Differentiation Of Liver Cells Research Articles

MiRNA-382-5p Suppresses the Expression of Farnesoid X Receptor to Promote Progression of Liver Cancer.

BackgroundThe dysregulation of microRNAs (miRNAs) and hepatotoxicity due to the aberrant accumulation of bile acids (BAs) are notorious causes that predispose an individual to the development of hepatocellular carcinoma (HCC). Farnesoid X receptor (FXR), encoded by NR1H4 gene, has been identified as a crucial BA receptor to maintain the homeostasis of BA pool and its expression is decreased in HCC. miR-382-5p plays an important role in the pathogenesis of many human malignancies and was reported to promote the proliferation and differentiation of normal liver cells and liver regeneration. However, there is still some controversy about its role in HCC microenvironment. This study aims to explore the expression pattern of miR-382-5p in HCC and its role in regulating FXR during the development of HCC.MethodsTissues collected from 30 HCC patients were subjected to extraction of total RNA and quantitative real-time PCR (qRT-PCR) for the analyses of miR-382-5p expression and NR1H4 mRNA levels, and their expressions were verified by analyzing the online HCC-related GSE datasets. The role of miR-382-5p in regulating cellular proliferation and expression of FXR in different HCC cell lines was analyzed by qRT-PCR, Western Blot, real-time cellular analysis (RTCA) and luciferase reporter assays. The role of miR-382-5p in regulating downstream genes of FXR in HCC cells was also analyzed.ResultsmiR-382-5p was upregulated in HCC tissues and inversely associated with the downregulation of NR1H4 mRNA levels. The luciferase reporter assay proved that miR-382-5p directly targeted the 3ʹ-untranslated region (3ʹ-UTR) of human NR1H4 mRNA. Overexpression of miR-382-5p led to a malignant proliferation of HCC cells by suppressing the expression of FXR. In contrast, blocking the endogenous miR-382-5p was sufficient to suppress the cellular proliferation rate of HCC through increasing FXR expression. Additionally, miR-382-5p inhibited the expression of some target genes of FXR, including SHP, FGF19 and SLC51A, and this inhibitory effect was FXR-dependent.ConclusionTherefore, miR-382-5p promotes the progression of HCC in vitro by suppressing FXR and could serve as a valuable therapeutic target for HCC treatment.

Synergistic effect of three-dimensionalcoculture and photobiomodulation therapy on vascularized liver spheroid formation by stem cells.

Despite studies reporting functional differentiation of liver cells, a three-dimensional, vascularized liver organ has yet to be developed from mesenchymal stem cells. We investigated whether treatment with photobiomodulation (PBM) before three-dimensional liver spheroid transplantation improved the recovery of liver function via stimulation of angiogenesis and hepatocyte differentiation. Liver spheroids composed of hepatic, endothelial,and mesenchymal cells were subjected to PBM therapy. To evaluate the in vivo therapeutic effect of the liver spheroids treated with PBM, phosphate-buffered saline, liver spheroid, and PBM-treated liver spheroid were transplanted into a damaged host liver using conventional chimeric mouse models. To further characterize the maturation of transplanted PBM-liver spheroid compared with the newly generated non-PBM-liver spheroid or human liver tissues, the expression profiles of mature liver signature genes were analyzed. Liver spheroids expressed hepatocyte growth factors, including vascular endothelial growth factor and angiogenic factors. The cells in liver spheroid compensated for the low viability and improved the function of hepatocytes. Here, we demonstrate the formation of vascularized and functional human liver spheroid from human adipose-derived stem cellsby transplantation of liver tissue created in vitro. Albumin secretion by PBM-treated liver spheroid was higher on Day 28 compared with liver spheroid-seeded transplant group. PBM-liver spheroids serve as individual vascularization units, promoting the simultaneous development of new microvascular networks at different locations inside the implanted tissue constructs. The vasculature in the liver spheroid transplants became functional by connecting to the host vessels within 48 h. These PBM-liver spheroids may be useful in designing artificial three-dimensional hepatic tissue constructs and in cell therapy with limited numbers of human hepatocytes.

Efficient Screening of Glycan-Specific Aptamers Using a Glycosylated Peptide as a Scaffold

