The Effect of Vitamin D3 and Valproic Acid on the Maturation of Human-Induced Pluripotent Stem Cell-Derived Enterocyte-Like Cells.

Cytochrome P450 3A4 (CYP3A4) is involved in first-pass metabolism in the small intestine and is heavily implicated in oral drug bioavailability and pharmacokinetics. We previously reported that vitamin D3 (VD3), a known CYP enzyme inducer, induces functional maturation of iPSC-derived enterocyte-like cells (iPSC-ent). Here, we identified a Notch activator and CYP modulator valproic acid (VPA), as a promotor for the maturation of iPSC-ent. We performed bulk RNA sequencing to investigate the changes in gene expression during the differentiation and maturation periods of these cells. VPA potentiated gene expression of key enterocyte markers ALPI, FABP2, and transporters such as SULT1B1. RNA-sequencing analysis further elucidated several function-related pathways involved in fatty acid metabolism, significantly upregulated by VPA when combined with VD3. Particularly, VPA treatment in tandem with VD3 significantly upregulated key regulators of enterohepatic circulation, such as FGF19, apical bile acid transporter SLCO1A2 and basolateral bile acid transporters SLC51A and SLC51B. To sum up, we could ascertain the genetic profile of our iPSC-ent cells to be specialized toward fatty acid absorption and metabolism instead of transporting other nutrients, such as amino acids, with the addition of VD3 and VPA in tandem. Together, these results suggest the possible application of VPA-treated iPSC-ent for modelling enterohepatic circulation.

Transient Methionine Deprivation Triggers Histone Modification and Potentiates Differentiation of Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) require high levels of methionine (Met). Met deprivation results in a rapid decrease in intracellular S-adenosyl-methionine (SAM), poising human iPSCs for differentiation and leading to the apoptosis of undifferentiated cells. Met deprivation triggers rapid metabolic changes, including SAM, followed by reversible epigenetic modifications. Here, we show that short-term Met deprivation impairs the pluripotency network through epigenetic modification in a 3D suspension culture. The trimethylation of lysine 4 on histone H3 (H3K4me3) was drastically affected compared with other histone modifications. Short-term Met deprivation specifically affects the transcription start site (TSS) region of genes, such as those involved in the transforming growth factor ß pathway and cholesterol biosynthetic process, besides key pluripotent genes such as NANOG and POU5F1. The expression levels of these genes decreased, correlating with the loss of H3K4me3 marks. Upon differentiation, Met deprivation triggers the upregulation of various lineage-specific genes, including key definitive endoderm genes, such as GATA6. Upon differentiation, loss of H3K27me3 occurs in many endodermal genes, switching from a bivalent to a monovalent (H3K4me3) state. In conclusion, Met metabolism maintains the pluripotent network with histone marks, and their loss potentiates differentiation.

A specific plasma amino acid profile in the Insulin2Q104del Kuma mice at the diabetic state and reversal from hyperglycemia.

The metabolites in the plasma serve as potential biomarkers of disease. We previously established an early-onset diabetes mouse model, Ins2+/Q104del Kuma mice, under a severe immune-deficient (Rag-2/Jak3 double-deficient in BALB/c) background. Here, we revealed the differences in plasma amino acid profiles between Kuma and the wild-type mice. We observed an early reduction in glucogenic and ketogenic amino acids, a late increase in branched-chain amino acids (BCAAs) and succinyl CoA-related amino acids, and a trend of increasing ketogenic amino acids in Kuma mice than in the wild-type mice. Kuma mice exhibited hyperglucagonemia at high blood glucose, leading to perturbations in plasma amino acid profiles. The reversal of blood glucose by islet transplantation normalized the increases of the BCAAs and several aspects of the altered metabolic profiles in Kuma mice. Our results indicate that the Kuma mice are a unique animal model to study the link between plasma amino acid profile and the progression of diabetes for monitoring the therapeutic effects.

Fetal androgen signaling defects affect pancreatic ß-cell mass and function, leading to glucose intolerance in high-fat diet-fed male rats.

