Shigeru B. H. Ko

Transient bursts of Zscan4 expression are accompanied by the rapid derepression of heterochromatin in mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) have a remarkable capacity to maintain normal genome stability and karyotype in culture. We previously showed that infrequent bursts of Zscan4 expression (Z4 events) are important for the maintenance of telomere length and genome stability in mESCs. However, the molecular details of Z4 events remain unclear. Here we show that Z4 events involve unexpected transcriptional derepression in heterochromatin regions that usually remain silent. During a Z4 event, we see rapid derepression and rerepression of heterochromatin leading to a burst of transcription that coincides with transient histone hyperacetylation and DNA demethylation, clustering of pericentromeric heterochromatin around the nucleolus, and accumulation of activating and repressive chromatin remodelling complexes. This heterochromatin-based transcriptional activity suggests that mESCs may maintain their extraordinary genome stability at least in part by transiently resetting their heterochromatin.

Deletion of Slc26a6 alters the stoichiometry of apical Cl−/HCO3− exchange in mouse pancreatic duct

To define the stoichiometry and molecular identity of the Cl(-)/HCO(3)(-) exchanger in the apical membrane of pancreatic duct cells, changes in luminal pH and volume were measured simultaneously in interlobular pancreatic ducts isolated from wild-type and Slc26a6-null mice. Transepithelial fluxes of HCO(3)(-) and Cl(-) were measured in the presence of anion gradients favoring rapid exchange of intracellular HCO(3)(-) with luminal Cl(-) in cAMP-stimulated ducts. The flux ratio of Cl(-) absorption/HCO(3)(-) secretion was ∼0.7 in wild-type ducts and ∼1.4 in Slc26a6(-/-) ducts where a different Cl(-)/HCO(3)(-) exchanger, most likely SLC26A3, was found to be active. Interactions between Cl(-)/HCO(3)(-) exchange and cystic fibrosis transmembrane conductance regulator (CFTR) in cAMP-stimulated ducts were examined by measuring the recovery of intracellular pH after alkali-loading by acetate prepulse. Hyperpolarization induced by luminal application of CFTRinh-172 enhanced HCO(3)(-) efflux across the apical membrane via SLC26A6 in wild-type ducts but significantly reduced HCO(3)(-) efflux in Slc26a6(-/-) ducts. In microperfused wild-type ducts, removal of luminal Cl(-), or luminal application of dihydro-4,4-diisothiocyanatostilbene-2,2-disulphonic acid to inhibit SLC26A6, caused membrane hyperpolarization, which was abolished in Slc26a6(-/-) ducts. In conclusion, we have demonstrated that deletion of Slc26a6 alters the apparent stoichiometry of apical Cl(-)/HCO(3)(-) exchange in native pancreatic duct. Our results are consistent with SLC26A6 mediating 1:2 Cl(-)/HCO(3)(-) exchange, and the exchanger upregulated in its absence, most probably SLC26A3, mediating 2:1 exchange.

Inflammation increases cells expressing ZSCAN4 and progenitor cell markers in the adult pancreas

We have recently identified the zinc finger and SCAN domain containing 4 (Zscan4), which is transiently expressed and regulates telomere elongation and genome stability in mouse embryonic stem (ES) cells. The aim of this study was to examine the expression of ZSCAN4 in the adult pancreas and elucidate the role of ZSCAN4 in tissue inflammation and subsequent regeneration. The expression of ZSCAN4 and other progenitor or differentiated cell markers in the human pancreas was immunohistochemically examined. Pancreas sections of alcoholic or autoimmune pancreatitis patients before and under maintenance corticosteroid treatment were used in this study. In the adult human pancreas a small number of ZSCAN4-positive (ZSCAN4⁺) cells are present among cells located in the islets of Langerhans, acini, ducts, and oval-shaped cells. These cells not only express differentiated cell markers for each compartment of the pancreas but also express other tissue stem/progenitor cell markers. Furthermore, the number of ZSCAN4⁺ cells dramatically increased in patients with chronic pancreatitis, especially in the pancreatic tissues of autoimmune pancreatitis actively regenerating under corticosteroid treatment. Interestingly, a number of ZSCAN4⁺ cells in the pancreas of autoimmune pancreatitis returned to the basal level after 1 yr of maintenance corticosteroid treatment. In conclusion, coexpression of progenitor cell markers and differentiated cell markers with ZSCAN4 in each compartment of the pancreas may indicate the presence of facultative progenitors for both exocrine and endocrine cells in the adult pancreas.