Abnormal glycan structures are valuable biomarkers for disease states; the development of glycan-specific binders is thereby significantly important. However, the structural homology and weak immunogenicity of glycans pose major hurdles in the evolution of antibodies, while the poor availability of complex glycans also has extremely hindered the selection of anti-glycan aptamers. Herein, we present a new approach to efficiently screen aptamers toward specific glycans with a complex structure, using a glycosylated peptide as a scaffold. In this method, using peptide-imprinted magnetic nanoparticles (MNPs) as a versatile platform, a glycopeptide tryptically digested from a native glycoprotein was selectively entrapped for positive selection, while a nonglycosylated analogue with an identical peptide sequence was synthesized for negative selection. Alternating positive and negative selection steps were carried out to guide the directed evolution of glycan-binding aptamers. As proof of the principle, the biantennary digalactosylated disialylated N-glycan A2G2S2, against which there have been no antibodies and lectins so far, was employed as the target. With the glycoprotein transferrin as a source of target glycan, two satisfied anti-A2G2S2 aptamers were selected within seven rounds. Since A2G2S2 is upregulated in cancerous liver cells, carboxyfluorescein (FAM)-labeled aptamers were prepared as fluorescent imaging reagents, and successful differentiation of cancerous liver cells over normal liver cells was achieved, which demonstrated the application feasibility of the selected aptamers. This approach obviated a tedious glycan preparation process and allowed favorable expose of the intrinsic flexible conformation of natural glycans. Therefore, it holds great promise for developing glycan-specific aptamers for challenging applications such as cancer targeting.

From Mother or Father: Uniparental Embryos Uncover Parent-of-Origin Effects in Humans.

In mammals, both parents make unique contributions to the offspring and maternal and paternal genomes are required for development. Two recent papers in Cell Stem Cell (Leng etal., 2019; Sagi etal., 2019) study uniparental embryos and uniparental embryonic stem cells to interrogate parent-of-origin effects in human embryogenesis.

Interferon gamma inhibits the differentiation of mouse adult liver and bone marrow hematopoietic stem cells by inhibiting the activation of notch signaling

BackgroundThe paradigm of hematopoietic stem and progenitor cells (HSPCs) has become accepted ever since the discovery of adult mouse liver hematopoietic stem cells and their multipotent characteristics that give rise to all blood cells. However, differences between bone marrow (BM) and liver hematopoietic stem cells and the hematopoietic microenvironment remain poorly understood. In addition, the regulation of the liver hematopoietic system remains unknown.MethodsClone formation assays were used to confirm that the proliferation of adult mouse liver and bone marrow HSPCs. Model mice with different interferon gamma (IFN-γ) levels and a co-culture system were used to detect the differentiation of liver HSPCs. The γ-secretase inhibitor (GSI) and the JAK/STAT inhibitor ruxolitinib and cell culture assays were used to explore the molecular mechanism by which IFN-γ impairs HSPC proliferation and differentiation.ResultsThe colony-forming activity of liver and bone marrow HSPCs was inhibited by IFN-γ. Model mice with different IFN-γ levels showed that the differentiation of liver HSPCs was impaired by IFN-γ. Using a co-culture system comprising liver HSPCs, we found that IFN-γ inhibited the development of liver hematopoietic stem cells into γδT cells. We then demonstrated that IFN-γ might impair liver HSPC differentiation by inhibiting the activation of the notch signaling via the JAK/STAT signaling pathway.ConclusionsIFN-γ inhibited the proliferation of liver-derived HSPCs. IFN-γ also impaired the differentiation of long-term hematopoietic stem cells (LT-HSCs) into short-term hematopoietic stem cells (ST-HSCs) and multipotent progenitors (MPPs) and the process from LSK (Lineage−Sca-1+c-Kit+) cells to γδT cells. Importantly, we proposed that IFN-γ might inhibit the activation of notch signaling through the JAK/STAT signaling pathway and thus impair the differentiation process of mouse adult liver and BM hematopoietic stem cells.

Interleukins-17 and 27 promote liver regeneration by sequentially inducing progenitor cell expansion and differentiation.

Liver progenitor cells (LPCs)/ductular reactions (DRs) are associated with inflammation and implicated in the pathogenesis of chronic liver diseases. However, how inflammation regulates LPCs/DRs remains largely unknown. Identification of inflammatory processes that involve LPC activation and expansion represent a key step in understanding the pathogenesis of liver diseases. In the current study, we found that diverse types of chronic liver diseases are associated with elevation of infiltrated interleukin (IL)‐17‐positive (+) cells and cytokeratin 19 (CK19)+ LPCs, and both cell types colocalized and their numbers positively correlated with each other. The role of IL‐17 in the induction of LPCs was examined in a mouse model fed a choline‐deficient and ethionine‐supplemented (CDE) diet. Feeding of wild‐type mice with the CDE diet markedly elevated CK19+Ki67+ proliferating LPCs and hepatic inflammation. Disruption of the IL‐17 gene or IL‐27 receptor, alpha subunit (WSX‐1) gene abolished CDE diet‐induced LPC expansion and inflammation. In vitro treatment with IL‐17 promoted proliferation of bipotential murine oval liver cells (a liver progenitor cell line) and markedly up‐regulated IL‐27 expression in macrophages. Treatment with IL‐27 favored the differentiation of bipotential murine oval liver cells and freshly isolated LPCs into hepatocytes. Conclusion: The current data provide evidence for a collaborative role between IL‐17 and IL‐27 in promoting LPC expansion and differentiation, respectively, thereby contributing to liver regeneration. (Hepatology Communications 2018;2:329‐343)

