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Background Pancreaticobiliary reflux (PBR) causes chronic inflammation of the gallbladder mucosa and changes in the bile components, which are known to promote gallstone formation. This study aimed to investigate the bile biochemistry changes in gallstone patients with PBR and provide new clues for research on the involvement of PBR in gallstone formation. Methods Patients undergoing surgery for gallstones between December 2020 and May 2021 were eligible for inclusion. The bile biochemistry (including amylase, lipase, triglyceride, cholesterol, free fatty acids [FFAs], alanine aminotransferase [ALT], aspartate aminotransferase [AST], alkaline phosphatase [ALP], and γ-glutamyl transferase [γ-GT]) of the included gallstone patients was analysed to determine correlations with PBR. Results In this study, 144 gallstone patients who underwent surgery were enrolled. Overall, 15.97 % of the patients had an increased bile amylase level, which was associated with older age and significantly higher bile levels of ALP, lipase, triglyceride, and FFAs. Positive correlations were observed between amylase and lipase, triglyceride, FFAs levels in the gallbladder bile. However, the bile levels of triglyceride, FFAs, and lipase were positively correlated with each other only in the PBR group and showed no significant correlation in the control (N) group. In addition, elevated bile FFAs levels were found to be an independent risk factor for gallbladder wall thickening. Conclusions In conclusion, PBR-induced increase in FFAs and triglyceride in the gallbladder bile is a cause of gallstone formation, and an increase in bile ALP suggests the presence of cholestasis in PBR.

Introduction: Occult pancreaticobiliary reflux (OPBR) causes chronic inflammation of the gallbladder and associated mucosal malignant changes, but its pathogenic mechanism remains unclear. Objective The aim of this study was to investigate the pathogenic mechanism in OPBR through untargeted metabolomic analysis of bile collected during surgery. Methods Patients undergoing surgery for gallstones between December 2020 and May 2021 were eligible for inclusion. There were 30 OPBR patients and 338 non-OPBR patients, and 30 non-OPBR patients were randomly selected as normal controls. Metabolomics analysis was performed based on bile samples from patients in the OPBR group. Results The principal component analysis by liquid chromatography-mass spectrometry revealed that 30 metabolites were significantly different between patients with OPBR compared to controls. Among these characteristic metabolites, palmitic acid and arachidonic acid are known to be involved in gallstone formation, methionine and lysophosphatidylcholine are known to be associated with carcinogenesis. Conclusions The present study suggested that an LC/MS-based metabolomic studies provide an efficient and promising method for identifying potential biomarkers of OPBR. The identified characteristic metabolites in the OPBR group were significantly different to the control group.

Objective N6-methyladenosine (m6A) RNA methylation involve in governing mechanism of tumor progression. We aimed to excavate the biological role and mechanism of m6A methyltransferase METTL3 in Cholangiocarcinoma (CCA). Methods METTL3 expression was determined by database and tissue microarray. The role of METTL3 in CCA was explored by loss- and gain-of-function experiments. m6A target of METTL3 was detected by RNA sequencing. The role of AKR1B10 in CCA was explored and the association between METTL3 and AKR1B10 was confirmed by rescue experiments. Result METTL3 expression was upregulated in CCA tissue and higher METTL3 expression was implicated a poor prognosis in CCA patients. Overexpression of METTL3 facilitated proliferation, migration, invasion, glucose uptake, and lactate production of CCA cells, whereas knockdown of METTL3 had the opposite effect. We further found that METTL3 deficiency inhibited tumor growth of CCA in vivo. RNA sequencing and MeRIP-qPCR confirmed that METTL3 enhanced AKR1B10 expression and m6A modification level. Further, METTL3 directly bind with AKR1B10 at an m6A modification site also confirmed. CCA tissue microarray showed that AKR1B10 expression was upregulated in CCA tissue and silencing of AKR1B10 suppressed the malignant phenotype mentioned-above in CCA. Notably, knockdown of AKR1B10 rescued the tumor-promoting effects induced by METTL3 overexpression. Conclusion Elevated METTL3 expression promotes tumor growth and glycolysis in CCA through m6A modification of AKR1B10, indicating that METTL3 is a potential target for blocking glycolysis to apply in CCA therapy.

The purpose of this study was to demonstrate the aberrant metabolism of bile acids in patients with cholesterol gallstone and explore for its underlying mechanisms. The composition of bile acids collected from the patients with cholelithiasis and the control individuals was analyzed by LC-MS. The expression of genes regulating the metabolism of bile acids was quantitatively determined by real-time PCR or Western blot analysis. Cholesterol saturation index of patients with gallstone was significantly higher than that of the controls. The concentrations of taurodeoxycholic acid and taurolithocholic acid in the bile of patients were significantly higher than that of the controls. When compared with the controls, it was remarkable in the patients that the mRNA expression of farnesoid X receptor (FXR) was lower, whereas that of organic anion transporting polypeptide (OATP1A2) was higher. However, the expressions of both mRNA and protein of cytochrome P-450 family 8 subfamily B member 1 (CYP8B1) did not differ between the patients and the controls. Although the protein level of CYP8B1 was significantly lower in the subjects with single nucleotide polymorphism (SNP) rs3732860(G), the composition of bile acids and the ratio of CA to CDCA remained unaltered in the patients with different SNP genotype of CYP8B1. In conclusion, the axis of FXR-OATP1A2 that physiologically regulated the reabsorption of bile acids might play an important role in the composition of bile acids and the development of gallstone. CYP8B1 gene was irrelevant to the altered composition of bile acids in patients with gallstone.NEW & NOTEWORTHY For the first time, our results indicate that the axis of farnesoid X receptor-organic anion transporter polypeptide 1A2 that physiologically regulates the reabsorption of bile acids might play an important role in the regulation of the composition of bile acids and make contribution to the development of cholelithiasis.