Transient ectopic expression of the histone demethylase JMJD3 accelerates the differentiation of human pluripotent stem cells

Harnessing epigenetic regulation is crucial for the efficient and proper differentiation of pluripotent stem cells (PSCs) into desired cell types. Histone H3 lysine 27 trimethylation (H3K27me3) functions as a barrier against cell differentiation through the suppression of developmental gene expression in PSCs. Here, we have generated human PSC (hPSC) lines in which genome-wide reduction of H3K27me3 can be induced by ectopic expression of the catalytic domain of the histone demethylase JMJD3 (called JMJD3c). We found that transient, forced demethylation of H3K27me3 alone triggers the upregulation of mesoendodermal genes, even when the culture conditions for the hPSCs are not changed. Furthermore, transient and forced expression of JMJD3c followed by the forced expression of lineage-defining transcription factors enabled the hPSCs to activate tissue-specific genes directly. We have also shown that the introduction of JMJD3c facilitates the differentiation of hPSCs into functional hepatic cells and skeletal muscle cells. These results suggest the utility of the direct manipulation of epigenomes for generating desired cell types from hPSCs for cell transplantation therapy and platforms for drug screenings. Summary: Forced expression of the catalytic domain of JMJD3 together with lineage-defining transcription factors can dramatically accelerate the differentiation of human PSCs.

Cytoplasmic expression of LGR5 in pancreatic adenocarcinoma

Background: CD133 has been identified as a cancer stem cell marker for pancreatic ductal adenocarcinoma. Although leucine-rich-repeat-containing G-protein-coupled receptor 5 (LGR5), a marker of intestinal stem cells, has been shown to be on a higher level of the stem cell hierarchy than CD133, the expression and function of LGR5 in pancreatic cancer tissue remains unclear. This study investigated tissue expression of LGR5 and CD133 in resected pancreatic cancer tissue. Methods: LGR5 and CD133 expression was immunohistochemically examined in 9 patients with pancreatic ductal adenocarcinoma who underwent resection. Results: LGR5 was expressed in the cytoplasm of pancreatic cancer cells in 4 of 9 cases. CD133 was not detected in cancerous tissue. In non-neoplastic tissue, LGR5 was expressed in the basolateral membrane of a subset of endocrine cells. Conversely, CD133 was expressed in the apical membrane of small duct cells. Co-localization of LGR5 and CD133 was not found in either neoplastic or non-neoplastic tissue. LGR5 expression in pancreatic cancer cells showed no statistically significant correlation with survival after surgery. Conclusion: We have demonstrated that LGR5 is expressed in the cytoplasm of pancreatic adenocarcinoma cells, and the basolateral membrane of a subset of endocrine cells of the human pancreas. Further investigation is required to clarify any prognostic significance of LGR5 expression.

Molecular Mechanisms of Pancreatic Stone Formation in Chronic Pancreatitis

Chronic pancreatitis (CP) is a progressive inflammatory disease in which the pancreatic secretory parenchyma is destroyed and replaced by fibrosis. The presence of intraductal pancreatic stone(s) is important for the diagnosis of CP; however, the precise molecular mechanisms of pancreatic stone formation in CP were left largely unknown. Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel expressed in the apical plasma membrane of pancreatic duct cells and plays a central role in HCO3- secretion. In previous studies, we have found that CFTR is largely mislocalized to the cytoplasm of pancreatic duct cells in all forms of CP and corticosteroids normalizes the localization of CFTR to the proper apical membrane at least in autoimmune pancreatitis. From these observations, we could conclude that the mislocalization of CFTR is a cause of protein plug formation in CP, subsequently resulting in pancreatic stone formation. Considering our observation that the mislocalization of CFTR also occurs in alcoholic or idiopathic CP, it is very likely that these pathological conditions can also be treated by corticosteroids, thereby preventing pancreatic stone formation in these patients. Further studies are definitely required to clarify these fundamental issues.

Identification of transcription factors that promote the differentiation of human pluripotent stem cells into lacrimal gland epithelium-like cells

Dry eye disease is the most prevalent pathological condition in aging eyes. One potential therapeutic strategy is the transplantation of lacrimal glands, generated in vitro from pluripotent stem cells such as human embryonic stem cells, into patients. One of the preceding requirements is a method to differentiate human embryonic stem cells into lacrimal gland epithelium cells. As the first step for this approach, this study aims to identify a set of transcription factors whose overexpression can promote the differentiation of human embryonic stem cells into lacrimal gland epithelium-like cells. We performed microarray analyses of lacrimal glands and lacrimal glands-related organs obtained from mouse embryos and adults, and identified transcription factors enriched in lacrimal gland epithelium cells. We then transfected synthetic messenger RNAs encoding human orthologues of these transcription factors into human embryonic stem cells and examined whether the human embryonic stem cells differentiate into lacrimal gland epithelium-like cells by assessing cell morphology and marker gene expression. The microarray analysis of lacrimal glands tissues identified 16 transcription factors that were enriched in lacrimal gland epithelium cells. We focused on three of the transcription factors, because they are expressed in other glands such as salivary glands and are also known to be involved in the development of lacrimal glands. We tested the overexpression of various combinations of the three transcription factors and PAX6, which is an indispensable gene for lacrimal glands development, in human embryonic stem cells. Combining PAX6, SIX1, and FOXC1 caused significant changes in morphology, i.e., elongated cell shape and increased expression (both RNAs and proteins) of epithelial markers such as cytokeratin15, branching morphogenesis markers such as BARX2, and lacrimal glands markers such as aquaporin5 and lactoferrin. We identified a set of transcription factors enriched in lacrimal gland epithelium cells and demonstrated that the simultaneous overexpression of these transcription factors can differentiate human embryonic stem cells into lacrimal gland epithelium-like cells. This study suggests the possibility of lacrimal glands regeneration from human pluripotent stem cells.Regenerative medicine: lacrimal gland epithelium in vitroOne possible approach to treat dry eye diseases is to transplant lacrimal glands generated in vitro from human embryonic stem cells into patients. As a first step, we developed a novel method to generate lacrimal gland epithelium-like cells. As a model, we first studied the gene expression patterns of mouse embryonic lacrimal gland and identified key transcription factors involved in the process. Subsequently, we introduced four transcription factors in the form of synthetic mRNAs into human embryonic stem cells and successfully generated lacrimal gland epithelium-like cells, which showed elongated cell shape and increased expression of markers for epithelia, branching morphogenesis, and lacrimal glands. This study suggests the possibility of treating dry eye diseases through regeneration of lacrimal gland from human pluripotent stem cells.