Heme Oxygenase 1 Inhibition Reverses Anemia in β-Thalassemia Mice

Thalassemias are a heterogeneous group of red blood cell (RBC) disorders ranging from a clinically severe phenotype requiring lifesaving transfusions (thalassemia major) to a relatively moderate symptomatic disorder, sometimes requiring transfusions (thalassemia intermedia). Though considered a major cause of morbidity and mortality worldwide, there is still no universally available cure for thalassemia major. The reason for this is, at least in part, due to the lack of full understanding of pathophysiology of thalassemia. The underlying basis of thalassemia pathology is the premature apoptotic destruction of erythroblasts causing ineffective erythropoeisis. In β-thalassemia, β-globin synthesis is diminished causing α-globin accumulation. Unpaired globin chains that accumulate in thalassemic erythroblasts are bound to heme. Moreover, in β-thalassemia an erythroid-specific protease destroys excess α-globin chains, likely leading to the generation of a pool of "free" heme in erythroblasts.Physiologically, heme can be degraded only via heme oxygenases (HO). Circulating erythrocytes contain the majority of heme destined for catabolism; this process takes place primarily in splenic and hepatic macrophages following erythrophagocytosis of senescent RBC. Heme oxygenase, in particular its heme-inducible isoform HO1, has been extensively studied in hepatocytes and many other non-erythroid cells. Recently, we have provided unequivocal evidence that this enzyme is present in erythroid progenitors as well as their differentiated progenies.1"Unshielded" heme is toxic, but this toxicity will likely be augmented, if HO1 releases iron from heme. We hypothesize that in β-thalassemic erythroblasts HO1-mediated release of iron from heme is the major culprit responsible for cellular damage. Additionally, it has been shown that prevention of heme-derived iron release from splenic and hepatic macrophages improves β-thalassemia phenotype2. Therefore, suppression of HO1-mediated heme catabolism from senescent RBC could be beneficial in reversing thalassemic phenotype.To test this hypothesis, we exploited the mouse model of β-thalassemia known as th3/th3; we obtained these mice from Dr. Stefano Rivella. Our data indicates that HO1 expression is increased in the liver of β-thalassemic mice as compared to wild type mice. Importantly, we observed that erythropoietin-mediated erythroid differentiation of fetal liver (FL) cells from β-thalassemic fetuses increased HO1 mRNA and protein levels to a higher degree than in their wild type counterparts. Ferritin levels were increased in β-thalassemic FL cells suggesting increased heme catabolism and iron release from the tetrapyrrole macrocycle. To investigate the contribution of HO1 to the pathology associated with β-thalassemia, wild type and thalassemic (th3/+) mice were injected intraperitoneally with 40 µmoles/kg/d of tin-protoporphyrin IX (SnPP, HO inhibitor) during a 4-weeks, 3-times a week. Our results show that β-thalassemic mice injected with SnPP have increased hemoglobin levels and red blood cell counts, and display a decrease in the spleen index, reticulocyte counts and liver iron content when compared to PBS-injected β-thalassemic mice. Furthermore, while hepcidin levels remain unchanged, liver ferroportin expression decreases in SnPP-injected β-thalassemic mice.Our results indicate that β-thalassemic erythroblasts have high levels of HO1, which would be expected to degrade any "free" heme. Further research is needed to determine whether iron liberated from heme by HO1 is directly responsible for the damage of β-thalassemic erythroblasts.1GarciaSantos D, et al. Heme oxygenase 1 is expressed in murine erythroid cells where it controls the level of regulatory heme. Blood 123 (14): 226977, 2014.2Nai A, et al. Deletion of TMPRSS6 attenuates the phenotype in a mouse model of β-thalassemia. Blood 119 (21): 5021, 2012. DisclosuresNo relevant conflicts of interest to declare.

Pathophysiology and Treatment of Beta-Thalassemia: Investigations of Heme Oxygenase 1 and Its Inhibitors