In this commentary we discuss a paper we published recently on the activities of the GTPase RhoA during neural differentiation of murine embryonic stem cells, and relate our findings to previous studies. We narrate how we found that RhoA impedes neural differentiation by inhibiting the production as well as the secretion of noggin, a soluble factor that antagonizes bone morphogenetic protein. We discuss how the questions we tried to address shaped the study, and how embryonic stem cells isolated from a genetically modified mouse model devoid of Syx, a RhoA-specific guanine exchange factor, were used to address them. We detail several signaling pathways downstream of RhoA that are hindered by the absence of Syx, and obstructed by retinoic acid, resulting in an increase of noggin production; we explain how the lower RhoA activity and, consequently, the sparser peri-junctional stress fibers in Syx−/− cells facilitated noggin secretion; and we report unpublished results showing that pharmacological inhibition of RhoA accelerates the neuronal differentiation of human embryonic stem cells. Finally, we identify signaling mechanisms in our recent study that warrant further study, and speculate on the possibility of manipulating RhoA signaling in combination with other pathways to drive the differentiation of neuronal subtypes.

SummaryAberrant hexanucleotide repeat expansions in C9orf72 are the most common genetic change underlying amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). RNA transcripts containing these expansions undergo repeat-associated non-ATG translation (RAN-T) to form five dipeptide repeat proteins (DPRs). DPRs are found as aggregates throughout the CNS of C9orf72-ALS/FTD patients, and some cause degeneration when expressed in vitro in neuronal cultures and in vivo in animal models. The spread of characteristic disease-related proteins drives the progression of pathology in many neurodegenerative diseases. While DPR toxic mechanisms continue to be investigated, the potential for DPRs to spread has yet to be determined. Using different experimental cell culture platforms, including spinal motor neurons derived from induced pluripotent stem cells from C9orf72-ALS patients, we found evidence for cell-to-cell spreading of DPRs via exosome-dependent and exosome-independent pathways, which may be relevant to disease.

It is well documented that adult neural stem cells (NSCs) residing in the subventricular zone (SVZ) and the subgranular zone (SGZ) are induced to proliferate and differentiate into new neurons after injury such as stroke and hypoxia. However, the role of injury‐related cues in driving this process and the means by which they communicate with NSCs remains largely unknown. Recently, the coupling of neurogenesis and angiogenesis and the extensive close contact between vascular cells and other niche cells, known as the neurovascular unit (NVU), has attracted interest. Further facilitating communication between blood and NSCs is a permeable blood‐brain‐barrier (BBB) present in most niches, making vascular cells a potential conduit between systemic signals, such as vascular endothelial growth factor (VEGF), and NSCs in the niche, which could play an important role in regulating neurogenesis. We show that the leaky BBB in stem cell niches of the intact and stroke brain can respond to circulating VEGF165 to drive induction of the Notch ligand DLL4 (one of the most important cues in angiogenesis) in endothelial cells (ECs), pericytes, and further induce significant proliferation and neurogenesis of stem cells. Stem Cells 2019;37:395–406, Circulating VEGF and DLL4 pathway in the stem cell niche. Circulating VEGF regulates NSC activity by direct or indirect routes via compromised blood‐brain barrier in the leaky stem cell niche.

Parkinson’s disease (PD) is characterized by the selective degeneration of dopamine (DA) neurons of the substantia nigra pars compacta (SN), while the neighboring ventral tegmental area (VTA) is relatively spared. The mechanisms underlying this selectivity are not fully understood. Here, we demonstrate a vital role for subregional astrocytes in the protection of VTA DA neurons. We found that elimination of astrocytes in vitro exposes a novel vulnerability of presumably protected VTA DA neurons to the PD mimetic toxin MPP(+), as well as exacerbation of SN DA neuron vulnerability. Conversely, VTA astrocytes protected both VTA and SN DA neurons from MPP(+) toxicity in a dose dependent manner, and this protection was mediated via a secreted molecule. RNAseq analysis of isolated VTA and SN astrocytes demonstrated a vast array of transcriptional differences between these two closely related populations demonstrating regional heterogeneity of midbrain astrocytes. We found that GDF15, a member of the TGFβ superfamily which is expressed 230-fold higher in VTA astrocytes than SN, recapitulates neuroprotection of both rat midbrain and iPSC-derived DA neurons, whereas its knockdown conversely diminished this effect. Neuroprotection was likely mediated through the GRFAL receptor expressed on DA neurons. Together; these results suggest that subregional differences in astrocytes underlie the selective degeneration or protection of DA neurons in PD.

Table S1. List of primers used for qRT-PCR. (DOCX 29 kb)

Recruitment of transcription factors (TFs) to repressed genes in euchromatin is essential to activate new transcriptional programs during cell differentiation. However, recruitment of all TFs, including pioneer factors, is impeded by condensed H3K27me3-containing chromatin. Single-cell and gene-specific analyses revealed that, during the first hours of induction of differentiation of mammalian embryonic stem cells (ESCs), accumulation of the repressive histone mark H3K27me3 is delayed after DNA replication, indicative of a decondensed chromatin structure in all regions of the replicating genome. This delay provides a critical "window of opportunity" for recruitment of lineage-specific TFs to DNA. Increasing the levels of post-replicative H3K27me3 or preventing S phase entry inhibited recruitment of new TFs to DNA and significantly blocked cell differentiation. These findings suggest that recruitment of lineage-specifying TFs occurs soon after replication and is facilitated by a decondensed chromatin structure. This insight may explain the developmental plasticity of stem cells and facilitate their exploitation for therapeutic purposes.

