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.

SCODE: an efficient regulatory network inference algorithm from single-cell RNA-Seq during differentiation.

MOTIVATION: The analysis of RNA-Seq data from individual differentiating cells enables us to reconstruct the differentiation process and the degree of differentiation (in pseudo-time) of each cell. Such analyses can reveal detailed expression dynamics and functional relationships for differentiation. To further elucidate differentiation processes, more insight into gene regulatory networks is required. The pseudo-time can be regarded as time information and, therefore, single-cell RNA-Seq data are time-course data with high time resolution. Although time-course data are useful for inferring networks, conventional inference algorithms for such data suffer from high time complexity when the number of samples and genes is large. Therefore, a novel algorithm is necessary to infer networks from single-cell RNA-Seq during differentiation. RESULTS: In this study, we developed the novel and efficient algorithm SCODE to infer regulatory networks, based on ordinary differential equations. We applied SCODE to three single-cell RNA-Seq datasets and confirmed that SCODE can reconstruct observed expression dynamics. We evaluated SCODE by comparing its inferred networks with use of a DNaseI-footprint based network. The performance of SCODE was best for two of the datasets and nearly best for the remaining dataset. We also compared the runtimes and showed that the runtimes for SCODE are significantly shorter than for alternatives. Thus, our algorithm provides a promising approach for further single-cell differentiation analyses. AVAILABILITY AND IMPLEMENTATION: The R source code of SCODE is available at https://github.com/hmatsu1226/SCODE. CONTACT: hirotaka.matsumoto@riken.jp. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Bicarbonate-rich fluid secretion predicted by a computational model of guinea-pig pancreatic duct epithelium.

KEY POINTS: The ductal system of the pancreas secretes large volumes of alkaline fluid containing HCO3- concentrations as high as 140 mm during hormonal stimulation. A computational model has been constructed to explore the underlying ion transport mechanisms. Parameters were estimated by fitting the model to experimental data from guinea-pig pancreatic ducts. The model was readily able to secrete 140 mm HCO3- . Its capacity to do so was not dependent upon special properties of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channels and solute carrier family 26 member A6 (SLC26A6) anion exchangers. We conclude that the main requirement for secreting high HCO3- concentrations is to minimize the secretion of Cl- ions. These findings help to clarify the mechanism responsible for pancreatic HCO3- secretion, a vital process that prevents the formation of protein plugs and viscous mucus in the ducts, which could otherwise lead to pancreatic disease. ABSTRACT: A computational model of guinea-pig pancreatic duct epithelium was developed to determine the transport mechanism by which HCO3- ions are secreted at concentrations in excess of 140 mm. Parameters defining the contributions of the individual ion channels and transporters were estimated by least-squares fitting of the model predictions to experimental data obtained from isolated ducts and intact pancreas under a range of experimental conditions. The effects of cAMP-stimulated secretion were well replicated by increasing the activities of the basolateral Na+ -HCO3- cotransporter (NBC1) and apical Cl- /HCO3- exchanger (solute carrier family 26 member A6; SLC26A6), increasing the basolateral K+ permeability and apical Cl- and HCO3- permeabilities (CFTR), and reducing the activity of the basolateral Cl- /HCO3- exchanger (anion exchanger 2; AE2). Under these conditions, the model secreted ∼140 mm HCO3- at a rate of ∼3 nl min-1  mm-2 , which is consistent with experimental observations. Alternative 1:2 and 1:1 stoichiometries for Cl- /HCO3- exchange via SLC26A6 at the apical membrane were able to support a HCO3- -rich secretion. Raising the HCO3- /Cl- permeability ratio of CFTR from 0.4 to 1.0 had little impact upon either the secreted HCO3- concentration or the volume flow. However, modelling showed that a reduction in basolateral AE2 activity by ∼80% was essential in minimizing the intracellular Cl- concentration following cAMP stimulation and thereby maximizing the secreted HCO3- concentration. The addition of a basolateral Na+ -K+ -2Cl- cotransporter (NKCC1), assumed to be present in rat and mouse ducts, raised intracellular Cl- and resulted in a lower secreted HCO3- concentration, as is characteristic of those species. We conclude therefore that minimizing the driving force for Cl- secretion is the main requirement for secreting 140 mm HCO3- .

Neural differentiation of human embryonic stem cells induced by the transgene-mediated overexpression of single transcription factors.