Thalassemias are a heterogeneous group of red blood cell disorders ranging from a clinically severe phenotype requiring life-saving transfusions (thalassemia major) to a relatively moderate symptomatic disorder, sometimes requiring transfusions (thalassemia intermedia). Thalassemia minor, the least severe form of the disorder, is characterized by minimal to mild symptoms. Though considered a major cause of morbidity and mortality worldwide, there is still no universally available cure for thalassemia major. The reason for this is, at least in part, due to the lack of full understanding of pathophysiology of thalassemia. The underlying basis of thalassemia pathology is the premature apoptotic destruction of erythroblasts causing ineffective erythropoeisis. Normally, the assembly of adult hemoglobin (consisting of a tetramer of two α- and two β-globin chains) features a very tight coordination of α- and β-globin chain synthesis. However, in β-thalassemia, β-globin synthesis is diminished causing α-globin accumulation; while in α-thalassemia the opposite scenario occurs. Unpaired globin chains that accumulate in thalassemic erythroblasts are bound to heme. In addition, in β-thalassemia an erythroid specific protease destroys excess α-globin chains, likely leading to the generation of a pool of "free" heme in erythroblasts. "Unshielded" heme is toxic, but this toxicity will likely be augmented, if heme oxygenase 1 (HO-1) can release iron from heme.So far, virtually no information about the expression of HO-1 in erythroblasts has been produced. However, we have recently provided unequivocal evidence that this enzyme is present in several model erythroid cells1. Based on this novel and important finding, we hypothesize that in β-thalassemic erythroblasts HO-1-mediated release of iron from heme is the major culprit responsible for cellular damage. To test this hypothesis, we exploited the mouse model of β-thalassemia known as th3/th3. Our data indicates that HO-1 expression is increased in the liver, spleen and kidney of β-thalassemic mice compared to wild-type mice. Importantly, we observed that erythropoietin-mediated erythroid differentiation of fetal liver (FL) cells isolated from β-thalassemic fetuses have increased levels of HO-1 at mRNA and protein levels as well as a decrease in phosphorylated eIF2-α levels. Ferritin levels were increased in β-thalassemic FL cells suggesting increased heme catabolism and iron release. To investigate the contribution of HO-1 to the pathology associated with β-thalassemia, wild-type and thalassemic (th3/+) mice were injected with 40 µmoles/kg/d of tin-protoporphyrin IX (SnPP, HO-1 inhibitor) during a 4-week period, 3 times a week. Our results show that β-thalassemic mice injected with SnPP display a decrease in the spleen index, hemoglobin levels, red blood cell counts, reticulocyte counts and liver iron content when compared to PBS injected β-thalassemic mice. Additionally, HO-1 inhibition reduced ineffective erytropoiesis in β-thalassemia mice. Our results indicate that β-thalassemic erythroblasts have inappropriately high levels of "free" heme that is continuously degraded by HO-1. Further research is needed to determine whether iron liberated from heme by HO-1 is directly responsible for the damage of β-thalassemic erythroblasts.1Garcia-Santos D, et al. Heme oxygenase 1 is expressed in murine erythroid cells where it controls the level of regulatory heme. Blood 123 (14): 2269-77, 2014. DisclosuresNo relevant conflicts of interest to declare.

Uncovering the Role of Heme Oxygenase 1 in the Pathophysiology of β-Thalassemia

Thalassemias are a heterogeneous group of red blood cell disorders ranging from a clinically severe phenotype requiring life-saving transfusions (thalassemia major) to a relatively moderate symptomatic disorder, sometimes requiring transfusions (thalassemia intermedia). Thalassemia minor, the least severe form of the disorder, is characterized by minimal to mild symptoms. While thalassemia minor and intermedia are vastly more prevalent than thalassemia major, the latter is often fatal when not treated. Though considered a major cause of morbidity and mortality worldwide, there is still no universally available cure for this severe form of thalassemia. A reason for this is at least in part due to the lack of full understanding of pathophsyiology of thalassemia. The underlying cause of pathology in thalassemia is the premature apoptotic destruction of erythroblasts causing ineffective erythropoeisis. Normally, the assembly of adult hemoglobin (consisting of a tetramer of two α- and two β-globin chains) features a very tight coordination of α- and β-globin chain synthesis. However, in β-thalassemia, β-globin synthesis is decelerated causing α-globin accumulation; while in α-thalassemia the opposite scenario occurs. Unpaired globin chains that accumulate in thalassemic erythroblasts are bound to heme. In addition, in β-thalassemia an erythroid specific protease destroys excess α-globin chains, likely leading to the generation of a pool of “free” heme in erythroblasts. “Free” heme is toxic, but this toxicity will likely be augmented, if heme oxygenase 1 (HO-1) can release iron from heme. To date, virtually no information about the expression of HO-1 in erythroblasts has been produced; however, we have recently provided unequivocal evidence that this enzyme is present in several model erythroid cells1. Based on this novel and important finding, we hypothesize that in β-thalassemic erythroblasts HO-1 mediated release of iron from heme is the major culprit responsible for cellular damage. To test this hypothesis we exploited the mouse model of β-thalassemia, th3/th3. Thus far, our data indicates that HO-1 expression is increased in liver, spleen and kidney of β-thalassemic mice compared to wild type mice. Importantly, we observed that Epo-mediated erythroid differentiation of fetal liver (FL) cells isolated from β-thalassemic fetuses, display increased levels of HO-1 at mRNA and protein levels as well as decreased phosphorylated eiF2-α. Ferritin levels are also increased in these cells suggesting increased heme catabolism and iron release. Altogether, these results indicate that β-thalassemic erythroblasts have inappropriately high levels of unbound heme that is continuously degraded by HO-1. Further research is needed to determine whether HO-1 liberated iron is responsible for the damage of β-thalassemic erythroblasts.1Garcia-Santos D, et al. Heme oxygenase 1 is expressed in murine erythroid cells where it controls the level of regulatory heme. Blood 123 (14): 2269-77, 2014. DisclosuresNo relevant conflicts of interest to declare.