We previously demonstrated that androgen signaling expands pancreatic ß-cell mass in the sexual maturation period (Am J Physiol Endocrinol Metab 314: E274-E286, 2018). The aim of this study was to elucidate whether fetal androgen signaling plays important roles in ß-cell mass development and ß-cell function in adulthood, defects of which are associated with type 2 diabetes mellitus. In the pancreas of male fetuses, androgen receptor (AR) was strongly expressed in the cytoplasm and at the cell membrane of Nkx6.1-positive ß-cell precursor cells but was markedly reduced in insulin-positive ß-cells. Administration of the anti-androgen flutamide to pregnant dams during late gestation reduced ß-cell mass and Ki67-positive proliferating ß-cells at birth in a male-specific manner without affecting body weight. The decrease of ß-cell mass in flutamide-exposed male rats was not recovered when rats were fed a standard diet, whereas it was fully recovered when rats were fed a high-fat diet (HFD), at 6 and 12 wk of age. Flutamide exposure in utero led to the development of glucose intolerance in male rats due to a decrease in insulin secretion when fed HFD but not standard diet. Insulin sensitivity did not differ between the two groups irrespective of diet. These results indicated that the action of fetal androgen contributed to ß-cell mass expansion in a sex-specific manner at birth and to the development of glucose intolerance by decreasing the secretion of insulin in HFD-fed male rats. Our data demonstrated the involvement of fetal androgen signaling in hypothesized sex differences in the developmental origins of health and disease by affecting pancreatic ß-cell function.

Vascular access management after percutaneous transluminal angioplasty using a calcium alginate sheet: a randomized controlled trial.

BACKGROUND: Management of vascular access (VA) is essential in hemodialysis (HD) patients. However, VA often fails and percutaneous transluminal angioplasty (PTA) is required. Conventional hemostasis at the puncture site is associated with complications. This study aimed to analyze the efficacy and safety of a hemostatic wound dressing made of calcium alginate at the puncture site of VA after PTA and evaluate other factors affecting hemostasis. METHODS: After PTA for VA, 200 HD patients were randomized to a calcium alginate sheet (CA) group (n = 100) or a no drug-eluting sheet (control) group (n = 100). We recorded time to hemostasis at the puncture site every 5 min, noting any complications. RESULTS: In the CA group, rates of hemostatic achievement at 5, 10, 15 and >15 min were 57, 25, 8 and 10%, respectively. In the control group, the rates were 39, 28, 14 and 19%, respectively. Rates of hemostatic achievement at 5 min were significantly higher in the CA group (P = 0.01). In logistic regression analysis, factors affecting hemostasis within 5 min were use of the CA sheet [odds ratio (OR) 2.33; 95% confidence interval (CI) 1.26-4.37], platelet count ≤100 000/µL (OR 0.19; 95% CI 0.04-0.69), number of antithrombotic tablets used per day ≥1 tablet (OR 0.50; 95% CI 0.26-0.94) and upper arm VA (OR 0.16; 95% CI 0.03-0.55). CONCLUSIONS: A CA sheet can safely reduce time to hemostasis at the puncture site after PTA, and should be considered for treating patients with a bleeding tendency.

Androgen signaling expands ß-cell mass in male rats and ß-cell androgen receptor is degraded under high-glucose conditions.

A deficient pancreatic ß-cell mass increases the risk of type 2 diabetes mellitus. Here, we investigated the effects of testosterone on the development of pancreatic ß-cell mass in male rats. The ß-cell mass of male rats castrated at 6 wk of age was reduced to ~30% of that of control rats at 16 wk of age, and castration caused glucose intolerance. Loss of ß-cell mass occurred because of decreases in islet density per pancreas and ß-cell cluster size. Castration was negatively associated with the number of Ki-67-positive ß-cells and positively associated with the number of TUNEL-positive ß-cells. These ß-cell changes could be prevented by testosterone treatment. In contrast, castration did not affect ß-cell mass in male mice. Androgen receptor (AR) localized differently in mouse and rat ß-cells. Testosterone enhanced the viability of INS-1 and INS-1 #6, which expresses high levels of AR, in rat ß-cell lines. siRNA-mediated AR knockdown or AR antagonism with hydroxyflutamide attenuated this enhancement. Moreover, testosterone did not stimulate INS-1 ß-cell viability under high d-glucose conditions. In INS-1 ß-cells, d-glucose dose dependently (5.5-22.2 mM) downregulated AR protein levels both in the presence and absence of testosterone. The intracellular calcium chelator (BAPTA-AM) could prevent this decrease in AR expression. AR levels were also reduced by a calcium ionophore (A23187), but not by insulin, in the absence of the proteasome inhibitor MG132. Our results indicate that testosterone regulates ß-cell mass, at least in part, by AR activation in the ß-cells of male rats and that the ß-cell AR is degraded under hyperglycemic conditions.