Pluripotent human embryonic stem cells (hESCs) can differentiate into multiple cell lineages, thus, providing one of the best platforms to study molecular mechanisms during cell differentiation. Recently, we have reported rapid and efficient differentiation of hESCs into functional neurons by introducing a cocktail of synthetic mRNAs encoding five transcription factors (TFs): NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. Here we further tested a possibility that even single transcription factors, when expressed ectopically, can differentiate hESCs into neurons. To this end, we established hESC lines in which each of these TFs can be overexpressed by the doxycycline-inducible piggyBac vector. The overexpression of any of these five TFs indeed caused a rapid and rather uniform differentiation of hESCs, which were identified as neurons based on their morphologies, qRT-PCR, and immunohistochemistry. Furthermore, calcium-imaging analyses and patch clamp recordings demonstrated that these differentiated cells are electrophysiologically functional. Interestingly, neural differentiations occurred despite the cell culture conditions that rather promote the maintenance of the undifferentiated state. These results indicate that over-expression of each of these five TFs can override the pluripotency-specific gene network and force hESCs to differentiate into neurons.

Functional characteristics of L1156F-CFTR associated with alcoholic chronic pancreatitis in Japanese.

Although cystic fibrosis is rare in Japanese, measurement of sweat Cl(-) has suggested mild dysfunction of cystic fibrosis transmembrane conductance regulator (CFTR) in some patients with chronic pancreatitis. In the present study, we have investigated the association of CFTR variants and chronic pancreatitis in Japanese and the functional characteristics of a Japanese- and pancreatitis-specific CFTR variant, L1156F. Seventy patients with alcoholic chronic pancreatitis, 18 patients with idiopathic chronic pancreatitis, and 180 normal subjects participated. All exons and their boundaries and promoter region of the CFTR gene were sequenced. Human embryonic kidney-293 cells were transfected with three CFTR variants (M470V, L1156F, and M470V+L1156F), and the protein expression was examined. Xenopus laevis oocytes were injected with the CFTR variants, and bicarbonate (HCO3 (-)) transport activity was examined. CFPAC-1 cells were transfected with the CFTR variants and Cl(-)/HCO3 (-) exchange activity was examined. Six variants (E217G, I556V, M470V, L1156F, Q1352H, and R1453W) were identified in the coding region of the CFTR gene. Cystic fibrosis-causing mutations were not found. The allele frequencies of L1156F and Q1352H in alcoholic chronic pancreatitis (5.0 and 7.9%) were significantly (P < 0.01) higher than those in normal subjects (0.6 and 1.9%). L1156F was linked with a worldwide CFTR variant, M470V. Combination of M470V and L1156F significantly reduced CFTR expression to ∼60%, impaired CFTR-mediated HCO3 (-)/Cl(-) transport activity to 50-60%, and impaired CFTR-coupled Cl(-)/HCO3 (-) exchange activity to 20-30%. The data suggest that the Japanese-specific CFTR variant L1156F causes mild dysfunction of CFTR and increases the risk of alcoholic chronic pancreatitis in Japanese.

ZSCAN4-binding motif-TGCACAC is conserved and enriched in CA/TG microsatellites in both mouse and human genomes.

The Zinc finger and SCAN domain containing 4 (ZSCAN4) protein, expressed transiently in pluripotent stem cells, gametes, and early embryos, extends telomeres, enhances genome stability, and improves karyotypes in mouse embryonic stem (mES) cells. To gain insights into the mechanism of ZSCAN4 function, we identified genome-wide binding sites of endogenous ZSCAN4 protein using ChIP-seq technology in mouse and human ES cells, where the expression of endogenous ZSCAN4 was induced by treating cells with retinoic acids or by overexpressing DUX4. We revealed that both mouse and human ZSCAN4 bind to the TGCACAC motif located in CA/TG microsatellite repeats, which are known to form unstable left-handed duplexes called Z-DNA that can induce double-strand DNA breaks and mutations. These ZSCAN4 binding sites are mostly located in intergenic and intronic regions of the genomes. By generating ZSCAN4 knockout in human ES cells, we showed that ZSCAN4 does not seem to be involved in transcriptional regulation. We also found that ectopic expression of mouse ZSCAN4 enhances the suppression of chromatin at ZSCAN4-binding sites. These results together suggest that some of the ZSCAN4 functions are mediated by binding to the error-prone regions in mouse and human genomes.

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.

Deletion of Slc26a6 alters the stoichiometry of apical Cl-/HCO-3 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.

