Temporal Dynamics of Spontaneous Ca2+ Transients, ERBB4, vGLUT1, GAD1, Connexin, and Pannexin Genes in Early Stages of Human Stem Cell Neurodifferentiation

Spontaneous Ca2+ transients drive stem cell proliferation and neurodifferentiation. Deciphering the relationship between neuronal and glial human genes on one side and spontaneous Ca2+ activity on the other side is essential for our understanding of normal brain development, and for insights into the pathogenesis of neurodegenerative and neurodevelopmental disorders. In the present study, forebrain neurons were derived from human embryonic and induced pluripotent stem cells (hESC-H9 and iPSC-15; 22q11.2 deletion) over a period of 21 days in vitro (DIV). Every 1–2 days, multisite optical imaging technique was applied to detect populations of cells with spontaneous Ca2+ transients. The expression levels of 14 genes of interest were analyzed by quantitative polymerase chain reaction (qPCR) on the same biological samples where physiological recordings were performed. The genes analyzed include: the schizophrenia candidate gene ERBB4, connexin (Cx) genes Cx26, Cx36, Cx43, Cx45, Cx47, pannexin-1 (PNX1), neuronal markers PAX6, vGLUT1, GAD1, TUBB3, glial lineage markers BLBP, GFAP, and housekeeping gene ACTB. We found that Ca2+ signals decrease in amplitude, decrease in duration, and increase in frequency during the first 21 days of human neurodifferentiation. The expression levels of ERBB4, PAX6, GAD1, vGLUT1, BLBP, Cx36, Cx45, and PNX1 were found to be strongly positively correlated with the percentage of cells exhibiting spontaneous Ca2+ transients (“Active Cells”). While expression of BLBP, Cx45, ERBB4, GAD1, PAX6, PNX1, and vGLUT1 were correlated with short-duration and long-amplitude Ca2+ transients, Cx43, TUBB3, and Cx47 were better correlated with long-duration and short-amplitude transients. The expression dynamics of Cx26 was unrelated to any aspect of spontaneous Ca2+ activity. Four genes showed an exponential time course with a distinct onset on a given DIV. The onset of PNX1, ERBB4, and vGLUT1 occurred before, while the onset of Cx36 occurred after the first action potentials were detected in early differentiating human neurons

Epigenetic Alterations in RASSF1A in Human Aberrant Crypt Foci

CpG island methylation (CIM) is an epigenetic mechanism for transcriptional silencing that occurs at various stages of colon tumorigenesis. CIM has been found in serrated adenomas and hyperplastic polyps. There is also evidence for hypermethylation in aberrant crypt foci (ACF) that are found in resected colons from cancer patients. Our study addresses promoter methylation of a tumor suppressor gene, RASSF1A, within the colonic epithelium of subjects undergoing screening colonoscopies in the absence of synchronous tumors. Patients included in this study were at elevated risk for colorectal cancer (CRC) based on family history, but without a previously occurring or synchronous colon carcinoma. ACF were identified using close-focus magnifying chromendoscopy and collected by biopsy in situ. We isolated ACF and adjacent normal colonic epithelium by laser capture microdissection (LCM) and studied methylation of the RASSF1A promoter region in ACF and in adjacent normal mucosa. Expression of RASSF1A was verified using quantitative real-time polymerase chain reaction (QRT–PCR). We found that 8.6% (3 out of 35) of ACF had K-ras mutations and 24% (6 out of 25) had RASSF1A hypermethylation. Our results demonstrate that RASSF1A hypermethylation and K-ras mutations are not mutually exclusive and are present in patients at elevated risk of CRC. Importantly, CIM of RASSF1A is an early epigenetic aberration, occurring in the absence of synchronous colon tumors and is not accompanied by field effects into the surrounding epithelium

Patch-clamp recordings and calcium imaging followed by single-cell PCR reveal the developmental profile of 13 genes in iPSC-derived human neurons

