[Highly Efficient Bioflocculation of Microalgae Using Mucor circinelloides].

Harvesting of microalgae is the major challenge in cost-efficient large-scale microalgal biomass production due to their low concentration and small cell size in the culture medium. The present paper aimed to study the efficiency of the filamentous fungus Mucor circinelloides spores suspensions to harvest the green unicellular microalga Chlorella pyrenoidosa grown in synthetic medium. Results showed that the optimal co-culture conditions were pH=6.0, 1.25 g·L-1 glucose and 1:250 fungi to microalgae ratio with harvest efficiency of 91.08%. In addition, the mentioned optimal conditions could be applied for actual sewage with harvest efficiency of 92.33%. Polysaccharide concentrations measured before and after 48 h of cultivation showed that the polysaccharide of C. pyrenoidosa cultured alone was increased by 0.047 g·L-1, while co-cultured mixture showed increase in polysaccharides by 0.019 g·L-1. The recorded decrease in polysaccharides concentration in the co-culture might be attributed to using of excreted polysaccharides by M. circinelloides to grow, confirming the symbiotic association of both organisms. Furthermore, with decreasing the pH, C. pyrenoidosa Zeta potential was stable, while it was increased from -37.7 mV to -9.87 mV in M. circinelloides, which indicated that charge neutralization was the mechanism of flocculation between algae and fungi.

Matrix Metalloproteinase 14 Overexpression Is Correlated with the Progression and Poor Prognosis of Nasopharyngeal Carcinoma.

BACKGROUND AND AIMS: Matrix metalloproteinase 14 (MMP14) has been identified to play a significant role in several types of cancers, but little is known about the significance of MMP14 in nasopharyngeal carcinoma (NPC) patients. The aim of this study was to explore the association of MMP14 expression with clinicopathologic features and prognosis in NPC. METHODS: MMP14 mRNA and protein expressions were examined in NPC and nasopharyngeal tissues through real-time PCR and immunohistochemistry. Meanwhile, the relationship of MMP14 expression levels with clinical features and prognosis of NPC patients was analyzed. RESULTS: MMP14 mRNA expression was markedly higher in NPC tissues than in nasopharyngeal epithelium tissues (p = 0.002). Using immunohistochemistry, staining for MMP14 protein was found in the normal nasopharyngeal epithelial cells and malignant epithelial cells, but increased expression of MMP14 was observed in NPC samples compared with normal nasopharyngeal epithelium samples (p = 0.027). In addition, high levels of MMP14 protein were positively correlated with the status of clinical stage (p = 0.009), N classification (p = 0.006), and distant metastasis (p = 0.005) of NPC patients. Patients with higher MMP14 expression had a significantly shorter overall survival time than did patients with low MMP14 expression. Multivariate analysis indicated that the level of MMP14 expression was an independent prognostic indicator (p < 0.001) for the survival of patients with NPC. CONCLUSIONS: MMP14 overexpression is a potentially unfavorable prognostic factor for NPC patients.

Purification of fetal liver stem/progenitor cells containing all the repopulation potential for normal adult rat liver.

BACKGROUND & AIMS: Previously, we showed high-level, long-term liver replacement after transplantation of unfractionated embryonic day (ED) 14 fetal liver stem/progenitor cells (FLSPC). However, for clinical applications, it will be essential to transplant highly enriched cells, while maintaining high repopulation potential. METHODS: Dlk-1, a member of the delta-like family of cell surface transmembrane proteins, is highly expressed in human and rodent fetal liver. Dlk-1(+) cells, isolated from ED14 fetal liver using immunomagnetic beads, were examined for their hepatic gene expression profile and characteristic properties in vitro and their proliferative and differentiation potential in vivo after transplantation into normal adult rat liver. RESULTS: Rat ED14 FLSPC were purified to 95% homogeneity and exhibited cell culture and gene expression characteristics expected for hepatic stem/progenitor cells. Rat ED14 FLSPC are alpha-fetoprotein(+)/cytokeratin-19(+) or alpha-fetoprotein(+)/cytokeratin-19(-) and contain all of the normal liver repopulation capacity found in fetal liver. Hematopoietic stem cells, a major component in crude fetal liver cell preparations that engraft in other organs, such as bone marrow, spleen, and lung, are totally removed by Dlk-1 selection, and Dlk-1 purified FLSPC repopulate only the liver. CONCLUSIONS: This is the first study reporting purification of hepatic stem/progenitor cells from fetal liver that are fully capable of repopulating the normal adult liver. This represents a major advance toward developing protocols that will be essential for clinical application of liver cell transplantation therapy.