Detection of a large heterozygous deletion and a splicing defect in the CFTR transcripts from nasal swab of a Japanese case of cystic fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in CFTR (CF transmembrane conductance regulator). Although CF is the most common hereditary disease in Caucasians, it is rare in Asian populations. Common disease-causing mutations of CFTR in Caucasians are rarely identified in Japanese patients with CF. In the present study, CFTR transcripts from nasal swab were analyzed in a Japanese boy, in addition to conventional PCR and direct sequence of all exons, their boundaries and promoter region of the CFTR gene. The boy was diagnosed with CF by chronic respiratory infection and the elevated sweat chloride level. None of the disease-causing mutations of CFTR was detected by the conventional analysis. Cloning and sequence of the CFTR transcripts revealed a heterozygous deletion spanning exons 16, 17a and 17b. The deletion was confirmed by multiplex ligation-dependent probe amplification and the direct sequence of the junction fragment obtained from the genomic DNA by primer walking, which revealed the mutation c.2908+1085_3367+260del7201. We also identified a splicing defect: deletion/skipping of exon 1 in the CFTR transcript from the other allele. The analysis of CFTR transcripts from nasal swab is recommended in the genetic analysis of CF in Japanese.

Three cases of bronchial asthma preceding IgG4-related autoimmune pancreatitis.

BACKGROUND: Autoimmune pancreatitis is characterized by diffuse swelling of the pancreas and a high serum immunoglobulin (Ig) G4 concentration. Histopathologically, dense infiltration of lymphocytes and IgG4-positive plasma cells with fibrosis are seen in the pancreas. Although allergic diseases complicating autoimmune pancreatitis have been reported, the clinical features of bronchial asthma complicated by autoimmune pancreatitis remain unclear. CASE SUMMARY: We report three cases of bronchial asthma preceding the onset of type 1 autoimmune pancreatitis by 3 months to 30 years. All three cases were males with high serum IgG, IgG4, and IgE concentrations. The radioallergosorbent tests were positive for common allergens such as mites and house dust. One case had a pulmonary manifestation that proved to be an inflammatory pseudotumor of the lung with an accumulation of IgG4-positive plasma cells. The asthma symptom was ameliorated by oral prednisolone therapy for autoimmune pancreatitis, and when the corticosteroid doses were reduced, asthma became worse in all three cases. DISCUSSION: It is possible that atopy and increased Th2 cell activity are related to a higher coincidence of IgG4-related diseases such as type 1 autoimmune pancreatitis. Because the present cases are few in number, further studies are necessary.

Corticosteroids correct aberrant CFTR localization in the duct and regenerate acinar cells in autoimmune pancreatitis.

BACKGROUND & AIMS: Corticosteroids are now widely accepted as a treatment for autoimmune pancreatitis (AIP). However, the molecular mechanism by which steroid treatment improves AIP remains largely unknown. The aim of this study was to elucidate cellular mechanisms by which corticosteroids improve both pancreatic exocrine function and histopathology in AIP. METHODS: Pancreatic exocrine function was evaluated by the secretin-stimulated function test and pancreatic biopsy specimens were processed for histologic analysis at the time of diagnosis and 3 months after initiation of steroid treatment. Expression and localization of proteins was assayed by immunohistochemistry. Analysis of immunoglobulin (Ig)G4-positive plasma cells was used to verify inflammation in AIP. RESULTS: The number of IgG4-positive plasma cells in pancreatic sections was decreased by steroid treatment, indicating reduced inflammation. Fluid, bicarbonate (HCO(3)(-)), and digestive enzyme secretions all were impaired in most patients with AIP. Corticosteroids improved both HCO(3)(-) and digestive enzyme secretion. A large fraction of the cystic fibrosis transmembrane conductance regulator (CFTR), which plays a central role in pancreatic duct HCO(3)(-) secretion, was mislocalized to the cytoplasm of duct cells before treatment. Corticosteroids corrected the localization of CFTR to the apical membrane, accounting for the improved HCO(3)(-) secretion. Steroid treatment resulted in regeneration of acinar cells, accounting for restored digestive enzyme secretion. CONCLUSIONS: Corticosteroids reduce inflammation and restore both digestive enzyme and HCO(3)(-) secretion in patients with AIP by regenerating acinar cells and correcting CFTR localization in pancreatic duct cells. Mislocalization of CFTR may explain aberrant HCO(3)(-) secretion in other forms of pancreatitis.

Effects of CFTR gene silencing by siRNA or the luminal application of a CFTR activator on fluid secretion from guinea-pig pancreatic duct cells.