Cisplatin is widely used as the standard gastric cancer treatment, but the relapse and metastasis are common as intrinsic or acquired drug resistance. CD133 has been widely known to be associated with chemoresistance in various cancer cells. In this study, we focused on investigating the function and mechanism of CD133 underlying cisplatin resistance in gastric cancer cell line KATO-III. We detected CD133 expression by using quantitative real-time polymerase chain reaction and Western blot and found that expression of CD133 was upregulated in cisplatin resistance of KATO-III cells (Cis-KATO-III) compared with KATO-III cells, indicating the role of CD133 in regulating cisplatin resistance of KATO-III cells. Then we sorted the Cis-KATO-III cells into CD133-positive (CD133+) pools and measured the proliferation and apoptosis after the cell is transfected with pc-CD133 and sh-CD133 by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay and flow cytometry. The results showed that the inhibition of CD133 inhibited the cell viability and promoted the cell apoptosis after cisplatin treatment. Furthermore, we found that inhibition of CD133 downregulated the expression of PI3K/AKT and promoted the expression of mammalian target of rapamycin, thus inhibited the autophagic activity in the Cis-KATO-III cells after cisplatin treatment. Besides, we also verified the effects of CD133 in vivo. The results indicated that inhibition of CD133 enhanced the Cis-KATO-III cell sensitivity to cisplatin by regulating PI3K/AKT/mTOR signaling pathway. In summary, our data provide new insight that CD133 activates the PI3K/AKT/mTOR signaling transduction pathway, resulting in activation of autophagy and cisplatin resistance of Cis-KATO-III cells. These results may offer a novel therapeutic target in cisplatin-resistant gastric cancer.

Background Dimethyl fumarate (DMF), working via its metabolite monomethylfumarate (MMF), acts as a potent antioxidant and immunomodulator in animal models of neurologic disease and in patients with multiple sclerosis. These properties and their translational potential led us to investigate whether DMF/MMF could also protect at-risk and/or dying neurons in models of ischemic stroke in vitro and in vivo. Although the antioxidant effects have been partially addressed, the benefits of DMF immunomodulation after ischemic stroke still need to be explored. Methods In vitro neuronal culture with oxygen-glucose deprivation and rats with middle cerebral artery occlusion were subjected to DMF/MMF treatment. Live/dead cell counting and LDH assay, as well as behavioral deficits, plasma cytokine assay, western blots, real-time PCR (Q-PCR) and immunofluorescence staining, were used to evaluate the mechanisms and neurological outcomes. Results We found that MMF significantly rescued cortical neurons from oxygen-glucose deprivation (OGD) in culture and suppressed pro-inflammatory cytokines produced by primary mixed neuron/glia cultures subjected to OGD. In rats, DMF treatment significantly decreased infarction volume by nearly 40 % and significantly improved neurobehavioral deficits after middle cerebral artery occlusion (MCAO). In the acute early phase (72 h after MCAO), DMF induced the expression of transcription factor Nrf2 and its downstream mediator HO-1, important for the protection of infarcted cells against oxidative stress. In addition to its antioxidant role, DMF also acted as a potent immunomodulator, reducing the infiltration of neutrophils and T cells and the number of activated microglia/macrophages in the infarct region by more than 50 % by 7–14 days after MCAO. Concomitantly, the levels of potentially harmful pro-inflammatory cytokines were greatly reduced in the plasma and brain and in OGD neuron/glia cultures. Conclusions We conclude that DMF is neuroprotective in experimental stroke because of its potent immunomodulatory and antioxidant effects and thus may be useful as a novel therapeutic agent to treat stroke in patients. Electronic supplementary material The online version of this article (doi:10.1186/s12974-016-0733-1) contains supplementary material, which is available to authorized users.

Backgound The purpose of this study was to assess the biological and clinical effects of n-acetyl-cysteine (NAC) in Parkinson’s disease (PD). Methods The overarching goal of this pilot study was to generate additional data about potentially protective properties of NAC in PD, using an in vitro and in vivo approach. In preparation for the clinical study we performed a cell tissue culture study with human embryonic stem cell (hESC)-derived midbrain dopamine (mDA) neurons that were treated with rotenone as a model for PD. The primary outcome in the cell tissue cultures was the number of cells that survived the insult with the neurotoxin rotenone. In the clinical study, patients continued their standard of care and were randomized to receive either daily NAC or were a waitlist control. Patients were evaluated before and after 3 months of receiving the NAC with DaTscan to measure dopamine transporter (DAT) binding and the Unified Parkinson’s Disease Rating Scale (UPDRS) to measure clinical symptoms. Results The cell line study showed that NAC exposure resulted in significantly more mDA neurons surviving after exposure to rotenone compared to no NAC, consistent with the protective effects of NAC previously observed. The clinical study showed significantly increased DAT binding in the caudate and putamen (mean increase ranging from 4.4% to 7.8%; p

Cell replacement therapy could be an important treatment strategy for Parkinson's disease (PD), which is caused by the degeneration of dopamine neurons in the midbrain (mDA). The success of this approach greatly relies on the discovery of an abundant source of cells capable of mDAergic function in the brain. With the paucity of available human fetal tissue, efforts have increasingly focused on renewable stem cells. Human induced pluripotent stem (hiPS) cells offer great promise in this regard. If hiPS cells can be differentiated into authentic mDA neuron, hiPS could provide a potential autologous source of transplant tissue when generated from PD patients, a clear advantage over human embryonic stem (hES) cells. Here, we report that mDA neurons can be derived from a commercially available hiPS cell line, IMR90 clone 4, using a modified hES differentiation protocol established in our lab. These cells express all the markers (Lmx1a, Aldh1a1, TH, TrkB), follow the same mDA lineage pathway as H9 hES cells, and have similar expression levels of DA and DOPAC. Moreover, when hiPS mDA progenitor cells are transplanted into 6-OHDA-lesioned PD rats, they survive long term and many develop into bona fide mDA neurons. Despite their differentiation and integration into the brain, many Nestin+ tumor-like cells remain at the site of the graft. Our data suggest that as with hES cells, selecting the appropriate population of mDA lineage cells and eliminating actively dividing hiPS cells before transplantation will be critical for the future success of hiPS cell replacement therapy in PD patients.