Mobilization with granulocyte colony-stimulating factor blocks medullar erythropoiesis by depleting F4/80+VCAM1+CD169+ER-HR3+Ly6G+ erythroid island macrophages in the mouse

Similarly to other tissues, the bone marrow contains subsets of resident tissue macrophages, which are essential to maintain bone formation, functional hematopoietic stem cell (HSC) niches, and erythropoiesis. Pharmacologic doses of granulocyte colony-stimulating factor (G-CSF) mobilize HSC in part by interfering with the HSC niche-supportive function of BM resident macrophages. Because bone marrow macrophages are key to both maintenance of HSC within their niche and erythropoiesis, we investigated the effect of mobilizing doses of G-CSF on erythropoiesis in mice. We now report that G-CSF blocks medullar erythropoiesis by depleting the erythroid island macrophages we identified as co-expressing F4/80, vascular cell adhesion molecule-1, CD169, Ly-6G, and the ER-HR3 erythroid island macrophage antigen. Both broad macrophage depletion, achieved by injecting clodronate-loaded liposomes, and selective depletion of CD169(+) macrophages, also concomitantly depleted F4/80(+)VCAM-1(+)CD169(+)ER-HR3(+)Ly-6G(+) erythroid island macrophages and blocked erythropoiesis. This more precise phenotypic definition of erythroid island macrophages will enable studies on their biology and function in normal settings and on diseases associated with anemia. Finally, this study further illustrates that macrophages are a potent relay of innate immunity and inflammation on bone, hematopoietic, and erythropoietic maintenance. Agents that affect these macrophages, such as G-CSF, are likely to affect these three processes concomitantly.

Global analysis of induced transcription factors and cofactors identifies Tfdp2 as an essential coregulator during terminal erythropoiesis

Key transcriptional regulators of terminal erythropoiesis, such as GATA-binding factor 1 (GATA1) and T-cell acute lymphocytic leukemia protein 1 (TAL1), have been well characterized, but transcription factors and cofactors and their expression modulations have not yet been explored on a global scale. Here, we use global gene expression analysis to identify 28 transcription factors and 19 transcriptional cofactors induced during terminal erythroid differentiation whose promoters are enriched for binding by GATA1 and TAL1. Utilizing protein-protein interaction databases to identify cofactors for each transcription factor, we pinpoint several co-induced pairs, of which E2f2 and its cofactor transcription factor Dp-2 (Tfdp2) were the most highly induced. TFDP2 is a critical cofactor required for proper cell cycle control and gene expression. GATA1 and TAL1 are bound to the regulatory regions of Tfdp2 and upregulate its expression and knockdown of Tfdp2 results in significantly reduced rates of proliferation as well as reduced upregulation of many erythroid-important genes. Loss of Tfdp2 also globally inhibits the normal downregulation of many E2F2 target genes, including those that regulate the cell cycle, causing cells to accumulate in S phase and resulting in increased erythrocyte size. Our findings highlight the importance of TFDP2 in coupling the erythroid cell cycle with terminal differentiation and validate this study as a resource for future work on elucidating the role of diverse transcription factors and coregulators in erythropoiesis.

Correlation between the overexpression of urokinase receptor isoform uPAR (D1D2) and hepatic cell malignant transformation

In order to provide an abundant source of specimen and to reveal the correlation between the overexpression of urokinase plasminogen activator receptor isoform uPAR (D1D2) and hepatic cell malignant transformation, the optimal liver cell culture method was selected from three cell culture methods to culture and separate out liver cells with a high density, high purity and high activity. The specimens were used to culture and assess the uPAR (D1D2) mRNA level in normal liver cells, liver cancer cells and para-carcinoma cells. In the present study, the correlation between the overexpression of uPAR (D1D2) and hepatic cell malignant transformation was discussed. When comparing the tissue block adherent method, liver cell grinding method and pancreatic enzyme digestion method, the liver tissue adherent method was found to be economical, simple and overall the optimal method for liver cell culture. This was used as a reference standard for cell culture. RT-PCR was used to determine isoform uPAR (D1D2) mRNA level in normal liver cells, para-carcinoma cells and liver cancer cells. The comparison of uPAR (D1D2) mRNA levels in normal liver cells, para-carcinoma cells and liver cancer cells, demonstrated that the brightness of the cells clearly increased in normal liver cells, para-carcinoma cells and liver cancer cells. The comparison of the cell grey values of three groups demonstrated a statistically significant difference (P<0.05). The liver tissue adherent method was able to produce liver cells with a high density, high purity and high activity, providing a sufficient source of specimen for our subsequent experiments. The electrophoresis results showed that: uPAR (D1D2) mRNA expression increased from normal liver cells to para-carcinoma cells to liver cancer cells, inferring that uPAR (D1D2) mRNA overexpression may be the result of changes in the conformation of the uPAR isoform. In addition, it is closely associated with abnormal cell signal transduction, which leads to clonal proliferation and abnormal differentiation of liver cells with malignant transformation in liver cells.