Erythropoietin facilitates definitive endodermal differentiation of mouse embryonic stem cells via activation of ERK signaling.

Artificially generated pancreatic ß-cells from pluripotent stem cells are expected for cell replacement therapy for type 1 diabetes. Several strategies are adopted to direct pluripotent stem cells toward pancreatic differentiation. However, a standard differentiation method for clinical application has not been established. It is important to develop more effective and safer methods for generating pancreatic ß-cells without toxic or mutagenic chemicals. In the present study, we screened several endogenous factors involved in organ development to identify the factor, which induced the efficiency of pancreatic differentiation and found that treatment with erythropoietin (EPO) facilitated the differentiation of mouse embryonic stem cells (ESCs) into definitive endoderm. At an early stage of differentiation, EPO treatment significantly increased Sox17 gene expression, as a marker of the definitive endoderm. Contrary to the canonical function of EPO, it did not affect the levels of phosphorylated JAK2 and STAT5, but stimulated the phosphorylation of ERK1/2 and Akt. The MEK inhibitor U0126 significantly inhibited EPO-induced Sox17 expression. The differentiation of ESCs into definitive endoderm is an important step for the differentiation into pancreatic and other endodermal lineages. This study suggests a possible role of EPO in embryonic endodermal development and a new agent for directing the differentiation into endodermal lineages like pancreatic ß-cells.

Mild electrical stimulation with heat shock guides differentiation of embryonic stem cells into Pdx1-expressing cells within the definitive endoderm.

BACKGROUND: Because of the increasing number of diabetic patients, it is important to generate pancreatic and duodenal homeobox gene 1 (Pdx1)-expressing cells, which are capable of differentiating into pancreatic endocrine ß cells. Mild electrical stimulation was reported to modulate the differentiation of ES cells into ectoderm-derived neuronal cells or mesoderm-derived cardiac cells. RESULTS: In this study, we report that mild electrical stimulation with heat shock (MET) potentiates the differentiation of ES cells into definitive endoderm-derived Pdx1-expressing cells. MET has no effect when applied to early definitive endoderm on differentiation day 5. A 1.87-fold increase in the proportion of Pdx1-expressing cells was observed when stimulation was applied to the late definitive endoderm one day prior to the immergence of Pdx1/GFP-expressing cells on differentiation day 7. Pdx1 mRNA was also up-regulated by MET. The potentiating effect of MET synergized with activin and basic fibroblast growth factor into Pdx1-expressing cells. Moreover, MET stimulation on late definitive endoderm up-regulated heat shock protein 72 and activated various kinases including Akt, extracellular signal-regulated kinase, p38, and c-jun NH2-terminal kinase in ES cells. CONCLUSIONS: Our findings indicate that MET induces the differentiation of Pdx1-expressing cells within the definitive endoderm in a time-dependent manner, and suggest useful application for regenerative medicine.

Late stage definitive endodermal differentiation can be defined by Daf1 expression.