Comparison of hepatic properties and transplantation of Thy-1(+) and Thy-1(-) cells isolated from embryonic day 14 rat fetal liver.

UNLABELLED: Thy-1, a marker of hematopoietic progenitor cells, is also expressed in activated oval cells of rat liver. Thy-1(+) cells are also in rat fetal liver and exhibit properties of bipotent hepatic epithelial progenitor cells in culture. However, no information is available concerning liver repopulation by Thy-1(+) fetal liver cells. Therefore, we isolated Thy-1(+) and Thy-1(-) cells from embryonic day (ED) 14 fetal liver and compared their gene expression characteristics in vitro and proliferative and differentiation potential after transplantation into adult rat liver. Fetal liver cells selected for Thy-1 expression using immunomagnetic microbeads were enriched from 5.2%-87.2% Thy-1(+). The vast majority of alpha fetoprotein(+), albumin(+), cytokine-19(+), and E-cadherin(+) cells were found in cultured Thy-1(-) cells, whereas nearly all CD45(+) cells were in the Thy-1(+) fraction. In normal rat liver, transplanted Thy-1(+) cells produced only rare, small DPPIV(+) cell clusters, very few of which exhibited a hepatocytic phenotype. In retrorsine-treated liver, transplanted Thy-1(+) fetal liver cells achieved a 4.6%-23.5% repopulation. In contrast, Thy-1(-) fetal liver cells substantially repopulated normal adult liver and totally repopulated retrorsine-treated liver. Regarding the stromal cell-derived factor (SDF)-1/chemokine (C-X-C motif) receptor 4 (CXCR4) axis for stem cell homing, Thy-1(+) and Thy-1(-) fetal hepatic epithelial cells equally expressed CXCR4. However, SDF-1alpha expression was augmented in bile ducts and oval cells in retrorsine/partial hepatectomy-treated liver, and this correlated with liver repopulation by Thy-1(+) cells. CONCLUSION: Highly enriched Thy-1(+) ED14 fetal liver cells proliferate and repopulate the liver only after extensive liver injury and represent a fetal hepatic progenitor cell population distinct from Thy-1(-) stem/progenitor cells, which repopulate the normal adult liver.

Properties of cryopreserved fetal liver stem/progenitor cells that exhibit long-term repopulation of the normal rat liver.

We have previously achieved a high level of long-term liver replacement by transplanting freshly isolated embryonic day (ED) 14 rat fetal liver stem/progenitor cells (FLSPCs). However, for most clinical applications, it will be necessary to use cryopreserved cells that can effectively repopulate the host organ. In the present study, we report the growth and gene expression properties in culture of rat FLSPCs cryopreserved for up to 20 months and the ability of cryopreserved FLSPCs to repopulate the normal adult rat liver. After thawing and placement in culture, cryopreserved FLSPCs exhibited a high proliferation rate: 49.7% Ki-67-positive on day 1 and 34.7% Ki-67-positive on day 5. The majority of cells were also positive for both alpha-fetoprotein and cytokeratin-19 (potentially bipotent) on day 5. More than 80% of cultured cells expressed albumin, the asialoglycoprotein receptor, and UDP-glucuronosyltransferase (unique hepatocyte-specific functions). Expression of glucose-6-phosphatase, carbamyl phosphate synthetase 1, hepatocyte nuclear factor 4alpha, tyrosine aminotransferase, and oncostatin M receptor mRNAs was initially negative, but all were expressed on day 5 in culture. After transplantation into the normal adult rat liver, cryopreserved FLSPCs proliferated continuously, regenerated both hepatocytes and bile ducts, and produced up to 15.1% (mean, 12.0% +/- 2.0%) replacement of total liver mass at 6 months after cell transplantation. These results were obtained in a normal liver background under nonselective conditions. This study is the first to show a high level of long-term liver replacement with cryopreserved fetal liver cells, an essential requirement for future clinical applications.

Purification and characterization of mouse fetal liver epithelial cells with high in vivo repopulation capacity.

