Hybrid poly-l-lactic acid/poly(ε-caprolactone) nanofibrous scaffold can improve biochemical and molecular markers of human induced pluripotent stem cell-derived hepatocyte-like cells.

A suitable alternative strategy for liver transplantation is the use of nanofibrous scaffolds together with stem cells. In this study, a random hybrid of poly-l-lactic acid (PLLA) and poly(ε-caprolactone) (PCL) was used as a three-dimensional (3D) culture for differentiation of hepatocyte-like cells and compared with routine culture (two-dimensional [2D]). The expression of the endodermal marker, forkhead box A2 (FOXA2), was assessed on Day 3 and the hepatic markers; albumin (ALB), α-1 antitrypsin (AAT), and cytokeratin-18 (CK-18) were evaluated on Day 18 using quantitative polymerase chain reaction qPCR. As well as, ALB, α-fetoprotein (AFP), and low-density lipoprotein (LDL) uptake were evaluated using immunocytochemistry; moreover, periodic acid-Schiff and Oil Red were done by cell staining. In addition, AFP and urea production were evaluated by chemiluminescence and colorimetric assays. Light and scanning electron microscopy (SEM) showed changes in the cells in 2D and 3D models. The gene expression of hepatic markers was significantly higher in the 3D cultures. In addition, immunocytochemistry and cell staining showed that ALB, AFP, LDL-uptake, periodic acid-Schiff, and Oil Red were expressed in both cells derived on 2D and 3D. Furthermore, the evaluation of AFP and urea secretion was significantly different between 2D and 3D strategies. These findings suggest that functionally cells cultured on a PLLA/PCL scaffold may be suitable for cell therapy and regenerative medicine.

Triple Staining Including FOXA2 Identifies Stem Cell Lineages Undergoing Hepatic and Biliary Differentiation in Cirrhotic Human Liver.

Recent investigations have reported many markers associated with human liver stem/progenitor cells, "oval cells," and identified "niches" in diseased livers where stem cells occur. However, there has remained a need to identify entire lineages of stem cells as they differentiate into bile ducts or hepatocytes. We have used combined immunohistochemical staining for a marker of hepatic commitment and specification (FOXA2 [Forkhead box A2]), hepatocyte maturation (Albumin and HepPar1), and features of bile ducts (CK19 [cytokeratin 19]) to identify lineages of stem cells differentiating toward the hepatocytic or bile ductular compartments of end-stage cirrhotic human liver. We identified large clusters of disorganized, FOXA2 expressing, oval cells in localized liver regions surrounded by fibrotic matrix, designated as "micro-niches." Specific FOXA2-positive cells within the micro-niches organize into primitive duct structures that support both hepatocytic and bile ductular differentiation enabling identification of entire lineages of cells forming the two types of structures. We also detected expression of hsa-miR-122 in primitive ductular reactions expected for hepatocytic differentiation and hsa-miR-23b cluster expression that drives liver cell fate decisions in cells undergoing lineage commitment. Our data establish the foundation for a mechanistic hypothesis on how stem cell lineages progress in specialized micro-niches in cirrhotic end-stage liver disease.

Human periapical cyst-mesenchymal stem cells differentiate into neuronal cells.

