737 in the side population cells of hepatocellular carcinomas is associated with poor prognosis

Analysis of loss of heterozygosity (LOH) is a useful method for finding genetic alterations in tumor and precancerous lesion tissues. In this study, we analyzed LOH of the tumor suppressor gene Tg737 in side population cells of human hepatocellular carcinomas. Side population cells were sorted and identification by flow cytometry from suspensions of hepatocarcinoma or normal liver cells generated from 95 hepatocellular carcinoma and normal tissues, respectively. DNA was extracted from the two groups of side population cells and peripheral blood specimens. Five microsatellite markers on the Tg737 gene were used to analyze the frequency of loss of heterozygosity in the side population cells of the hepatocellular carcinoma. Twenty-four (25.30%) tumor samples had a large deletion in more than three microsatellite markers. The highest frequency of loss of heterozygosity was observed with the G64212 marker (78.75%) and the SHGC-57879 marker (75.95%). Statistical analysis of the correlation between loss of heterozygosity of Tg737 and clinicopathological features indicated a strong correlation between the two markers associated with the highest frequency of loss of heterozygosity and survival. The results indicate that loss of heterozygosity of the tumor suppressor gene Tg737 may play an important role in the carcinogenetic mechanism of liver cancer stem cells. In addition, the independent association between loss of heterozygosity at the SHGC-57879 and G64212 markers and worsened short-term survival in patients could be used as a novel prognostic predictor. Further studies of side population cells may contribute to the establishment of novel therapeutic strategies for hepatocellular carcinoma.

Swainsonine inhibits growth and potentiates the cytotoxic effect of paclitaxel in hepatocellular carcinoma in vitro and in vivo

Swainsonine, an extract from Astragalus membranaceus, exhibits broad inhibition of growth and pro-apoptotic activity in a number of tumor types. However, the underlying mechanism involved remains unclear. To investigate the effects and mechanisms of swainsonine on hepatocellular carcinoma (HCC), we performed experiments on HepG2, SMCC7721, Huh7 and MHCC97-H human hepatoma and HL-7702 human hepatocyte cells. We observed that swainsonine significantly inhibited the viability of human hepatoma cells in a dose- and time-dependent manner, but did not affect human hepatocytes. Due to their highly proliferative and tumorigenic nature, we selected MHCC97-H cells as a model system to examine. Swainsonine significantly inhibited MHCC97-H cell growth by causing cell cycle arrest at the G0/G1 phase and the induction of apoptosis. Blockage of G0/G1 phase was accompanied by a decrease in cyclins (D1 and E) and cyclin-dependent kinases (Cdk2 and Cdk4) and an increase in the Cdk inhibitors p21 and p27. Furthermore, swainsonine enhanced the apoptosis of MHCC97-H cells with the induction of the upregulation of Bax and the downregulation of Bcl-2, whereas the expressionof Fas and Fas-L remained almost unchanged. These changes were accompanied by the enhanced cytoplasmic accumulation of nuclear factor κB (NF-κB) with a concomitant decrease in the nuclear fraction. Importantly, swainsonine also potentiated the cytotoxic effects of paclitaxel in vitro and in vivo, in part, by restricting the paclitaxel-induced nuclear accumulation of NF-κB. Taken together, these results suggest that swainsonine may be an important agent against HCC via directly inhibiting HCC cell growth and enhancing the responsiveness of HCC cells to paclitaxel.

Efficient Enrichment of Hepatic Cancer Stem-Like Cells from a Primary Rat HCC Model via a Density Gradient Centrifugation-Centered Method

Background Because few definitive markers are available for hepatic cancer stem cells (HCSCs), based on physical rather than immunochemical properties, we applied a novel method to enrich HCSCs. Methodology After hepatic tumor cells (HTCs) were first isolated from diethylinitrosamine-induced F344 rat HCC model using percoll discontinuous gradient centrifugation (PDGC) and purified via differential trypsinization and differential attachment (DTDA), they were separated into four fractions using percoll continuous gradient centrifugation (PCGC) and sequentially designated as fractions I–IV (FI–IV). Morphological characteristics, mRNA and protein levels of stem cell markers, proliferative abilities, induced differentiation, in vitro migratory capacities, in vitro chemo-resistant capacities, and in vivo malignant capacities were determined for the cells of each fraction. Findings As the density of cells increased, 22.18%, 11.62%, 4.73% and 61.47% of primary cultured HTCs were segregated in FI–FIV, respectively. The cells from FIII (density between 1.041 and 1.062 g/ml) displayed a higher nuclear-cytoplasmic ratio and fewer organelles and expressed higher levels of stem cell markers (AFP, EpCAM and CD133) than cells from other fractions (P<0.01). Additionally, in vitro, the cells from FIII showed a greater capacity to self-renew, differentiate into mature HTCs, transit across membranes, close scratches, and carry resistance to chemotherapy than did cells from any other fraction; in vivo, injection of only 1×104 cells from FIII could generate tumors not only in subcutaneous tissue but also in the livers of nude mice. Conclusions Through our novel method, HCSC-like cells were successfully enriched in FIII. This study will greatly contribute to two important areas of biological interest: CSC isolation and HCC therapy.