Osteoprotegerin Regulates Pancreatic β-Cell Homeostasis upon Microbial Invasion.

Osteoprotegerin (OPG), a decoy receptor for receptor activator of NF-κB ligand (RANKL), antagonizes RANKL’s osteoclastogenic function in bone. We previously demonstrated that systemic administration of lipopolysaccharide (LPS) to mice elevates OPG levels and reduces RANKL levels in peripheral blood. Here, we show that mice infected with Salmonella, Staphylococcus, Mycobacteria or influenza virus also show elevated serum OPG levels. We then asked whether OPG upregulation following microbial invasion had an effect outside of bone. To do so, we treated mice with LPS and observed OPG production in pancreas, especially in β-cells of pancreatic islets. Insulin release following LPS administration was enhanced in mice lacking OPG, suggesting that OPG inhibits insulin secretion under acute inflammatory conditions. Consistently, treatment of MIN6 pancreatic β-cells with OPG decreased their insulin secretion following glucose stimulation in the presence of LPS. Finally, our findings suggest that LPS-induced OPG upregulation is mediated in part by activator protein (AP)-1 family transcription factors, particularly Fos proteins. Overall, we report that acute microbial infection elevates serum OPG, which maintains β-cell homeostasis by restricting glucose-stimulated insulin secretion, possibly preventing microbe-induced exhaustion of β-cell secretory capacity.

Efficient differentiation of human pluripotent stem cells into skeletal muscle cells by combining RNA-based MYOD1-expression and POU5F1-silencing

Direct generation of skeletal muscle cells from human pluripotent stem cells (hPSCs) would be beneficial for drug testing, drug discovery, and disease modelling in vitro. Here we show a rapid and robust method to induce myogenic differentiation of hPSCs by introducing mRNA encoding MYOD1 together with siRNA-mediated knockdown of POU5F1 (also known as OCT4 or OCT3/4). This integration-free approach generates functional skeletal myotubes with sarcomere-like structure and a fusion capacity in several days. The POU5F1 silencing facilitates MYOD1 recruitment to the target promoters, which results in the significant activation of myogenic genes in hPSCs. Furthermore, deep sequencing transcriptome analyses demonstrated that POU5F1-knockdown upregulates the genes associated with IGF- and FGF-signaling and extracellular matrix that may also support myogenic differentiation. This rapid and direct differentiation method may have potential applications in regenerative medicine and disease therapeutics for muscle disorders such as muscular dystrophy.

Expression analysis of the endogenous Zscan4 locus and its coding proteins in mouse ES cells and preimplantation embryos

Mouse Zinc finger and SCAN domain containing 4 (Zscan4) is encoded in multiple copies of Zscan4 genes, which are expressed in late two-cell stage preimplantation embryos and in 1–5% of the embryonic stem (ES) cell population at a given time. Due to the highly identical nucleotide sequences of multiple copies of Zscan4 paralogs and pseudogenes in the mouse Zscan4 genomic cluster, previous analyses have been done using exogenous transgenes under the regulation of Zscan4c promoter. In this manuscript, we generated knock-in mouse ES cell lines and mouse lines, in which the expression of endogenous Zscan4c, one of the Zscan4 genes, can be specifically monitored with a green fluorescent protein variant, Emerald. Interestingly, we found that only ∼30% of Zscan4-immunopositive ES cells were Emerald positive, suggesting that even when the Zscan4 locus is active, not all Zscan4 genes are expressed synchronously. We also carried out mass spectrometry of protein complexes associated with endogenous Zscan4 proteins. Taken together, our genetic engineering at an endogenous Zscan4c gene provides the first clue for the expression and function of each gene copy of Zscan4 locus in a physiological context.