It was recently reported that human periapical cysts (hPCys), a commonly occurring odontogenic cystic lesion of inflammatory origin, contain mesenchymal stem cells (MSCs) with the capacity for self-renewal and multilineage differentiation. In this study, periapical inflammatory cysts were compared with dental pulp to determine whether this tissue may be an alternative accessible tissue source of MSCs that retain the potential for neurogenic differentiation. Flow cytometry and immunofluorescence analysis indicated that hPCy-MSCs and dental pulp stem cells spontaneously expressed the neuron-specific protein ß-III tubulin and the neural stem-/astrocyte-specific protein glial fibrillary acidic protein (GFAP) in their basal state before differentiation occurs. Furthermore, undifferentiated hPCy-MSCs showed a higher expression of transcripts for neuronal markers (ß-III tubulin, NF-M, MAP2) and neural-related transcription factors (MSX-1, Foxa2, En-1) as compared with dental pulp stem cells. After exposure to neurogenic differentiation conditions (neural media containing epidermal growth factor [EGF], basic fibroblast growth factor [bFGF], and retinoic acid), the hPCy-MSCs showed enhanced expression of ß-III tubulin and GFAP proteins, as well as increased expression of neurofilaments medium, neurofilaments heavy, and neuron-specific enolase at the transcript level. In addition, neurally differentiated hPCy-MSCs showed upregulated expression of the neural transcription factors Pitx3, Foxa2, Nurr1, and the dopamine-related genes tyrosine hydroxylase and dopamine transporter. The present study demonstrated for the first time that hPCy-MSCs have a predisposition toward the neural phenotype that is increased when exposed to neural differentiation cues, based on upregulation of a comprehensive set of proteins and genes that define neuronal cells. In conclusion, these results provide evidence that hPCy-MSCs might be another optimal source of neural/glial cells for cell-based therapies to treat neurologic diseases.

Molecular mechanisms of hepatic apoptosis regulated by nuclear factors.

Apoptosis is a prominent characteristic in the pathogenesis of liver disease. The mechanism of hepatic apoptosis is not well understood. Hepatic apoptosis alters relative levels of nuclear factors such as Foxa2, NF-κB, C/EBPß, and p53. Regulation of nuclear factors modulates the degree of hepatic apoptosis and the progression of liver disease. Nuclear factors have distinctive mechanisms to mediate hepatic apoptosis. The modification of nuclear factors is a novel therapeutic strategy for liver disease as demonstrated by pre-clinical models and clinical trials.

Expression and regulation of Foxa2 in the rat uterus during early pregnancy.

The forkhead box a (Foxa) protein family has been found to play important roles in mammals. Recently, the expression of Foxa2 was reported in the mouse uterus, and it was reported to be involved in regulation of implantation. However, the regulation of Foxa2 expression in the uterus is still poorly understood. Therefore, the present study was conducted to investigate the expressional profiles of Foxa2 in the rat uterus during the estrus cycle and pregnancy. Furthermore, the effect of steroid hormones and Hedgehog protein on the expression of Foxa2 was analyzed in vivo and in vitro. In this study, the level of expression of Foxa2 was low in the rat uterus during the different stages of the estrus cycle. However, the expression increased transiently during early pregnancy at 3.5 days post coitus (dpc) and decreased at 5.5 dpc. In ovariectomized rats, P4 treatment had no effect on the expression of Foxa2 compared with the expression in control animals. Moreover, the expression of Foxa2 in cultured epithelial cells was not increased by P4 treatment in vitro. However, Foxa2 expression was significantly decreased in the rat uterus after 24 h of E2 treatment. Treatment of cells with a recombinant Hedgehog protein significantly increased the expression of Foxa2. These results suggest that the expression of Foxa2 may transiently increase just before the implantation and it may be regulated by E2 and Hedgehog protein.

Hedgehog signaling regulates FOXA2 in esophageal embryogenesis and Barrett's metaplasia.

Metaplasia can result when injury reactivates latent developmental signaling pathways that determine cell phenotype. Barrett's esophagus is a squamous-to-columnar epithelial metaplasia caused by reflux esophagitis. Hedgehog (Hh) signaling is active in columnar-lined, embryonic esophagus and inactive in squamous-lined, adult esophagus. We showed previously that Hh signaling is reactivated in Barrett's metaplasia and overexpression of Sonic hedgehog (SHH) in mouse esophageal squamous epithelium leads to a columnar phenotype. Here, our objective was to identify Hh target genes involved in Barrett's pathogenesis. By microarray analysis, we found that the transcription factor Foxa2 is more highly expressed in murine embryonic esophagus compared with postnatal esophagus. Conditional activation of Shh in mouse esophageal epithelium induced FOXA2, while FOXA2 expression was reduced in Shh knockout embryos, establishing Foxa2 as an esophageal Hh target gene. Evaluation of patient samples revealed FOXA2 expression in Barrett's metaplasia, dysplasia, and adenocarcinoma but not in esophageal squamous epithelium or squamous cell carcinoma. In esophageal squamous cell lines, Hh signaling upregulated FOXA2, which induced expression of MUC2, an intestinal mucin found in Barrett's esophagus, and the MUC2-processing protein AGR2. Together, these data indicate that Hh signaling induces expression of genes that determine an intestinal phenotype in esophageal squamous epithelial cells and may contribute to the development of Barrett's metaplasia.