Heme Oxygenase 1 Plays a Role In The Pathophysiology Of β-Thalassemia

Thalassemias are a heterogeneous group of red blood cell disorders ranging from a clinically severe phenotype requiring life-saving transfusions (thalassemia major) to a relatively moderate symptomatic disorder, sometimes requiring transfusions (thalassemia intermedia). Thalassemia minor, the least severe form of the disorder, is characterized by minimal to mild symptoms. While thalassemia minor and intermedia are vastly more prevalent than thalassemia major, the latter is often fatal when not treated. Though considered a major cause of morbidity and mortality worldwide, there is still no universally available cure for this severe form of thalassemia. A reason for this is at least in part due to the lack of full understanding of pathophsyiology of thalassemia. The underlying cause of pathology in thalassemia is the premature apoptotic destruction of erythroblasts causing ineffective erythropoeisis. Normally, the assembly of adult hemoglobin (consisting of a tetramer of two α- and two β-globin chains) features a very tight coordination of α- and β-globin chain synthesis. However, in β-thalassemia, β-globin synthesis is decelerated causing α-globin accumulation; while in α-thalassemia the opposite scenario occurs. Unpaired globin chains that accumulate in thalassemic erythroblasts are bound to heme. In addition, in β-thalassemia an erythroid specific protease destroys excess α-globin chains, likely leading to the generation of a pool of “free” heme in erythroblasts. “Free” heme is toxic, but this toxicity will likely be augmented, if heme oxygenase 1 (HO-1) can release iron from heme. To date, virtually no information about the expression of HO-1 in erythroblasts has been produced; however, we have recently provided unequivocal evidence that this enzyme is present in several model erythroid cells1. Based on this novel and important finding, we hypothesize that in β-thalassemic erythroblasts HO-1 mediated release of iron from heme is the major culprit responsible for cellular damage. To test this hypothesis we exploited the mouse model of β-thalassemia, th3/+. Thus far, our data indicates that HO-1 expression is increased in liver, spleen and kidney of β-thalassemic mice compared to wild type mice. Importantly, we observed that Epo-mediated erythroid differentiation of fetal liver (FL) cells isolated from β-thalassemic fetuses, display increased levels of HO-1 as well as decreased phosphorylated eiF2-α. These results indicate that β-thalassemic erythroblasts have inappropriately high levels of unbound heme that is continuously degraded by HO-1. Further research is needed to determine whether HO-1 liberated iron is responsible for the damage of β-thalassemic erythroblasts.1Garcia Santos D, Schranzhofer M, Bogo Chies JA, Ponka P. Heme Oxygenase 1 plays an unexpected role during erythroid differentiation. Blood (ASH Annual Meeting Abstracts) 118: 344, 2011. Disclosures:No relevant conflicts of interest to declare.

Upregulated MicroRNA-92b Regulates the Differentiation and Proliferation of EpCAM-Positive Fetal Liver Cells by Targeting C/EBPß

microRNAs (miRNAs) are short noncoding RNAs that negatively regulate gene expression. Although recent evidences have been indicated that their aberrant expression may play an important role in cancer stem cells, the mechanism of their deregulation in neoplastic transformation of liver cancer stem cells (LCSCs) has not been explored. In our study, the HCC model was established in F344 rats by DEN induction. The EpCAM+ cells were sorted out from unfractionated fetal liver cells and liver cancer cells using the FACS analysis and miRNA expression profiles of two groups were screened through microarray platform. Gain-of-function studies were performed in vitro and in vivo to determine the role of miR-92b on proliferation and differentiation of the hepatic progenitors. In addition, luciferase reporter system and gene function analysis were used to predict miR-92b target. we found that miR-92b was highly downregulated in EpCAM+ fetal liver cells in expression profiling studies. RT-PCR analysis demonstrated reverse correlation between miR-92b expression and differentiation degree in human HCC samples. Overexpression of miR-92b in EpCAM+ fetal liver cells significantly increased proliferation and inhibited differentiation as well as in vitro and in vivo studies. Moreover, we verified that C/EBPß is a direct target of miR-92b and contributes to its effects on proliferation and differentiation. We conclude that aberrant expression of miR-92b can result in proliferation increase and differentiation arrest of hepatic progenitors by targeting C/EBPß.