AIMS: The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP regulated chloride channel expressed in the apical plasma membrane of pancreatic duct cells where it plays an important role in fluid secretion. The purpose of this study was to elucidate the role of the CFTR chloride channel on ion and fluid secretion from the guinea-pig pancreas by manipulating the expression of CFTR by RNA interference or by luminal application of a CFTR selective activator, MPB91, in isolated cultured pancreatic ducts. MATERIALS AND METHODS: Using cDNA isolated from the guinea-pig small intestine, fragments of the CFTR gene were generated by polymerase chain reaction and directly sequenced. Two different RNA duplexes for small interference RNA (siRNA) were designed from the sequence obtained. Fluid secretion from the isolated guinea-pig pancreatic ducts was measured using video-microscopy. The amount of CFTR chloride channel or AQP1 water channel expressed in pancreatic ducts was examined by immunoblotting with antibodies against CFTR or AQP1, respectively. RESULTS: Guinea-pig CFTR consists of 1481 amino acid residues. An additional glutamine residue was found to be inserted between amino acid residues 403 and 404 of human CFTR. Forskolin-stimulated fluid secretion from intact pancreatic ducts was significantly higher in the presence of MPB91 compared to fluid secretion in the absence of MPB91. Both basal and forskolin-stimulated fluid secretion in pancreatic ducts transfected with CFTR specific siRNAs were reduced by ∼50% compared to fluid secretion from ducts transfected with scrambled negative control dsRNAs. The amount of CFTR and AQP1 proteins was reduced to 34% and 45% of control, respectively. CONCLUSIONS: The activity of the CFTR chloride channel or the amount of CFTR protein expressed determines the rate of fluid secretion from the isolated guinea-pig pancreatic ducts.

Gating of CFTR by the STAS domain of SLC26 transporters.

Chloride absorption and bicarbonate secretion are vital functions of epithelia, as highlighted by cystic fibrosis and diseases associated with mutations in members of the SLC26 chloride-bicarbonate exchangers. Many SLC26 transporters (SLC26T) are expressed in the luminal membrane together with CFTR, which activates electrogenic chloride-bicarbonate exchange by SLC26T. However, the ability of SLC26T to regulate CFTR and the molecular mechanism of their interaction are not known. We report here a reciprocal regulatory interaction between the SLC26T DRA, SLC26A6 and CFTR. DRA markedly activates CFTR by increasing its overall open probablity (NP(o)) sixfold. Activation of CFTR by DRA was facilitated by their PDZ ligands and binding of the SLC26T STAS domain to the CFTR R domain. Binding of the STAS and R domains is regulated by PKA-mediated phosphorylation of the R domain. Notably, CFTR and SLC26T co-localize in the luminal membrane and recombinant STAS domain activates CFTR in native duct cells. These findings provide a new understanding of epithelial chloride and bicarbonate transport and may have important implications for both cystic fibrosis and diseases associated with SLC26T.

Synthetic mRNA-based differentiation method enables early detection of Parkinson's phenotypes in neurons derived from Gaucher disease-induced pluripotent stem cells.

Gaucher disease, the most prevalent metabolic storage disorder, is caused by mutations in the glucocerebrosidase gene GBA1, which lead to the accumulation of glucosylceramide (GlcCer) in affected cells. Gaucher disease type 1 (GD1), although defined as a nonneuronopathic subtype, is accompanied by an increased risk of Parkinson's disease. To gain insights into the association of progressive accumulation of GlcCer and the Parkinson's disease phenotypes, we generated dopaminergic (DA) neurons from induced pluripotent stem cells (iPSCs) derived from a GD1 patient and a healthy donor control, and measured GlcCer accumulation by liquid chromatography-mass spectrometry. We tested two DA neuron differentiation methods: a well-established method that mimics a step-wise developmental process from iPSCs to neural progenitor cells, and to DA neurons; and a synthetic mRNA-based method that overexpresses a transcription factor in iPSCs. GD1-specific accumulation of GlcCer was detected after 60 days of differentiation by the former method, whereas it was detected after only 10 days by the latter method. With this synthetic mRNA-based rapid differentiation method, we found that the metabolic defect in GD1 patient cells can be rescued by the overexpression of wild-type GBA1 or the treatment with an inhibitor for GlcCer synthesis. Furthermore, we detected the increased phosphorylation of α-synuclein, a biomarker for Parkinson's disease, in DA neurons derived from a GD1 patient, which was significantly decreased by the overexpression of wild-type GBA1. These results suggest that synthetic mRNA-based method accelerates the analyses of the pathological mechanisms of Parkinson's disease in GD1 patients and possibly facilitates drug discovery processes.