Pyocyanin-induced mucin production is associated with redox modification of FOXA2.

BACKGROUND: The redox-active pyocyanin (PCN) is a toxic, secondary metabolite secreted by the respiratory pathogen Pseudomonas aeruginosa (PA). Previously, we have shown that mouse lungs chronically exposed to PCN develop goblet cell hyperplasia and metaplasia (GCHM) and mucus hypersecretion, fibrosis and emphysema. These pathological features are commonly found in the airways of several chronic lung diseases, including cystic fibrosis (CF), as well as in mouse airways deficient in the forkhead box A2 (FOXA2), a transcriptional repressor of goblet GCHM and mucus biosynthesis. Furthermore, PCN inhibits FOXA2 by activating the pro-GCHM signaling pathways Stat6 and EGFR. However, it is not known whether PCN-generated reactive oxygen (ROS) and nitrogen (RNS) species posttranslationally modify and inactivate FOXA2. METHODS: We examined the posttranslational modifications of FOXA2 by PCN using specific antibodies against oxidation, nitrosylation, acetylation and ubiquitination. Electrophoretic mobility shift assay (EMSA) was used to examine the ability of modified FOXA2 to bind the promoter of MUC5B mucin gene. In addition, we used quantitative real time PCR, ELISA, immunofluorescence and mouse lung infection to assess whether the loss of FOXA2 function caused GCHM and mucin overexpression. Finally, we examined the restoration of FOXA2 function by the antioxidant glutathione (GSH). RESULTS: We found that PCN-generated ROS/RNS caused nitrosylation, acetylation, ubiquitination and degradation of FOXA2. Modified FOXA2 had reduced ability to bind the promoter of the MUC5B gene. The antioxidant GSH alleviated the modification of FOXA2 by PCN, and inhibited the overexpression of MUC5AC and MUC5B mucins. CONCLUSION: These results suggest that PCN-mediated posttranslational modifications of FOXA2 are positively correlated with GCHM and overexpression of airway mucins. Furthermore, antioxidant treatment restores the function of FOXA2 to attenuate GCHM and mucus hypersecretion.

Microarray analysis of papillary thyroid cancers in Korean.

BACKGROUND/AIMS: Papillary thyroid cancer (PTC) is the most common malignancy of the thyroid gland. It involves several molecular mechanisms. The BRAF V600E mutation has been identified as the most common genetic abnormality in PTC. Moreover, it is known to be more prevalent in Korean PTC patients than in patients from other countries. We investigated distinct genetic profiles in Korean PTC through cDNA microarray analysis. METHODS: Transcriptional profiles of five PTC samples and five paired normal thyroid tissue samples were generated using cDNA microarrays. The tumors were genotyped for BRAF mutations. The results of the cDNA microarray gene expression analysis were confirmed by real-time PCR and immunohistochemistry analysis of 35 PTC patients. RESULTS: Four of the five patients whose PTC tissues were subjected to microarray analysis were found to carry the BRAF V600E mutation. Microarrays analysis of the five PTC tissue samples showed the expression of 96 genes to be increased and that of 16 genes decreased. Real-time reverse transcription-polymerase chain reaction (RT-PCR) confirmed increased expression of SLC34A2, TM7SF4, COMP, KLK7, and KCNJ2 and decreased expression of FOXA2, SLC4A4, LYVE-1, and TFCP2L1 in PTC compared with normal tissue. Of these genes, TFCP2L1, LYVE-1, and KLK7 were previously unidentified in PTC microarray analysis. Notably, Foxa2 activity in PTC was reduced, as shown by its cytoplasmic localization, in immunohistochemical analyses. CONCLUSIONS: These findings demonstrate both similarities and differences between our results and previous reports. In Korean cases of PTC, Foxa2 activity was reduced with its cytoplasmic accumulation. Further studies are needed to confirm the relationship between FOXA2 and BRAF mutations in Korean cases of PTC.