BACKGROUND: Definitive endoderm (DE) gives rise to the respiratory apparatus and digestive tract. Sox17 and Cxcr4 are useful markers of the DE. Previously, we identified a novel DE marker, Decay accelerating factor 1(Daf1/CD55), by identifying DE specific genes from the expression profile of DE derived from mouse embryonic stem cells (ESCs) by microarray analysis, and in situ hybridization of early embryos. Daf1 is expressed in a subpopulation of E-cadherin + Cxcr4+ DE cells. The characteristics of the Daf1-expressing cells during DE differentiation has not been examined. RESULTS: In this report, we utilized the ESC differentiation system to examine the characteristics of Daf1-expressing DE cells. We found that Daf1 expression could discriminate late DE from early DE. Early DE cells are Daf1-negative (DE-) and late DE cells are Daf1-positive (DE+). We also found that Daf1+ late DE cells show low proliferative and low cell matrix adhesive characteristics. Furthermore, the purified SOX17(low) early DE cells gave rise to Daf1+ Sox17(high) late DE cells. CONCLUSION: Daf1-expressing late definitive endoderm proliferates slowly and show low adhesive capacity.

Neural cells play an inhibitory role in pancreatic differentiation of pluripotent stem cells.

Pancreatic endocrine ß-cells derived from embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have received attention as screening systems for therapeutic drugs and as the basis for cell-based therapies. Here, we used a 12-day ß-cell differentiation protocol for mouse ES cells and obtained several hit compounds that promoted ß-cell differentiation. One of these compounds, mycophenolic acid (MPA), effectively promoted ES cell differentiation with a concomitant reduction of neuronal cells. The existence of neural cell-derived inhibitory humoral factors for ß-cell differentiation was suggested using a co-culture system. Based on gene array analysis, we focused on the Wnt/ß-catenin pathway and showed that the Wnt pathway inhibitor reversed MPA-induced ß-cell differentiation. Wnt pathway activation promoted ß-cell differentiation also in human iPS cells. Our results showed that Wnt signaling activation positively regulates ß-cell differentiation, and represent a downstream target of the neural inhibitory factor.

VMAT2 identified as a regulator of late-stage ß-cell differentiation.

Cell replacement therapy for diabetes mellitus requires cost-effective generation of high-quality, insulin-producing, pancreatic ß cells from pluripotent stem cells. Development of this technique has been hampered by a lack of knowledge of the molecular mechanisms underlying ß-cell differentiation. The present study identified reserpine and tetrabenazine (TBZ), both vesicular monoamine transporter 2 (VMAT2) inhibitors, as promoters of late-stage differentiation of Pdx1-positive pancreatic progenitor cells into Neurog3 (referred to henceforth as Ngn3)-positive endocrine precursors. VMAT2-controlled monoamines, such as dopamine, histamine and serotonin, negatively regulated ß-cell differentiation. Reserpine or TBZ acted additively with dibutyryl adenosine 3',5'-cyclic AMP, a cell-permeable cAMP analog, to potentiate differentiation of embryonic stem (ES) cells into ß cells that exhibited glucose-stimulated insulin secretion. When ES cell-derived ß cells were transplanted into AKITA diabetic mice, the cells reversed hyperglycemia. Our protocol provides a basis for the understanding of ß-cell differentiation and its application to a cost-effective production of functional ß cells for cell therapy.

High oxygen condition facilitates the differentiation of mouse and human pluripotent stem cells into pancreatic progenitors and insulin-producing cells.

Pluripotent stem cells have potential applications in regenerative medicine for diabetes. Differentiation of stem cells into insulin-producing cells has been achieved using various protocols. However, both the efficiency of the method and potency of differentiated cells are insufficient. Oxygen tension, the partial pressure of oxygen, has been shown to regulate the embryonic development of several organs, including pancreatic ß-cells. In this study, we tried to establish an effective method for the differentiation of induced pluripotent stem cells (iPSCs) into insulin-producing cells by culturing under high oxygen (O2) conditions. Treatment with a high O2 condition in the early stage of differentiation increased insulin-positive cells at the terminus of differentiation. We found that a high O2 condition repressed Notch-dependent gene Hes1 expression and increased Ngn3 expression at the stage of pancreatic progenitors. This effect was caused by inhibition of hypoxia-inducible factor-1α protein level. Moreover, a high O2 condition activated Wnt signaling. Optimal stage-specific treatment with a high O2 condition resulted in a significant increase in insulin production in both mouse embryonic stem cells and human iPSCs and yielded populations containing up to 10% C-peptide-positive cells in human iPSCs. These results suggest that culturing in a high O2 condition at a specific stage is useful for the efficient generation of insulin-producing cells.