Membrane Bioreactor for Expansion and Differentiation of Embryonic Liver Cells

There is a growing demand for the expansion and differentiation of stem cells for cell therapies, tissue engineering, and model systems for drug screening. Current methods for stem cell production are based on the use of batch tissue culture flasks, which have several drawbacks. In this paper we report on the use of a crossed hollow fiber membrane bioreactor for the expansion and differentiation of embryonic liver cells, which have been used as an alternative model of human liver progenitor cells. The bioreactor is based on two bundles of fiber (PEEK-WC HF and PES-HF) which are cross-assembled in an alternating manner. This bioreactor geometry ensures high mass exchange of nutrients and metabolites, which is important for cell proliferation and differentiation. The membrane bioreactor, thanks to its optimized fluid dynamics and mass transport and the adequate surface properties of hollow fibers, was able to guide the expansion and differentiation of liver progenitor cells in mature hepatocytes as demonstrated by their expression of liver specific functions in terms of urea synthesis, albumin production, and diazepam drug biotransformation.

Dynamic Regulation of 5-Hydroxymethylcytosine At the γ-Globin Promoter During Erythroid Differentiation

AbstractAbstract 824DNA methylation is a key element responsible for γ-globin gene repression in adult erythroid cells. Our laboratory previously observed that the γ-globin gene promoter region was demethylated in a progressive manner as γ-globin expression was activated during erythroid differentiation of primary baboon pre-switch fetal liver cells and, to a lesser extent, of adult baboon bone marrow (BM) cells (Singh et al Exp Hematol 35:48-55, 2007). The mechanism responsible for DNA demethylation of the γ-globin promoter during erythroid differentiation remains unknown. Recent studies have shown that DNA demethylation in the early embryo is mediated by the “sixth base” 5-hydroxymethylcytosine (5-hmC) whose formation from 5-methylcytosine is catalyzed by the enzymatic activity of the three TET proteins. To investigate the hypothesis that 5-hmC mediates DNA demethylation of the γ-globin promoter during erythroid differentiation, levels of 5-hmC at Msp I (CCGG) sites within the γ- and ε-globin promoters and γ-globin IVS II region in DNA isolated from peripheral WBC, purified terminal erythroid precursors, and FACS-purified adult bone marrow subpopulations enriched for different stages of differentiation (CD117+ CD36-, CD117+ CD36+, and CD117-CD36+), were analyzed using a T4 glucosylase-MspI quantitative real time PCR assay. Levels of 5-hmC associated with the γ-globin promoter MspI site were 11.6 fold higher (p<.0001) in terminal erythroblasts (n=9) compared to peripheral blood WBC (n=4) while 5-hmC levels associated with the ε-globin promoter were 11.8 fold higher (p<.0001) in terminal erythroid precursors (n=13) compared to peripheral WBC (n=4). Levels of 5-hmC associated with the γ-globin promoter (n=9) were 9.4 fold higher (p<.0001) than with the IVS II region of the γ-globin gene (n=8) in terminal erythroid precursors suggesting that elevated levels of 5-hmC within the β-globin gene complex in terminal erythroid precursors may be localized to promoter regions. Genomic 5-hmC levels, analyzed by HPLC-MS, were similar in WBC and terminal erythroid precursors. Within purified BM subpopulations enriched for different stages of erythroid differentiation, levels of 5-hmC associated with the γ-globin promoter were 3.2 to 4.1 fold higher in the CD117+CD36+ subpopulation enriched in erythroid colony forming cells (n=4) than in CD117+CD36- (n=3; p<.01), more differentiated CD117-CD36+ (n=3; p<.02), and terminal erythroid precursor (p<.001) subpopulations. Similar differences in levels of 5-hmC associated with the ε-globin promoter were also observed between these subpopulations. Enrichment of 5-hmC within the β-globin locus in the CD117+CD36+ and terminal erythroid precursors compared to peripheral WBC was confirmed by 5-hmC affinity selection and PCR analysis. In addition, similar differences in genomic 5-hmC levels between these subpopulations were also observed by HPLC-MS analysis. Bisulfite sequence analysis showed that the changes in 5-hmC levels at the γ-globin promoter were temporally associated with loss of DNA methylation within the γ-globin promoter as erythroid differentiation progressed. TET gene expression analysis showed that TET2 expression was 3 fold less (p<.01) while TET3 expression was >7 fold higher (p<.05) in terminal erythroid precursors compared to the CD117+CD36- subpopulation. These results strongly suggest that differences in 5-hmC associated with the ε- and γ-globin promoters are the result of wider dynamic alterations of genomic 5-hmC levels during erythroid differentiation that may be mediated by differences in TET gene expression and support the hypothesis that 5-hmC is involved in the mechanism responsible for DNA demethylation of the γ-globin promoter during erythroid differentiation. Disclosures:Godley:Celgene: Research Funding.