Microarray Analysis of Papillary Thyroid Cancers in Korean

BACKGROUND/AIMS: Papillary thyroid cancer (PTC) is the most common malignancy of the thyroid gland. It involves several molecular mechanisms. The BRAF V600E mutation has been identified as the most common genetic abnormality in PTC. Moreover, it is known to be more prevalent in Korean PTC patients than in patients from other countries. We investigated distinct genetic profiles in Korean PTC through cDNA microarray analysis. METHODS: Transcriptional profiles of five PTC samples and five paired normal thyroid tissue samples were generated using cDNA microarrays. The tumors were genotyped for BRAF mutations. The results of the cDNA microarray gene expression analysis were confirmed by real-time PCR and immunohistochemistry analysis of 35 PTC patients. RESULTS: Four of the five patients whose PTC tissues were subjected to microarray analysis were found to carry the BRAF V600E mutation. Microarrays analysis of the five PTC tissue samples showed the expression of 96 genes to be increased and that of 16 genes decreased. Real-time reverse transcription-polymerase chain reaction (RT-PCR) confirmed increased expression of SLC34A2, TM7SF4, COMP, KLK7, and KCNJ2 and decreased expression of FOXA2, SLC4A4, LYVE-1, and TFCP2L1 in PTC compared with normal tissue. Of these genes, TFCP2L1, LYVE-1, and KLK7 were previously unidentified in PTC microarray analysis. Notably, Foxa2 activity in PTC was reduced, as shown by its cytoplasmic localization, in immunohistochemical analyses. CONCLUSIONS: These findings demonstrate both similarities and differences between our results and previous reports. In Korean cases of PTC, Foxa2 activity was reduced with its cytoplasmic accumulation. Further studies are needed to confirm the relationship between FOXA2 and BRAF mutations in Korean cases of PTC.

Molecular interactions between HNF4a, FOXA2 and GABP identified at regulatory DNA elements through ChIP-sequencing.

Gene expression is regulated by combinations of transcription factors, which can be mapped to regulatory elements on a genome-wide scale using ChIP experiments. In a previous ChIP-chip study of USF1 and USF2 we found evidence also of binding of GABP, FOXA2 and HNF4a within the enriched regions. Here, we have applied ChIP-seq for these transcription factors and identified 3064 peaks of enrichment for GABP, 7266 for FOXA2 and 18783 for HNF4a. Distal elements with USF2 signal was frequently bound also by HNF4a and FOXA2. GABP peaks were found at transcription start sites, whereas 94% of FOXA2 and 90% of HNF4a peaks were located at other positions. We developed a method to accurately define TFBS within peaks, and found the predicted sites to have an elevated conservation level compared to peak centers; however the majority of bindings were not evolutionary conserved. An interaction between HNF4a and GABP was seen at TSS, with one-third of the HNF4a positive promoters being bound also by GABP, and this interaction was verified by co-immunoprecipitations.

Tumour necrosis factor-alpha attenuates insulin action on phosphoenolpyruvate carboxykinase gene expression and gluconeogenesis by altering the cellular localization of Foxa2 in HepG2 cells.

Circulating tumour necrosis factor-alpha (TNFalpha) levels, which are elevated in obesity-associated insulin resistance and diabetes, inhibit insulin signalling at several points in the signalling cascade. The liver is critical in maintaining circulating glucose levels and, in a preliminary investigation using the human hepatoma (HepG2) cell line in this study, we demonstrated the role of TNFalpha in the regulation of this phenomenon and determined the underlying molecular mechanisms. As the transcription factor Foxa2 has been implicated, in part, in the regulation of gluconeogenic genes, we studied the effects of TNFalpha and/or insulin on its cellular status in hepatocytes, followed by an assessment of its occupancy on the phosphoenolpyruvate carboxykinase (PEPCK) promoter. Preincubation of cells with TNFalpha, followed by insulin, significantly prevented insulin-mediated nuclear exclusion of Foxa2 and substantially increased its nuclear concentration. Foxa2 was subsequently found to occupy its binding element on the PEPCK promoter. TNFalpha alone, however, did not alter the status of cellular Foxa2 or its occupancy on the PEPCK promoter. TNFalpha preincubation also significantly attenuated insulin-induced inhibition of the expression of gluconeogenic enzymes and hepatic glucose production. Insulin inhibition of PEPCK expression and the preventive effect of TNFalpha could be partially but significantly restored in the presence of Foxa2 siRNA. Several other well-known mediators of insulin action in the liver in general and of gluconeogenic genes in particular include Foxo1, PGC-1 and SREBP-1c. Our results indicate that another transcription factor, Foxa2, is at least partly responsible for the attenuating effect of TNFalpha on insulin action on PEPCK expression and glucose production in HepG2 cells.