Recent studies have provided important insight into the homeoprotein LIM homeobox transcription factor 1α (Lmx1a) and its role in the commitment of cells to a midbrain dopamine (mDA) fate in the developing mouse. We show here that Lmx1a also plays a pivotal role in the mDA differentiation of human embryonic stem (hES) cells. Thus, as indicated by small interfering RNA experiments, the transient early expression of Lmx1a is necessary for the coordinated expression of all other dopamine (DA)-specific phenotypic traits as hES cells move from multipotent human neural progenitor cells (hNPs) to more restricted precursor cells in vitro. Moreover, only Lmx1a-specified hNPs have the potential to differentiate into bona fide mDA neurons after transplantation into the 6-hydroxydopamine-treated rat striatum. In contrast, cortical human neuronal precursor cells (HNPCs) and mouse subventricular zone cells do not express Lmx1a or become mDA neurons even when placed in an environment that fosters their DA differentiation in vitro or in vivo. These findings suggest that Lmx1a may be critical to the development of mDA neurons from hES cells and that, along with other key early DA markers (i.e., Aldh1a1), may prove to be extremely useful for the selection of appropriately staged and suitably mDA-specified hES cells for cell replacement in Parkinson's disease.

Neurogenesis in the dentate gyrus (DG) declines severely by middle age, potentially because of age-related changes in the DG microenvironment. We hypothesize that providing fresh glial restricted progenitors (GRPs) or neural stem cells (NSCs) to the aging hippocampus via grafting enriches the DG microenvironment and thereby stimulates the production of new granule cells from endogenous NSCs. The GRPs isolated from the spinal cords of embryonic day 13.5 transgenic F344 rats expressing human alkaline phosphatase gene and NSCs isolated from embryonic day 9 caudal neural tubes of Sox-2:EGFP transgenic mice were expanded in vitro and grafted into the hippocampi of middle-aged (12 months old) F344 rats. Both types of grafts survived well, and grafted NSCs in addition migrated to all layers of the hippocampus. Phenotypic characterization revealed that both GRPs and NSCs differentiated predominantly into astrocytes and oligodendrocytic progenitors. Neuronal differentiation of graft-derived cells was mostly absent except in the dentate subgranular zone (SGZ), where some of the migrated NSCs but not GRPs differentiated into neurons. Analyses of the numbers of newly born neurons in the DG using 5′-bromodeoxyuridine and/or doublecortin assays, however, demonstrated considerably increased dentate neurogenesis in animals receiving grafts of GRPs or NSCs in comparison with both naïve controls and animals receiving sham-grafting surgery. Thus, both GRPs and NSCs survive well, differentiate predominantly into glia, and stimulate the endogenous NSCs in the SGZ to produce more new dentate granule cells following grafting into the aging hippocampus. Grafting of GRPs or NSCs therefore provides an attractive approach for improving neurogenesis in the aging hippocampus. Disclosure of potential conflicts of interest is found at the end of this article.

Multipotent neural stem cells (NSC) have been derived from human embryonic stem cells (hESC) as well as isolated from fetal tissues. However, there have been few exclusive markers of NSC identified to date, and the differences between NSC from various sources are poorly understood. Although cells isolated from these two sources share many important characteristics, it is not clear how closely they are related in terms of gene expression. Here, we compare the gene expression profiles of 11 lines of NSC derived from hESC (ES_NSC), four lines of NSC isolated from fetus (F_NSC), and two lines of restricted progenitors in order to characterize these cell populations and identify differences between NSC derived from these two sources. We showed that ES_NSC were clustered together with high transcriptional similarities but were distinguished from F_NSC, oligodendrocyte precursor cells, and astrocyte precursor cells. There were 17 genes expressed in both ES_NSC and F_NSC whose expression was not identified in restricted neural progenitors. Furthermore, the major differences between ES_NSC and F_NSC were mostly observed in genes related to the key neural differentiation pathways. Here, we show that comparison of global gene expression profiles of ES_NSC, F_NSC, and restricted neural progenitor cells makes it possible to identify some of the common characteristics of NSC and differences between similar stem cell populations derived from hESCs or isolated from fetal tissue. Disclosure of potential conflicts of interest is found at the end of this article.

The identification of neural stem cells (NSCs) in situ has been prevented by the inability to identify a marker consistently expressed in all adult NSCs and is thus generally accomplished using the in vitro neurosphere-forming assay. The high-mobility group transcription factor Sox2 is expressed in embryonic neural epithelial stem cells; because these cells are thought to give rise to the adult NSC population, we hypothesized that Sox2 may continue to be expressed in adult NSCs. Using Sox2:EGFP transgenic mice, we show that Sox2 is expressed in neurogenic regions along the rostral-caudal axis of the central nervous system throughout life. Furthermore, all neurospheres derived from these neurogenic regions express Sox2, suggesting that Sox2 is indeed expressed in adult NSCs. We demonstrate that NSCs are heterogeneous within the adult brain, with differing capacities for cell production. In vitro, all neurospheres express Sox2, but the expression of markers common to early progenitor cells within individual neurospheres varies; this heterogeneity of NSCs is mirrored in vivo. For example, both glial fibrillary acidic protein and NG2 are expressed within individual neurospheres, but their expression is mutually exclusive; likewise, these two markers show distinct staining patterns within the Sox2+ regions of the brain's neurogenic regions. Thus, we propose that the expression of Sox2 is a unifying characteristic of NSCs in the adult brain, but that not all NSCs maintain the ability to form all neural cell types in vivo.

The mechanisms that determine whether neural stem cells remain in a proliferative state or differentiate into neurons or glia are largely unknown. Here we establish a pivotal role for gap junction-mediated intercellular communication in determining the proliferation and survival of mouse neural progenitor cells (NPCs). When cultured in the presence of basic fibroblast growth factor (bFGF), NPCs express the gap junction protein connexin 43 and are dye-coupled. Upon withdrawal of bFGF, levels of connexin 43 and dye coupling decrease, and the cells cease proliferating and differentiate into neurons; the induction of gap junctions by bFGF is mediated by p42/p44 mitogen-activated protein kinases. Inhibition of gap junctions abolishes the ability of bFGF to maintain NPCs in a proliferative state resulting in cell differentiation or cell death, while overexpression of connexin 43 promotes NPC self-renewal in the absence of bFGF. In addition to promoting their proliferation, gap junctions are required for the survival of NPCs. Gap junctional communication is therefore both necessary and sufficient to maintain NPCs in a self-renewing state.