Generation and Profiling of 2,135 Human ESC Lines for the Systematic Analyses of Cell States Perturbed by Inducing Single Transcription Factors.

Transcription factors (TFs) play a pivotal role in determining cell states, yet our understanding of the causative relationship between TFs and cell states is limited. Here, we systematically examine the state changes of human pluripotent embryonic stem cells (hESCs) by the large-scale manipulation of single TFs. We establish 2,135 hESC lines, representing three clones each of 714 doxycycline (Dox)-inducible genes including 481 TFs, and obtain 26,998 microscopic cell images and 2,174 transcriptome datasets-RNA sequencing (RNA-seq) or microarrays-48 h after the presence or absence of Dox. Interestingly, the expression of essentially all the genes, including genes located in heterochromatin regions, are perturbed by these TFs. TFs are also characterized by their ability to induce differentiation of hESCs into specific cell lineages. These analyses help to provide a way of classifying TFs and identifying specific sets of TFs for directing hESC differentiation into desired cell types.

Pancreas-specific aquaporin 12 null mice showed increased susceptibility to caerulein-induced acute pancreatitis.

Aquaporin 12 (AQP12) is the most recently identified member of the mammalian AQP family and is specifically expressed in pancreatic acinar cells. In vitro expression studies have revealed that AQP12 is localized at intracellular sites. To determine the physiological roles of AQP12 in the pancreas, we generated knockout mice for this gene (AQP12-KO). No obvious differences were observed under normal conditions between wild-type (WT) and AQP12-KO mice in terms of growth, blood chemistry, pancreatic fluid content, or histology. However, when we induced pancreatitis through the administration of a cholecystokinin-8 (CCK-8) analog, the AQP12-KO mice showed more severe pathological damage to this organ than WT mice. Furthermore, when we analyzed exocytosis in the pancreatic acini using a two-photon excitation imaging method, the results revealed larger exocytotic vesicles (vacuoles) in the acini of AQP12-KO mice at a high CCK-8 dose (100 nM). From these results, we conclude that AQP12 may function in the mechanisms that control the proper secretion of pancreatic fluid following rapid and intense stimulation.

High glucose inhibits HCO3(-) and fluid secretion in rat pancreatic ducts.

Cellular mechanisms underlying the impairment of pancreatic fluid and electrolyte secretion in diabetes were examined using interlobular ducts isolated from rat pancreas. Fluid secretion was assessed by monitoring changes in luminal volume. HCO3(-) uptake across the basolateral membrane was estimated from the recovery of intracellular pH following an acid load. Exposure to high glucose concentrations inhibited fluid secretion and reduced the rate of basolateral HCO3(-) uptake in secretin-stimulated ducts isolated from normal rats. In ducts isolated from streptozotocin-treated diabetic rats, fluid secretion and basolateral HCO3(-) uptake were also severely impaired but could be largely reversed by incubation in normal-glucose solutions. Sodium-dependent glucose cotransporter 1 (SGLT1), glucose transporter (GLUT)1, GLUT2, and GLUT8 transcripts were detected by reverse transcriptase polymerase chain reaction in isolated ducts. Raising the luminal glucose concentration in microperfused ducts caused a depolarization of the membrane potential, consistent with the presence of SGLT1 at the apical membrane. Unstimulated ducts filled with high-glucose solutions lost luminal fluid by a phlorizin-sensitive mechanism, indicating that pancreatic ducts are capable of active glucose reabsorption from the lumen via SGLT1. In ducts exposed to high glucose concentrations, continuous glucose diffusion to the lumen and active reabsorption via SGLT1 would lead to elevation of intracellular Na+ concentration and sustained depolarization of the apical membrane. These two factors would tend to inhibit the basolateral uptake and apical efflux of Cl(-) and HCO3(-) and could therefore account for the impaired fluid and electrolyte secretion that is observed in diabetes.

Histological diagnosis of autoimmune pancreatitis using EUS-guided trucut biopsy: a comparison study with EUS-FNA.