Identification of DNA-dependent protein kinase as a cofactor for the forkhead transcription factor FoxA2.

Forkhead factors are important regulators of animal development and homeostasis. They are among the earliest to bind quiescent genes, which they activate in conjunction with other transcription factors. Many liver-specific genes are under the control of FoxA2, a liver-enriched forkhead protein. Here we confirmed by chromatin immunoprecipitation that FoxA2 is one of the factors bound to the promoter-proximal enhancer of the gene encoding apolipoprotein AI (a component of high density lipoprotein) and that it functions in synergy with the nuclear receptor hepatocyte nuclear factor-4alpha. Furthermore, toward identifying additional cofactors that could potentially regulate FoxA2 activity, we identified DNA-dependent protein kinase (DNA-PK) as a FoxA2-associated factor upon affinity purification of epitope-tagged FoxA2. We show that FoxA2, found to be a phosphoprotein in vivo, is also an efficient substrate for DNA-PK, which targets serine 283. This residue is contained within a conserved serine-glutamine phosphorylation signal for DNA-PK, located within the C-terminal third of the polypeptide, just distal to its winged-helix DNA binding domain. We establish that this residue is critical for FoxA2 function because FoxA2 bearing a mutation at this site is severely compromised in its ability to activate a reporter gene under the control of its cognate DNA-binding site (apoAI site B). Complementary experiments rule out that this mutation compromises the ability of FoxA2 to either translocate to the nucleus or to bind site B. We therefore conclude that DNA-PK-dependent phosphorylation of FoxA2 plays a critical role in its transcriptional activation function per se.

The correlation of hepatocyte nuclear factor 4 alpha and 3 beta with hepatitis B virus replication in the liver of chronic hepatitis B patients.

Hepatocyte nuclear factors 4 alpha (HNF4alpha) and 3 beta (HNF3beta) are members of a group of liver-enriched transcription factors (LETFs) that play important roles in regulating the replication of hepatitis B virus (HBV). Using cell culture and animal models, we showed that HNF4alpha supports HBV replication in nonhepatic cells and HNF3beta inhibits HBV replication. However, the expression of HNF4alpha and HNF3beta in the liver tissue of chronic HBV-infected patients and the relationship between the levels of HNF4alpha and HNF3beta and HBV replication are unclear. In this study, liver biopsy specimens from 86 chronic HBV-infected patients were collected. The expression levels of HNF4alpha, HNF3beta, hepatitis B surface antigen (HBsAg) and hepatitis B core antigen (HBcAg) were detected by an immunohistochemical technique and the level of HBV DNA was checked by in situ hybridization with serial sections from liver biopsy tissue samples. We show here that samples with higher levels of HNF4alpha expression also have higher levels of HBsAg, HBcAg and HBV DNA. In contrast, in samples with higher levels of HNF3beta expression, levels of HBsAg, HBcAg and HBV DNA were lower. There was a positive correlation between HNF4alpha expression and HBV replication, and a negative correlation between HNF3beta expression and HBV replication, in the liver of chronic HBV-infected patients. This suggests that HNF4alpha and HNF3beta likely participate in HBV replication in patients with HBV infection, or that HBV replication may somehow influence the expression of HNF4alpha and HNF3beta in the liver.

Origin of androgen-insensitive poorly differentiated tumors in the transgenic adenocarcinoma of mouse prostate model.