A synthetic nanofibrillar matrix promotes in vitro hepatic differentiation of embryonic stem cells and induced pluripotent stem cells.

Embryonic stem (ES) cells recapitulate normal developmental processes and serve as an attractive source for routine access to a large number of cells for research and therapies. We previously reported that ES cells cultured on M15 cells, or a synthesized basement membrane (sBM) substratum, efficiently differentiated into an endodermal fate and subsequently adopted fates of various digestive organs, such as the pancreas and liver. Here, we established a novel hepatic differentiation procedure using the synthetic nanofiber (sNF) as a cell culture scaffold. We first compared endoderm induction and hepatic differentiation between murine ES cells grown on sNF and several other substrata. The functional assays for hepatocytes reveal that the ES cells grown on sNF were directed into hepatic differentiation. To clarify the mechanisms for the promotion of ES cell differentiation in the sNF system, we focused on the function of Rac1, which is a Rho family member protein known to regulate the actin cytoskeleton. We observed the activation of Rac1 in undifferentiated and differentiated ES cells cultured on sNF plates, but not in those cultured on normal plastic plates. We also show that inhibition of Rac1 blocked the potentiating effects of sNF on endoderm and hepatic differentiation throughout the whole differentiation stages. Taken together, our results suggest that morphological changes result in cellular differentiation controlled by Rac1 activation, and that motility is not only the consequence, but is also able to trigger differentiation. In conclusion, we believe that sNF is a promising material that might contribute to tissue engineering and drug delivery.

[Methionine metabolism regulates maintenance and differentiation of human ES/iPS cells].

Embryonic stem (ES) and induced pluripotent stem (iPS) cells are pluripotent and can give rise to all cell types. ES/iPS cells have a unique transcriptional circuit that sustains the pluripotent state. These cells also possess a characteristically high rate of proliferation as well as an abbreviated G1 phase. These unique molecular properties distinguish ES and iPS cells from somatic cells. Mouse ES/iPS cells are in a high-flux metabolic state, with a high dependence on threonine catabolism. However, little is known about amino acid metabolism in human ES/iPS cells. Recently, we reported that human ES/iPS cells require high amounts of methionine (Met) and express high levels of Met metabolism enzymes (Shriaki N, et al: Cell Metabolism, 2014). Met deprivation results in a rapid decrease in intracellular S-adenosyl-methionine (SAM), triggering the activation of p53 signaling, reducing pluripotent marker gene NANOG expression, and poising human ES/iPS cells for differentiation, follow by potentiated differentiation into all three germ layers. However, when exposed to prolonged Met deprivation, the cells went to apoptosis. In this review, we explain the importance of SAM in Met metabolism and its relationship with pluripotency, cell survival, and differentiation of human ES/iPS cells.

Wnt and Notch signals guide embryonic stem cell differentiation into the intestinal lineages.

The studies of differentiation of mouse or human embryonic stem cells (hESCs) into specific cell types of the intestinal cells would provide insights to the understanding of intestinal development and ultimately yield cells for the use in future regenerative medicine. Here, using an in vitro differentiation procedure of pluripotent stem cells into definitive endoderm (DE), inductive signal pathways' guiding differentiation into intestinal cells was investigated. We found that activation of Wnt/ß-catenin and inhibition of Notch signaling pathways, by simultaneous application of 6-bromoindirubin-3'-oxime (BIO), a glycogen synthase kinase-3ß inhibitor, and N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester (DAPT), a known γ-secretase inhibitor, efficiently induced intestinal differentiation of ESCs cultured on feeder cell. BIO and DAPT patterned the DE at graded concentrations. Upon prolonged culture on feeder cells, all four intestinal differentiated cell types, the absorptive enterocytes and three types of secretory cells (goblet cells, enteroendocrine cells, and Paneth cells), were efficiently differentiated from mouse and hESC-derived intestinal epithelium cells. Further investigation revealed that in the mouse ESCs, fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) signaling act synergistically with BIO and DAPT to potentiate differentiation into the intestinal epithelium. However, in hESCs, FGF signaling inhibited, and BMP signaling did not affect differentiation into the intestinal epithelium. We concluded that Wnt and Notch signaling function to pattern the anterior-posterior axis of the DE and control intestinal differentiation.