Multiple classes of precursor cells have been isolated and characterized from the developing spinal cord including multipotent neuroepithelial (NEP) stem cells and lineage-restricted precursors for neurons (NRPs) and glia (GRPs). We have compared the survival, differentiation and integration of multipotent NEP cells with lineage-restricted NRPs and GRPs using cells isolated from transgenic rats that express the human placental alkaline phosphatase gene. Our results demonstrate that grafted NEP cells survive poorly, with no cells observed 3 days after transplant in the adult hippocampus, striatum and spinal cord, indicating that most CNS regions are not compatible with transplants of multipotent cells derived from fetal CNS. By contrast, at 3 weeks and 5 weeks post-engraftment, lineage-restricted precursors showed selective migration along white-matter tracts and robust survival in all three CNS regions. The grafted precursors expressed the mature neuronal markers NeuN and MAP2, the astrocytic marker GFAP, the oligodendrocytic markers RIP, NG2 and Sox-10, and the synaptic marker synaptophysin. Similar behavior was observed when these precursors were transplanted into the injured spinal cord. Predifferentiated, multipotent NEP cells also survive and integrate, which indicates that lineage-restricted CNS precursors are well suited for transplantation into the adult CNS and provide a promising cellular replacement candidate.

We have characterized several potential stem cell markers and defined the membrane properties of rat fetal (E10.5) neural stem cells (NSC) by immunocytochemistry, electrophysiology and microarray analysis. Immunocytochemical analysis demonstrates specificity of expression of Sox1, ABCG2/Bcrp1, and shows that nucleostemin labels both progenitor and stem cell populations. NSCs, like hematopoietic stem cells, express high levels of aldehyde dehydrogenase (ALDH) as assessed by Aldefluor labeling. Microarray analysis of 96 transporters and channels showed that Glucose transporter 1 (Glut1/Slc2a1) expression is unique to fetal NSCs or other differentiated cells. Electrophysiological examination showed that fetal NSCs respond to acetylcholine and its agonists, such as nicotine and muscarine. NSCs express low levels of tetrodotoxin (TTX) sensitive and insensitive sodium channels and calcium channels while expressing at least three kinds of potassium channels. We find that gap junction communication is mediated by connexin (Cx)43 and Cx45, and is essential for NSC survival and proliferation. Overall, our results show that fetal NSCs exhibit a unique signature that can be used to determine their location and assess their ability to respond to their environment.

Fetal neural stem cells (NSCs) have received great attention not only for their roles in normal development but also for their potential use in the treatment of neurodegenerative disorders. To develop a robust method of assessing the state of stem cells, we have designed, tested, and validated a rodent NSC array. This array consists of 260 genes that include cell type-specific markers for embryonic stem (ES) cells and neural progenitor cells as well as growth factors, cell cycle-related genes, and extracellular matrix molecules known to regulate NSC biology. The 500-bp polymerase chain reaction products amplified and validated by using gene-specific primers were arrayed along with positive controls. Blanks were included for quality control, and some genes were arrayed in duplicate. No cross-hybridization was detected. The quality of the arrays and their sensitivity were also examined by using probes prepared by conventional reverse transcriptase or by using amplified probes prepared by linear polymerase replication (LPR). Both methods showed good reproducibility, and probes prepared by LPR labeling appeared to detect expression of a larger proportion of expressed genes. Expression detected by either method could be verified by RT-PCR with high reproducibility. Using these stem cell chips, we have profiled liver, ES, and neural cells. The cell types could be readily distinguished from each other. Nine markers specific to mouse ES cells and 17 markers found in neural cells were verified as robust markers of the stem cell state. Thus, this focused neural stem array provides a convenient and useful tool for detection and assessment of NSCs and progenitor cells and can reliably distinguish them from other cell populations.

Nitric oxide (NO) is believed to act as an intercellular signal that regulates synaptic plasticity in mature neurons. We now report that NO also regulates the proliferation and differentiation of mouse brain neural progenitor cells (NPCs). Treatment of dissociated mouse cortical neuroepithelial cluster cell cultures with the NO synthase inhibitor L-NAME or the NO scavenger hemoglobin increased cell proliferation and decreased differentiation of the NPCs into neurons, whereas the NO donor sodium nitroprusside inhibited NPC proliferation and increased neuronal differentiation. Brain-derived neurotrophic factor (BDNF) reduced NPC proliferation and increased the expression of neuronal NO synthase (nNOS) in differentiating neurons. The stimulatory effect of BDNF on neuronal differentation of NPC was blocked by L-NAME and hemoglobin, suggesting that NO produced by the latter cells inhibited proliferation and induced neuronal differentiation of neighboring NPCs. A similar role for NO in regulating the switch of neural stem cells from proliferation to differentiation in the adult brain is suggested by data showing that NO synthase inhibition enhances NPC proliferation and inhibits neuronal differentiation in the subventricular zone of adult mice. These findings identify NO as a paracrine messenger stimulated by neurotrophin signaling in newly generated neurons to control the proliferation and differentiation of NPC, a novel mechanism for the regulation of developmental and adult neurogenesis.