Epithelial cells in embryonic day (ED) 12.5 murine fetal liver were separated from hematopoietic cell populations using fluorescence-activated cell sorting (FACS) and were characterized by immunocytochemistry using a broad set of antibodies specific for epithelial cells (alpha-fetoprotein [AFP], albumin [ALB], pancytokeratin [PanCK], Liv2, E-cadherin, Dlk), hematopoietic/endothelial cells (Ter119, CD45, CD31), and stem/progenitor cells (c-Kit, CD34, Sca-1). AFP(+)/ALB(+) cells represented approximately 2.5% of total cells and were positive for the epithelial-specific surface markers Liv2, E-cadherin, and Dlk, but were clearly separated and distinct from hematopoietic cells (Ter119(+)/CD45(+)). Fetal liver epithelial cells (AFP(+)/E-cadherin(+)) were Sca-1(+) but showed no expression of hematopoietic stem cell markers c-Kit and CD34. These cells were enriched by FACS sorting for E-cadherin to a purity of 95% as defined by co-expression of AFP and PanCK. Purified fetal liver epithelial cells formed clusters in cell culture and differentiated along the hepatocytic lineage in the presence of dexamethasone, expressing glucose-6-phosphatase (G6P) and tyrosine amino transferase. Wild-type ED12.5 murine fetal liver cells were transplanted into adult dipeptidyl peptidase IV knockout mice and differentiated into mature hepatocytes expressing ALB, G6P, and glycogen, indicating normal biochemical function. Transplanted cells became fully incorporated into the hepatic parenchymal cords and showed up to 80% liver repopulation at 2 to 6 months after cell transplantation. In conclusion, we isolated and highly purified a population of epithelial cells from the ED12.5 mouse fetal liver that are clearly separate from hematopoietic cells and differentiate into mature, functional hepatocytes in vivo with the capacity for efficient liver repopulation. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34+ stem cell recruitment to the liver.

Hematopoietic stem cells rarely contribute to hepatic regeneration, however, the mechanisms governing their homing to the liver, which is a crucial first step, are poorly understood. The chemokine stromal cell-derived factor-1 (SDF-1), which attracts human and murine progenitors, is expressed by liver bile duct epithelium. Neutralization of the SDF-1 receptor CXCR4 abolished homing and engraftment of the murine liver by human CD34+ hematopoietic progenitors, while local injection of human SDF-1 increased their homing. Engrafted human cells were localized in clusters surrounding the bile ducts, in close proximity to SDF-1-expressing epithelial cells, and differentiated into albumin-producing cells. Irradiation or inflammation increased SDF-1 levels and hepatic injury induced MMP-9 activity, leading to both increased CXCR4 expression and SDF-1-mediated recruitment of hematopoietic progenitors to the liver. Unexpectedly, HGF, which is increased following liver injury, promoted protrusion formation, CXCR4 upregulation, and SDF-1-mediated directional migration by human CD34+ progenitors, and synergized with stem cell factor. Thus, stress-induced signals, such as increased expression of SDF-1, MMP-9, and HGF, recruit human CD34+ progenitors with hematopoietic and/or hepatic-like potential to the liver of NOD/SCID mice. Our results suggest the potential of hematopoietic CD34+/CXCR4+cells to respond to stress signals from nonhematopoietic injured organs as an important mechanism for tissue targeting and repair.

Repopulation of rat liver by fetal hepatoblasts and adult hepatocytes transduced ex vivo with lentiviral vectors.

Recent studies have shown that nondividing primary cells, such as hepatocytes, can be efficiently transduced in vitro by human immunodeficiency virus-based lentivirus vectors. Other studies have reported that, under certain conditions, the liver can be repopulated with transplanted hepatocytes. In the present study, we combined these procedures to develop a model system for ex vivo gene therapy by repopulating rat livers with hepatocytes and hepatoblasts transduced with a lentivirus vector expressing a reporter gene, green fluorescent protein (GFP). Long-term GFP expression in vivo (up to 4 months) was achieved when the transgene was driven by the liver-specific albumin enhancer/promoter but was silenced when the cytomegalovirus (CMV) enhancer/promoter was used. Transplanted cells were massively amplified ( approximately 10 cell doublings) under the influence of retrorsine/partial hepatectomy, and both repopulation and continued transgene expression in individual cells were documented by dual expression of a cell transplantation marker, dipeptidyl peptidase IV (DPPIV), and GFP. In this system, maintenance or expansion of the transplanted cells did not depend on expression of the transgene, establishing that positive selection is not required to maintain transgene expression following multiple divisions of transplanted, lentivirus-transduced hepatic cells. In conclusion, fetal hepatoblasts (liver stem/progenitor cells) can serve as efficient vehicles for ex vivo gene therapy and suggest that liver-based genetic disorders that do not shorten hepatocyte longevity or cause liver damage, such as phenylketonuria, hyperbilirubinemias, familial hypercholesterolemia, primary oxalosis, and factor IX deficiency, among others, might be amenable to treatment by this approach.