Low serum testosterone is associated with atherosclerosis in postmenopausal women undergoing hemodialysis.

BACKGROUND: Low serum testosterone levels have been recently linked to endothelial dysfunction, arterial stiffness, and worse outcomes in male hemodialysis patients. We tested the hypothesis that low serum testosterone levels are also associated with atherosclerosis risk factors in postmenopausal women undergoing hemodialysis. METHODS: We measured serum testosterone in 115 confirmed postmenopausal ethnically Japanese women undergoing hemodialysis with mean age of 68.1 ± 10.6 years and median dialysis vintage of 73 months. The severity of atherosclerosis was evaluated by carotid intima-media thickness (cIMT) and cardio-ankle vascular index (CAVI). In addition, we also included a control cohort of 32 age-matched postmenopausal women without chronic kidney disease. RESULTS: Serum testosterone was significantly lower in women undergoing hemodialysis than in age-matched controls. Women undergoing hemodialysis who had undetectable testosterone concentration presented higher cIMT and higher CAVI than women undergoing hemodialysis with testosterone concentration above detection limits (P < 0.05 for all). Multiple logistic regression analyses confirmed the independence of these associations. CONCLUSION: Serum testosterone levels in postmenopausal women undergoing hemodialysis are abnormally low and associated with features of atherosclerosis.

Characterization of heterozygous ATTR Tyr114Cys amyloidosis-specific induced pluripotent stem cells.

Hereditary transthyretin (TTR) amyloidosis (ATTRv amyloidosis) is autosomal dominant and caused by mutation of TTR gene. Heterozygous ATTR Tyr114Cys (p.Tyr134Cys) amyloidosis is a lethal disease with a life expectancy of about 10 years after onset of the disease. However, the molecular pathogenesis of ATTR Tyr114Cys amyloidosis is still largely unknown. In this study, we took advantage of disease-specific induced pluripotent stem (iPS) cells and generated & characterized the heterozygous ATTR Tyr114Cys amyloidosis-specific iPS cells (Y114C iPS cells), to determine whether Y114C iPS cells could be useful for elucidating the pathogenesis of ATTR Tyr114Cys amyloidosis. We successfully differentiated heterozygous Y114C iPS cells into hepatocyte like cells (HLCs) mainly producing TTR protein. On day 27 after differentiation, the expression of hepatocyte maker albumin was detected, and TTR expression was significantly increased in HLCs differentiated from Y114C iPS cells. LC-MS/MS analysis showed that both WT TTR & ATTR Y114C protein were indeed expressed in the HLCs differentiated from Y114C iPS cells. Notably, the number of detected peptides derived from ATTR Y114C protein was lower than that of WT TTR protein, indeed indicating the clinical phenotype of ATTR Tyr114Cys amyloidosis. Taken together, we first reported the heterozygous Y114C iPS cells generated from patient with ATTR Tyr114Cys amyloidosis, and suggested that Y114C iPS cells could be a potential pathological tool, which may contribute to elucidating the molecular pathogenesis of heterozygous ATTR Tyr114Cys amyloidosis.

Gut-liver microphysiological systems revealed potential crosstalk mechanism modulating drug metabolism.