To access and compare gene expression in fetal neuroepithelial cells (NEPs) and progenitor cells, we have used microarrays containing approximately 500 known genes related to cell cycle regulation, apoptosis, growth and differentiation. We have identified 152 genes that are expressed in NEPs and 209 genes expressed by progenitor cells. The majority of genes (141) detected in NEPs are also present in progenitor populations. There are 68 genes specifically expressed in progenitors with little or no expression in NEPs, and a few genes that appear to be present exclusively in NEPs. Using cell sorting, RT-PCR, in situ hybridization or immunocytochemistry, we have examined the segregation of expression to neuronal and glial progenitors, and identified several that appeared to be enriched in neuronal (e.g. CDK5, neuropilin, EphrinB2, FGF11) or glial (e.g. CXCR4, RhoC, CD44, tenascin C) precursors. Our data provide a first report of gene expression profiles of neural stem and progenitor cells at early stages of development, and provide evidence for the potential roles of specific cell cycle regulators, chemokines, cytokines and extracellular matrix molecules in neural development and lineage segregation.

Gallbladder cancer (GBC) is the most common and aggressive type of biliary tract cancer. The study of potential treatments for GBC has recently focused on microRNAs, a class of small non-coding RNAs, which post-transcriptionally regulate gene expression during various crucial cell processes. The present study aimed to investigate the role of microRNA-146b (miR-146b) in GBC. Human GBC tissue and adjacent normal gallbladder tissue sections were surgically removed and miR-146b-5p expres- sion and the development and pathological characteristics of GBC were investigated. miR-146b-5p expression was reduced in GBC tissue compared with that in adjacent tissue, and a significant correlation was observed between miR-146b-5p expression levels and carcinoma size and devel- opment. miR-146b-5p overexpression in the SGC-996 GBC cell line inhibited cell growth through enhanced apoptosis and G1 phase arrest. Furthermore, it was demonstrated that epidermal growth factor receptor (EGFR) was regulated directly by miR-146b-5p and was essential as a mediator of the biological effects of miR-146b-5p in GBC. Enforced expression of EGFR reversed the ability of miR-146b-5p to inhibit proliferation. In conclusion, the present study indi- cated that the mechanism of action of miR-146b-5p in GBC involves the regulation of EGFR expression.

Parkinson disease (PD) is the second leading neurodegenerative disease in the US. As there is no known cause or cure for PD, researchers continue to investigate disease mechanisms and potential new therapies in cell culture and in animal models of PD. In PD, one of the most profoundly affected neuronal populations is the tyrosine hydroxylase (TH)-expressing dopaminergic (DA) neurons of the substantia nigra pars compacta (SNpc). These DA-producing neurons undergo degeneration while neighboring DA-producing cells of the ventral tegmental area (VTA) are largely spared. To aid in these studies, The Michael J. Fox Foundation (MJFF) partnered with Thomas Jefferson University and Taconic Inc. to generate new transgenic rat lines carrying the human TH gene promoter driving EGFP using a 11 kb construct used previously to create a hTH-GFP mouse reporter line. Of the five rat founder lines that were generated, three exhibited high level specific GFP fluorescence in DA brain structures (ie. SN, VTA, striatum, olfactory bulb, hypothalamus). As with the hTH-GFP mouse, none of the rat lines exhibit reporter expression in adrenergic structures like the adrenal gland. Line 12141, with its high levels of GFP in adult DA brain structures and minimal ectopic GFP expression in non-DA structures, was characterized in detail. We show here that this line allows for anatomical visualization and microdissection of the rat midbrain into SNpc and/or VTA, enabling detailed analysis of midbrain DA neurons and axonal projections after toxin treatment in vivo. Moreover, we further show that embryonic SNpc and/or VTA neurons, enriched by microdissection or FACS, can be used in culture or transplant studies of PD. Thus, the hTH-GFP reporter rat should be a valuable tool for Parkinson's disease research.

Production of new neurons throughout adulthood has been well characterized in two brain regions, the subventricular zone (SVZ) of the anterolateral ventricle and the subgranular zone (SGZ) of the hippocampus. The neurons produced from these regions arise from neural stem cells (NSCs) found in highly regulated stem cell niches. We recently showed that midline structures called circumventricular organs (CVOs) also contain NSCs capable of neurogenesis and/or astrogliogenesis in vitro and in situ (Bennett et al.). The present study demonstrates that NSCs derived from two astrogliogenic CVOs, the median eminence and organum vasculosum of the lamina terminalis of the nestin-GFP mouse, possess the potential to integrate into the SVZ and differentiate into cells with a neuronal phenotype. These NSCs, following expansion and BrdU-labeling in culture and heterotopic transplantation into a region proximal to the SVZ in adult mice, migrate caudally to the SVZ and express early neuronal markers (TUC-4, PSA-NCAM) as they migrate along the rostral migratory stream. CVO-derived BrdU(+) cells ultimately reach the olfactory bulb where they express early (PSA-NCAM) and mature (NeuN) neuronal markers. Collectively, these data suggest that although NSCs derived from the ME and OVLT CVOs are astrogliogenic in situ, they produce cells phenotypic of neurons in vivo when placed in a neurogenic environment. These findings may have implications for neural repair in the adult brain.

Although embryonic stem (ES) cells can generate dopamine (DA) neurons that are potentially useful as a cell replacement therapy in Parkinson's disease (PD), associated ethical and practical concerns remain major stumbling blocks to their eventual use in humans. In this study, we examined human amniotic fluid stem (hAFS) cells derived from routine amniocenteses for their potential to give rise to DA neurons in vitro and following transplantation into the 6-hydroxydopamine-lesioned rat brain. We show that undifferentiated hAFS cells constitutively expressed mRNAs and proteins typical of stem cells but also cell derivatives of all three germ layers, including neural progenitors/neurons (nestin, beta-tubulin III, neurofilament). Additionally, these cells expressed mRNAs of an immature DA phenotype (Lmx1a, Pitx-3, Nurr1, Aldh1a1) but not the corresponding proteins. Importantly, treatment with DA differentiation factors using a variety of protocols did not further promote the development of fully differentiated DA neurons from hAFS cells. Thus, Lmx1a, Aldh1a1, AADC, TH, and DAT proteins were not detected in hAFS cells in culture or after transplantation into the PD rat brain. Moreover, by 3 weeks after implantation, there were no surviving AFS cells in the graft, likely as a result of an acute immunorejection response, as evidenced by the abundant presence of CD11+ macrophage/microglia and reactive GFAP+ astrocytes in the host brain. Taken together, these results suggest that further studies will be needed to improve differentiation procedures in culture and to prolong cell survival in vivo if hAFS cells are to be useful as replacement cells in PD.