PURPOSE: The aim of this study was to evaluate the feasibility and safety of endoscopic ultrasonography (EUS)-guided trucut biopsy (TCB) for diagnosis of autoimmune pancreatitis (AIP). METHODS: Fourteen patients with suspected AIP based on imaging studies underwent both EUS-guided fine-needle aspiration (FNA) and EUS-TCB for diagnosis of AIP and exclusion of pancreatic cancer (PC). According to the revised Japanese clinical diagnostic criteria, AIP was diagnosed in eight while the remaining six patients had pancreatitis of other etiologies. Pathologically, AIP was defined as lymphoplasmacytic sclerosing pancreatitis (LPSP), and sub-divided into two types: definite LPSP (d-LPSP) showing fulspectrum of LPSP and probable LPSP (p-LPSP) without obliterative phlebitis or abundant (>10 cells/hpf) IgG4-positive plasmacytes infiltration. RESULTS: PC was excluded in all patients. EUS-FNA resulted in three of eight patients with AIP were reported as p-LPSP, one was reported as normal, and 4 were inconclusive. One of six with non-autoimmune pancreatitis was diagnosed as p-LPSP on EUS-FNA, one as idiopathic chronic pancreatitis (ICP) and four were inconclusive. By using EUS-TCB, all AIP patients were diagnosed as LPSP (4 d-LPSP and 4 p-LPSP). Of the six patients with non-autoimmune pancreatitis, three were diagnosed as LPSP (1 d-LPSP and 2 p-LPSP) and three showed ICP on TCB. No complications were identified in any patient with either EUS-FNA or TCB. CONCLUSION: EUS-TCB is a safe and accurate procedure for obtaining a histological diagnosis in patients with suspected AIP. EUS-TCB can serve as a rescue technique in cases of AIP lacking typical findings.

Induction of human pluripotent stem cells into kidney tissues by synthetic mRNAs encoding transcription factors.

The derivation of kidney tissues from human pluripotent stem cells (hPSCs) and its application for replacement therapy in end-stage renal disease have been widely discussed. Here we report that consecutive transfections of two sets of synthetic mRNAs encoding transcription factors can induce rapid and efficient differentiation of hPSCs into kidney tissues, termed induced nephron-like organoids (iNephLOs). The first set - FIGLA, PITX2, ASCL1 and TFAP2C, differentiated hPSCs into SIX2+SALL1+ nephron progenitor cells with 92% efficiency within 2 days. Subsequently, the second set - HNF1A, GATA3, GATA1 and EMX2, differentiated these cells into PAX8+LHX1+ pretubular aggregates in another 2 days. Further culture in both 2-dimensional and 3-dimensional conditions produced iNephLOs containing cells characterized as podocytes, proximal tubules, and distal tubules in an additional 10 days. Global gene expression profiles showed similarities between iNephLOs and the human adult kidney, suggesting possible uses of iNephLOs as in vitro models for kidneys.

Asian diagnostic criteria for autoimmune pancreatitis: consensus of the Japan-Korea Symposium on Autoimmune Pancreatitis.

In 2002, the Japan Pancreas Society (JPS) was the first in the world to propose diagnostic criteria for autoimmune pancreatitis (AIP). Since the concept of AIP has changed with the accumulation of AIP cases, the Research Committee of Intractable Pancreatic Diseases (RCIPD) provided by the Ministry of Health, Labour and Welfare of Japan and the JPS issued revised clinical diagnostic criteria of AIP in 2006. The Asan Medical Center of Korea also proposed diagnostic criteria for AIP in 2006. However, there are subtle but clinically challenging differences between the Japanese and Korean criteria. This inconsistency makes it difficult to compare data in studies from different centers and elucidate the characteristics of AIP. To reach a consensus on AIP, the RCIPD and the Korean Society of Pancreatobiliary Diseases established the following Asian criteria for the diagnosis of AIP: I-1. Imaging studies of pancreatic parenchyma show a diffuse/segmental/focally enlarged gland, occasionally with a mass and/or a hypoattenuation rim. I-2. Imaging studies of pancreaticobiliary ducts show diffuse/segmental/focal pancreatic ductal narrowing, often with stenosis of the bile duct. (Both I-1 and I-2 are required for diagnosis). II. Elevated level of serum IgG or IgG4, and detection of autoantibodies. III. Common lymphoplasmacytic infiltration and fibrosis, with abundant IgG4-positive cell infiltration. AIP should be diagnosed when criterion I and one of the other two criteria are satisfied, or when histology shows the presence of lymphoplasmacytic sclerosing pancreatitis in the resected pancreas. A diagnostic trial of steroid therapy can be applied carefully by expert pancreatologists only in patients fulfilling criterion I alone with negative diagnostic work-up results for pancreatobiliary cancer.