Molecular genetic studies are typically performed on homogenized biological samples, resulting in contamination from non-neuronal cells. To improve expression profiling of neurons we combined patch recordings with single-cell PCR. Two iPSC lines (healthy subject and 22q11.2 deletion) were differentiated into neurons. Patch electrode recordings were performed on 229 human cells from Day-13 to Day-88, followed by capture and single-cell PCR for 13 genes: ACTB, HPRT, vGLUT1, βTUBIII, COMT, DISC1, GAD1, PAX6, DTNBP1, ERBB4, FOXP1, FOXP2, and GIRK2. Neurons derived from both iPSC lines expressed βTUBIII, fired action potentials, and experienced spontaneous depolarizations (UP states) ~2 weeks before vGLUT1, GAD1 and GIRK2 appeared. Multisite calcium imaging revealed that these UP states were not synchronized among hESC-H9-derived neurons. The expression of FOXP1, FOXP2 and vGLUT1 was lost after 50 days in culture, in contrast to other continuously expressed genes. When gene expression was combined with electrophysiology, two subsets of genes were apparent; those irrelevant to spontaneous depolarizations (including vGLUT1, GIRK2, FOXP2 and DISC1) and those associated with spontaneous depolarizations (GAD1 and ERBB4). The results demonstrate that in the earliest stages of neuron development, it is useful to combine genetic analysis with physiological characterizations, on a cell-to-cell basis

Pigment Epithelium-Derived Factor (PEDF) Inhibits Wnt/β-catenin Signaling in the LiverSummary

Background & Aims: Pigment epithelium-derived factor (PEDF) is a secretory protein that inhibits multiple tumor types. PEDF inhibits the Wnt coreceptor, low-density lipoprotein receptor-related protein 6 (LRP6), in the eye, but whether the tumor-suppressive properties of PEDF occur in organs such as the liver is unknown. Methods: Wnt-dependent regulation of PEDF was assessed in the absence and presence of the Wnt coreceptor LRP6. Whole genome expression analysis was performed on PEDF knockout (KO) and control livers (7 months). Interrogation of Wnt/β-catenin signaling was performed in whole livers and human hepatocellular carcinoma (HCC) cell lines after RNA interference of PEDF and restoration of a PEDF-derived peptide. Western diet feeding for 6 to 8 months was used to evaluate whether the absence of PEDF was permissive for HCC formation (n = 12/group). Results: PEDF levels increased in response to canonical Wnt3a in an LRP6-dependent manner but were suppressed by noncanonical Wnt5a protein in an LRP6-independent manner. Gene set enrichment analysis (GSEA) of PEDF KO livers revealed induction of pathways associated with experimental and human HCC and a transcriptional profile characterized by Wnt/β-catenin activation. Enhanced Wnt/β-catenin signaling occurred in KO livers, and PEDF delivery in vivo reduced LRP6 activation. In human HCC cells, RNA interference of PEDF led to increased levels of activated LRP6 and β-catenin, and a PEDF 34-mer peptide decreased LRP6 activation and β-catenin signaling, and reduced Wnt target genes. PEDF KO mice fed a Western diet developed sporadic well-differentiated HCC. Human HCC specimens demonstrated decreased PEDF staining compared with hepatocytes. Conclusions: PEDF is an endogenous inhibitor of Wnt/β-catenin signaling in the liver. Keywords: Extracellular Matrix, PEDF, Wnt/β-Cateni

cPLA2 Is Protective Against COX Inhibitor–Induced Intestinal Damage

Cytosolic phospholipase A2 (cPLA2) is the rate-limiting enzyme responsible for the generation of prostaglandins (PGs), which are bioactive lipids that play critical roles in maintaining gastrointestinal (GI) homeostasis. There has been a long-standing association between administration of cyclooxygenase (COX) inhibitors and GI toxicity. GI injury is thought to be induced by suppressed production of GI-protective PGs as well as direct injury to enterocytes. The present study sought to determine how pan-suppression of PG production via a genetic deletion of cPLA2 impacts the susceptibility to COX inhibitor–induced GI injury. A panel of COX inhibitors including celecoxib, rofecoxib, sulindac, and aspirin were administered via diet to cPLA2− / − and cPLA2+ / + littermates. Administration of celecoxib, rofecoxib, and sulindac, but not aspirin, resulted in acute lethality (within 2 weeks) in cPLA2− / − mice, but not in wild-type littermates. Histomorphological analysis revealed severe GI damage following celecoxib exposure associated with acute bacteremia and sepsis. Intestinal PG levels were reduced equivalently in both genotypes following celecoxib exposure, indicating that PG production was not likely responsible for the differential sensitivity. Gene expression profiling in the small intestines of mice identified drug-related changes among a panel of genes including those involved in mitochondrial function in cPLA2− / − mice. Further analysis of enterocytic mitochondria showed abnormal morphology as well as impaired ATP production in the intestines from celecoxib-exposed cPLA2− / − mice. Our data demonstrate that cPLA2 appears to be an important component in conferring protection against COX inhibitor–induced enteropathy, which may be mediated through affects on enterocytic mitochondria