BackgroundWe initiated differentiation of human embryonic stem cells (hESCs) into dopamine neurons, obtained a purified population of neuronal precursor cells by cell sorting, and determined patterns of gene transcription.MethodologyDopaminergic differentiation of hESCs was initiated by culturing hESCs with a feeder layer of PA6 cells. Differentiating cells were then sorted to obtain a pure population of PSA-NCAM-expressing neuronal precursors, which were then analyzed for gene expression using Massive Parallel Signature Sequencing (MPSS). Individual genes as well as regions of the genome which were activated were determined.Principal findingsA number of genes known to be involved in the specification of dopaminergic neurons, including MSX1, CDKN1C, Pitx1 and Pitx2, as well as several novel genes not previously associated with dopaminergic differentiation, were expressed. Notably, we found that a specific region of the genome located on chromosome 11p15.5 was highly activated. This region contains several genes which have previously been associated with the function of dopaminergic neurons, including the gene for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, IGF2, and CDKN1C, which cooperates with Nurr1 in directing the differentiation of dopaminergic neurons. Other genes in this region not previously recognized as being involved in the functions of dopaminergic neurons were also activated, including H19, TSSC4, and HBG2. IGF2 and CDKN1C were also found to be highly expressed in mature human TH-positive dopamine neurons isolated from human brain samples by laser capture.ConclusionsThe present data suggest that the H19-IGF2 imprinting region on chromosome 11p15.5 is involved in the process through which undifferentiated cells are specified to become neuronal precursors and/or dopaminergic neurons.

Background The identification of molecular pathways of differentiation of embryonic stem cells (hESC) is critical for the development of stem cell based medical therapies. In order to identify biomarkers and potential regulators of the process of differentiation, a high quality microarray containing 16,659 seventy base pair oligonucleotides was used to compare gene expression profiles of undifferentiated hESC lines and differentiating embryoid bodies. Results Previously identified "stemness" genes in undifferentiated hESC lines showed down modulation in differentiated cells while expression of several genes was induced as cells differentiated. In addition, a subset of 194 genes showed overexpression of greater than ≥ 3 folds in human embryoid bodies (hEB). These included 37 novel and 157 known genes. Gene expression was validated by a variety of techniques including another large scale array, reverse transcription polymerase chain reaction, focused cDNA microarrays, massively parallel signature sequencing (MPSS) analysis and immunocytochemisty. Several novel hEB specific expressed sequence tags (ESTs) were mapped to the human genome database and their expression profile characterized. A hierarchical clustering analysis clearly depicted a distinct difference in gene expression profile among undifferentiated and differentiated hESC and confirmed that microarray analysis could readily distinguish them. Conclusion These results present a detailed characterization of a unique set of genes, which can be used to assess the hESC differentiation.

The precise lineage between neural stem cells and mature astrocytes remains poorly defined. To examine astrocyte development, we have characterized glial precursors from neural tissue derived from early embryonic ages. We show that CD44 identifies an astrocyte-restricted precursor cell (ARP) that is committed to generating astrocytes in vitro and in vivo in both rodent and human tissue. CD44+ cells arise later in development than neuronal-restricted precursors (NRPs) or tripotential glial-restricted precursors (GRPs). ARPs are distinguished from GRP and NRP cells by their antigenic profile and differentiation ability. ARPs can be generated from GRP cells in mass or clonal cultures and in vivo after transplantation, suggesting a sequential differentiation of neuroepithelial stem cells (NEPs) to GRPs to ARPs and then to astrocytes. The properties of ARPs are different from other astrocyte precursors described previously in their expression of CD44 and S-100h and absence of other lineage markers. Using a CD44 misexpression transgenic mouse model (CNP-CD44 mouse), we show that CD44 overexpression in vivo and in vitro decreases the number of mature glia and increases the number of O4+/GFAP+ cells tenfold. Misexpression of CD44 in culture inhibits oligodendrocytes and arrests cells at the precursor state. In summary, our data provide strong evidence for the existence of a CD44+ ARP in the developing nervous system. Published by Elsevier Inc.

Stem cells have been identified and characterized in a variety of tissues. In this review we examine possible shared properties of stem cells. We suggest that irrespective of their lineal origin, stem cells have to respond in similar ways to regulate self-renewal and differentiation and it is likely that cell-cycle control, asymmetry/differentiation controls, cellular protective and DNA repair mechanisms, and associated apoptosis/senescence signaling pathways all might be expected to be more highly regulated in stem cells, likely by similar mechanisms. We review the literature to suggest a set of candidate stemness genes that may serve as universal stem cell markers. While we predict many similarities, we also predict that differences will exist between stem cell populations and that when transdifferentiation is considered genes expected to be both similar and different need to be examined.

Multipotent neural stem cells (NSCs) present in the developing neural tube (E10.5, neuroepithelial cells; NEP) were examined for the expression of candidate stem cell markers, and the expression of these markers was compared with later appearing precursor cells (E14.5) that can be distinguished by the expression of embryonic neural cell adhesion molecule (E-NCAM) and A2B5. NEP cells possess gap junctions, express connexins, and appear to lack long cilia. Most candidate markers, including Nestin, Presenilin, Notch, and Numb, were expressed by both NEP cells as well as other cell populations. Fibroblast growth factor receptor 4 (FGFR4), Frizzled 9 (Fz9), and SRY box-containing gene 2 (Sox2) as assessed by immunocytochemistry and in situ hybridization are markers that appear to distinguish NSCs from other precursor cells. Neither Hoechst 33342 nor rhodamine-123 staining, telomerase (Tert) expression, telomerase activity, or breakpoint cluster region protein 1 (Bcrp1) transporter expression could be used to distinguish NEP stem cells from other dividing cells. NEP cells, however, lacked expression of several lineage markers that are expressed by later appearing cells. These included absence of expression of CD44, E-NCAM, A2B5, epidermal growth factor receptor (EGFR), and platelet-derived growth factor receptor-alpha (PDGFRα), suggesting that negative selection using cell surface epitopes could be used to isolate stem cell populations from mixed cultures of cells. Using mixed cultures of cells isolated from E14.5 stage embryos, we show that NEP cells can be enriched by depleting differentiating cells that express E-NCAM or A2B5 immunoreactivity. Overall, our results show that a spectrum of markers used in combination can reliably distinguish multipotent NSCs from other precursor cells as well as differentiated cells present in the CNS.