The small intestine and liver play important role in determining oral drug's fate. Both organs are also interconnected through enterohepatic circulation, which imply there are crosstalk through circulating factors such as signaling molecules or metabolites that may affect drug metabolism. Coculture of hepatocytes and intestinal cells have shown to increase hepatic drug metabolism, yet its crosstalk mechanism is still unclear. In this study, we aim to elucidate such crosstalk by coculturing primary human hepatocytes harvested from chimeric mouse (PXB-cells) and iPSc-derived intestinal cells in a microphysiological systems (MPS). Perfusion and direct oxygenation from the MPS were chosen and confirmed to be suitable features that enhanced PXB-cells albumin secretion, cytochrome P450 (CYP) enzymes activity while also maintaining barrier integrity of iPSc-derived intestine cells. Results from RNA-sequencing showed significant upregulation in gene ontology terms related to fatty acids metabolism in PXB-cells. One of such fatty acids, arachidonic acid, enhanced several CYP enzyme activity in similar manner as coculture. From the current evidences, it is speculated that the release of bile acids from PXB-cells acted as stimuli for iPSc-derived intestine cells to release lipoprotein which was ultimately taken by PXB-cells and enhanced CYP activity.

Tissue-specific demethylation in CpG-poor promoters during cellular differentiation.

Epigenetic regulation is essential in determining cellular phenotypes during differentiation. Although tissue-specific DNA methylation has been studied, the significance of methylation variance for tissue phenotypes remains unresolved, especially for CpG-poor promoters. Here, we comprehensively studied methylation levels of 27 578 CpG sites among 21 human normal tissues from 12 anatomically different regions using an epigenotyping beadarray system. Remarkable changes in tissue-specific DNA methylation were observed within CpG-poor promoters but not CpG-rich promoters. Of note, tissue-specific hypomethylation is accompanied by an increase in gene expression, which gives rise to specialized cellular functions. The hypomethylated regions were significantly enriched with recognition motifs for transcription factors that regulate cell-type-specific differentiation. To investigate the dynamics of hypomethylation events, we analyzed methylation levels of the entire APOA1 gene locus during in vitro differentiation of embryonic stem cells toward the hepatic lineage. A decrease in methylation was observed after day 13, coinciding with alpha-fetoprotein detection, in the vicinity of its transcription start sites (TSSs), and extends up to ∼200 bp region encompassing the TSS at day 21, equivalent to the hepatoblastic stage. This decrease is even more pronounced in the adult liver, where the entire APOA1 gene locus is hypomethylated. Furthermore, when we compared the methylation status of induced pluripotent stem (iPS) cells with their parental cell, IMR-90, we found that fibroblast-specific hypomethylation is restored to a fully methylated state in iPS cells after reprogramming. These results illuminate tissue-specific methylation dynamics in CpG-poor promoters and provide more comprehensive views on spatiotemporal gene regulation in terminal differentiation.

Recovery from diabetes in neonatal mice after a low-dose streptozotocin treatment.

Administration of streptozotocin (STZ) induces destruction of ß-cells and is widely used as an experimental animal model of type I diabetes. In neonatal rat, after low-doses of STZ-mediated destruction of ß-cells, ß-cells regeneration occurs and reversal of hyperglycemia was observed. However, in neonatal mice, ß-cell regeneration seems to occur much slowly compared to that observed in the rat. Here, we described the time dependent quantitative changes in ß-cell mass during a spontaneous slow recovery of diabetes induced in a low-dose STZ mice model. We then investigated the underlying mechanisms and analyzed the cell source for the recovery of ß-cells. We showed here that postnatal day 7 (P7) female mice treated with 50 mg/kg STZ underwent the destruction of a large proportion of ß-cells and developed hyperglycemia. The blood glucose increased gradually and reached a peak level at 500 mg/dl on day 35-50. This was followed by a spontaneous regeneration of ß-cells. A reversal of non-fasting blood glucose to the control value was observed within 150 days. However, the mice still showed impaired glucose tolerance on day 150 and day 220, although a significant improvement was observed on day 150. Quantification of the ß-cell mass revealed that the ß-cell mass increased significantly between day 100 and day 150. On day 150 and day 220, the ß-cell mass was approximately 23% and 48.5% of the control, respectively. Of the insulin-positive cells, 10% turned out to be PCNA-positive proliferating cells. Our results demonstrated that, ß-cell duplication is one of the cell sources for ß-cell regeneration.