Designation of enzyme activity of glycine-N-acyltransferase family genes and depression of glycine-N-acyltransferase in human hepatocellular carcinoma

The human glycine-N-acyltransferase (hGLYAT) gene and two related-genes (GLYATL1 and GLYATL2) were isolated. Human GLYAT, GLYATL1, and GLYATL2 cDNAs were isolated and shown to encode polypeptides of 295, 302, and 294 amino acids, respectively. GLYAT catalyzes glycine-N-acyltransfer reaction with benzoyl-CoA acting as a typical aralkyl transferase, while GLYATL1 catalyzed glutamine-N-acyltransfer reaction with phenylacetyl-CoA as an arylacetyl transferase. GLYAT was shown to be expressed specifically in the liver and kidney, and the cellular localization of GLYAT protein was restricted to the mitochondria. Interestingly, labeling using highly affinity purified anti-GLYAT antibody revealed that GLYAT expression was suppressed in all hepatocellular carcinomas, but not in other liver diseases. hGLYAT repression in cancerous cells in the liver was controlled at the transcriptional level. hGLYAT is a good candidate as a novel marker of hepatocellular carcinoma and may be a key molecule in the transition between differentiation and carcinogenesis of liver cells.

Efficient Generation of Hepatoblasts From Human ES Cells and iPS Cells by Transient Overexpression of Homeobox Gene HEX

Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the potential to differentiate into all cell lineages, including hepatocytes, in vitro. Induced hepatocytes have a wide range of potential application in biomedical research, drug discovery, and the treatment of liver disease. However, the existing protocols for hepatic differentiation of PSCs are not very efficient. In this study, we developed an efficient method to induce hepatoblasts, which are progenitors of hepatocytes, from human ESCs and iPSCs by overexpression of the HEX gene, which is a homeotic gene and also essential for hepatic differentiation, using a HEX-expressing adenovirus (Ad) vector under serum/feeder cell-free chemically defined conditions. Ad-HEX-transduced cells expressed α-fetoprotein (AFP) at day 9 and then expressed albumin (ALB) at day 12. Furthermore, the Ad-HEX-transduced cells derived from human iPSCs also produced several cytochrome P450 (CYP) isozymes, and these P450 isozymes were capable of converting the substrates to metabolites and responding to the chemical stimulation. Our differentiation protocol using Ad vector-mediated transient HEX transduction under chemically defined conditions efficiently generates hepatoblasts from human ESCs and iPSCs. Thus, our methods would be useful for not only drug screening but also therapeutic applications.

Biodegradable and synthetic membranes for the expansion and functional differentiation of rat embryonic liver cells

The insufficient availability of donor organs for orthotopic liver transplantation worldwide has urgently increased the requirement for new therapies for acute and chronic liver disease. The creation of an unlimited source of donor cells for hepatocyte transplantation therapy and pharmaceutical applications may be the isolation and expansion of liver progenitor cells or stem cells. Here we report the expansion and functional differentiation of rat embryonic liver cells on biodegradable and synthetic polymeric membranes in comparison with traditional substrates, such as collagen and polystyrene culture dishes. Membranes prepared from chitosan and modified polyetheretherketone were used for the culture of liver progenitor cells derived from rat embryonic liver. Cells proliferated, with a significant increase in their number within 8–11 days. The cells displayed functional differentiation showing urea synthesis, albumin production and diazepam biotransformation on all substrates investigated. In particular, on a chitosan membrane liver-specific functions were expressed at significantly higher levels for prolonged times compared with other synthetic membranes, utilizing traditional substrates (collagen and PSCD) as references. These results demonstrate that chitosan membranes offer cells favourable conditions to promote the expansion and functional differentiation of embryonic liver cells that could be effectively used in liver tissue engineering and in pharmaceutical applications.

Three-Dimensional Perfusion Bioreactor Culture Supports Differentiation of Human Fetal Liver Cells

The ability of human fetal liver cells to survive, expand, and form functional tissue in vitro is of high interest for the development of bioartificial extracorporeal liver support systems, liver cell transplantation therapies, and pharmacologic models. Conventional static two-dimensional culture models seem to be inadequate tools. We focus on dynamic three-dimensional perfusion technologies and developed a scaled-down bioreactor, providing decentralized mass exchange with integral oxygenation. Human fetal liver cells were embedded in a hyaluronan hydrogel within the capillary system to mimic an in vivo matrix and perfusion environment. Metabolic performance was monitored daily, including glucose consumption, lactate dehydrogenase activity, and secretion of alpha-fetoprotein and albumin. At culture termination cells were analyzed for proliferation and liver-specific lineage-dependent cytochrome P450 (CYP3A4/3A7) gene expression. Occurrence of hepatic differentiation in bioreactor cultures was demonstrated by a strong increase in CYP3A4/3A7 gene expression ratio, lower alpha-fetoprotein, and higher albumin secretion than in conventional Petri dish controls. Cells in bioreactors formed three-dimensional structures. Viability of cells was higher in bioreactors than in control cultures. In conclusion, the culture model implementing three-dimensionality, constant perfusion, and integral oxygenation in combination with a hyaluronan hydrogel provides superior conditions for liver cell survival and differentiation compared to conventional culture.