Following castration, the transgenic adenocarcinoma of mouse prostate (TRAMP) model demonstrates rapid development of SV40-Tag-driven poorly differentiated tumors that express neuroendocrine cell markers. The cell population dynamics within the prostates of castrated TRAMP mice were characterized by analyzing the incorporation of 5-bromodeoxyuridine (BrdUrd) and the expression of SV40-Tag, synaptophysin, and androgen receptor (AR). Fourteen days postcastration, the remaining epithelial cells and adenocarcinoma cells were nonproliferative and lacked detectable SV40-Tag or synaptophysin expression. In contrast, morphologically distinct intraglandular foci were identified which expressed SV40-Tag, synaptophysin, and Ki67, but that lacked AR expression. These proliferative SV40-Tag and synaptophysin-expressing intraglandular foci were associated with the rare BrdUrd-retaining cells. These foci expanded rapidly in the postcastration prostate environment, in contrast to the AR- and SV40-Tag-expressing adenocarcinoma cells that lost SV40-Tag expression and underwent apoptosis after castration. Intraglandular foci of synaptophysin-expressing cells were also observed in the prostates of intact TRAMP mice at a comparable frequency; however, they did not progress to rapidly expanding tumors until much later in the life of the mice. This suggests that the foci of neuroendocrine-like cells that express SV40-Tag and synaptophysin, but lack AR, arise independent of androgen-deprivation and represent the source of the poorly differentiated tumors that are the lethal phenotype in the TRAMP model.

N-cadherin is a useful marker for the progenitor of cardiomyocytes differentiated from mouse ES cells in serum-free condition.

Cardiomyocytes are known to differentiate spontaneously from embryonic stem (ES) cells when they formed aggregates, so called "embryoid bodies", in the presence of serum. In this study, we explored the induction of cardiomyocytes from mouse ES cells in chemically defined serum-free medium by using a mesoderm-inducing factor, BMP4. Comparing the different inductive methods, we found a candidate cell surface marker, N-cadherin, for cardiomyocyte progenitors from ES cells. N-cadherin-positive cells highly expressed cardiogenic markers, Nkx2.5, Tbx5, and Isl1, and showed a high differentiation rate into cardiomyocyte lineage. These results indicate that N-cadherin can be a useful cell surface marker for the progenitors of cardiomyocyte differentiated from ES cells in the serum-free culture.

Expression and role of Foxa proteins in prostate cancer.

The molecular mechanism(s) for prostate cancer progression to androgen independence are poorly understood. We have recently shown that Foxa1 and Foxa2 proteins are differentially expressed in epithelial cells during murine prostate development, growth, and adult function. Currently, the role of Foxa proteins in prostate cancer development and progression is unknown. Foxa protein expression was investigated in the LPB-Tag LADY mouse prostate cancer models, in human prostate cancer specimens, and various prostate cancer cell lines using Western blot and immunostaining analysis. In vitro transient transfection, studies were performed to investigate Foxa/prostate-specific gene regulation. Foxa1 was strongly expressed in areas of prostatic intraepithelial neoplasia (PIN) in both the androgen dependent 12T-7f and in the metastatic, androgen independent 12T-10 LADY models. Prominent Foxa1 and Foxa2 expression was observed in 12T-10 invasive undifferentiated neuroendocrine carcinomas, in the hormone independent and metastasizing 12T-10 derived, NE-10 allograft tumors, and in all metastatic lesions isolated from 12T-10 mice. Foxa1 protein expression was always observed in human prostate carcinomas, regardless of Gleason grade score, while Foxa2 was only detected in neuroendocrine small cell carcinomas and in some high Gleason score adenocarcinomas. Foxa proteins were also differentially expressed in three prostate cancer cell lines. Importantly, in vitro functional assays demonstrated that Foxa2 could activate androgen-dependent prostate-specific genes in an androgen receptor and ligand-independent manner. These results suggest that Foxa proteins are important in prostate carcinogenesis. In particular, Foxa2 may be involved in progression of prostate cancer to androgen independence. As such, Foxa proteins may represent novel targets for therapeutic intervention.