Ovarian cancer is ranked as the 4th most common type of cancer among women in the United States. Lysophosphatidic acid (LPA) is the diagnostic marker of ovarian cancer. It is significantly elevated in 90% of ovarian cancer patients. Moreover, LPA receptors are aberrantly up-regulated in ovarian cancer patients. LPA is a type of G protein-coupled receptor ligand. In our previous studies, we have demonstrated that a signaling axis consisting of beta-arrestin 2 and CARMA3 (CARD and MAGUK domain-containing protein 3) is required in LPA-induced nuclear factor kappa B (NF-κB) activation in mouse embryonic fibroblast cells. NF-κB is an important transcription factor, which has been proven to mediate LPA-induced ovarian cancer migration and invasion. In this study, we further investigated whether the beta-arrestin2-CARMA3 signaling axis plays a significant role in LPA-induced ovarian cancer cell migration and invasion. Using beta-arrestin 2 and CARMA3 shRNA, we knockdowned their protein expression levels in ovarian cancer cell lines. Consistent with previous reports, we found that down-regulation of beta-arrestin 2 and CARMA3 abolished LPA-induced IKK activity and NF-κB activation in ovarian cancer cells. In addition, in vitro transwell migration and matrigel invasion assays demonstrated that beta-arrestin 2 and CARMA3 shRNA significantly impaired LPA-induced ovarian cancer cell motility and invasiveness. Together, our results provide the evidence that beta-arrestin 2 and CARMA3 serve as critical regulators in LPA-induced, NF-κB-mediated ovarian cancer migration and invasion. Therefore, we speculate that beta-arrestin 2 and CARMA3 may represent attractive therapeutic targets for ovarian cancer and many other types of malignancies. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B22

Ovarian cancer is ranked as the 4th most common type of cancer among women in the United States. One of the biomarkers of ovarian cancer is lysophosphatidic acid (LPA). LPA is a type of G protein-coupled receptor ligand. It is strikingly elevated in 90% of ovarian cancer patients. Additionally, LPA receptors are aberrantly up-regulated in ovarian cancer patients. We have reported previously that a signaling axis consisting of beta-arrestin 2 and CARMA3 (CARD and MAGUK domain-containing protein 3) is indispensable in LPA-induced nuclear factor kappa B (NF-kappaB) activation in mouse embryonic fibroblast cells. NF-kappaB is an important transcription factor, which has been suggested to mediate LPA-induced ovarian cancer migration and invasion. However, it remains unknown whether the beta-arrestin2-CARMA3 signaling axis plays an important role in LPA-induced ovarian cancer cell migration and invasion. In the present study, using beta-arrestin 2 and CARMA3 shRNA, we knockdowned their protein expression levels in ovarian cancer cell lines. Consistent with previous reports, we found that down-regulation of beta-arrestin 2 and CARMA3 abolished LPA-induced IKK activity and NF-kappaB activation in ovarian cancer cells. In addition, in vitro transwell migration and matrigel invasion assays demonstrated that beta-arrestin 2 and CARMA3 shRNA significantly impaired LPA-induced ovarian cancer cell motility and invasiveness. Together, our results provide the evidence that beta-arrestin 2 and CARMA3 serve as critical regulators in LPA-induced, NF-kappaB-mediated ovarian cancer migration and invasion. Therefore, we speculate that beta-arrestin 2 and CARMA3 may represent attractive therapeutic targets for ovarian cancer and many other types of malignancies. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5286.

Ovarian cancer is the fourth leading cause of cancer death among women in the United States. One of the biomarkers of ovarian cancer is lysophosphatidic acid (LPA). LPA is a type of G protein-coupled receptor ligand. It is strikingly elevated in 90% of ovarian cancer patients. Additionally, LPA receptors are aberrantly up-regulated in ovarian cancer patients. We have reported previously that a signaling axis consisting of beta-arrestin 2 and CARMA3 (CARD and MAGUK domain-containing protein 3) is indispensable in LPA-induced nuclear factor kappa B (NF-kappaB) activation in mouse embryonic fibroblast cells. NF-kappaB is an important transcription factor, which has been suggested to mediate LPA-induced ovarian cancer migration and invasion. However, it remains unknown whether the beta-arrestin2-CARMA3 signaling axis plays an important role in LPA-induced ovarian cancer cell migration and invasion. In the present study, using beta-arrestin 2 and CARMA3 shRNA, we knockdowned their protein expression levels in ovarian cancer cell lines. Consistent with previous reports, we found that down-regulation of beta-arrestin 2 and CARMA3 abolished LPA-induced IKK activity and NF-kappaB activation in ovarian cancer cells. In addition, in vitro transwell migration and matrigel invasion assays demonstrated that beta-arrestin 2 and CARMA3 shRNA significantly impaired LPA-induced ovarian cancer cell motility and invasiveness. Together, our results provide the evidence that β-arrestin 2 and CARMA3 serve as critical regulators in LPA-induced, NF-kappaB-mediated ovarian cancer migration and invasion. Therefore, we speculate that β-arrestin 2 and CARMA3 may represent attractive therapeutic targets for ovarian cancer and many other types of malignancies. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B41.