BK channel dysfunction disrupts attention-controlled behaviors and altered perseverative responses in murine instrumental learning.

This study examined the effect of knockout of KCNMA1 gene, coding for the BK channel, on cognitive and attentional functions in mice, with an aim to better understand its implications for human neurodevelopmental disorders. The study used the 3-choice serial reaction time task (3-CSRTT) to assess the learning performance, attentional abilities, and repetitive behaviors in mice lacking the KCNMA1 gene (KCNMA1-/-) compared to wild-type (WT) controls. Results showed no significant differences in learning accuracy between the two groups. However, KCNMA1-/- mice were more prone to omitting responses to stimuli. In addition, when the timing of cue presentation was randomized, the KCNMA1-/- showed premature responses. Notably, these mice also demonstrated a marked reduction in perseverative responses, which include repeated nose-poke behaviors following decisions. These findings highlight the involvement of the KCNMA1 gene in managing attention, impulsivity, and potentially moderating repetitive actions.

Treatment with lysophosphatidic acid improves glomerulonephritis through the suppression of macrophage activation in a murine model of systemic lupus erythematosus.

OBJECTIVES: Several therapeutic agents have been developed and used for the clinical treatment of systemic lupus erythematosus (SLE). In cases where SLE is accompanied by severe organ failures, such as neuropsychiatric lupus erythematosus (NPSLE) and acute onset of lupus nephritis, the use of potent immunosuppressive drugs, such as cyclophosphamide, is necessary. However, potent immunosuppressive drugs are known to increase infection risks. Thus, the development of therapeutic agents with novel mechanisms is urgently required. Previously, we reported that treatment with lysophosphatidic acid (LPA) prevents depression-like behaviours by suppressing microglial activation in MRL/lpr mice. In this study, we examined whether the treatment with LPA improves glomerulonephritis by affecting systemic immunity in MRL/lpr mice. METHODS: Eighteen-week-old MRL/lpr mice were treated with a vehicle or LPA for 3 weeks. After treatment, the glomerular inflammation and damage parameters were compared between the 2 groups. Moreover, we examined the effects of LPA on immune cells by flow cytometry using isolated splenocytes. RESULTS: LPA treatment in MRL/lpr mice significantly reduced the daily urinary albumin content and suppressed the CD68-positive cells and Periodic acid-Schiff (PAS)-positive areas in the glomeruli. The treatment also suppressed plasma anti-dsDNA antibodies and inflammatory cytokines in MRL/lpr mice. Although LPA did not significantly affect the total number of splenocytes, the treatment significantly reduced CD11b+Ly6G-Ly6C- cells (mature macrophages), as well as CD11b+Ly6G-Ly6C-CD68+ cells (activated mature macrophages). CONCLUSIONS: These results suggest that LPA may improve glomerulonephritis by suppressing macrophage activation in MRL/lpr mice.

A preliminary therapeutic study of the effects of molecular hydrogen on intestinal dysbiosis and small intestinal injury in high-fat diet-loaded senescence-accelerated mice.

OBJECTIVES: Aging and excessive fat intake may additively induce dysbiosis of the gut microbiota and intestinal inflammatory damage. Here, we analyzed microbiota dysbiosis and intestinal injury in high-fat diet-loaded senescence-accelerated mice (SAMP8). Additionally, we examined whether treatment with molecular hydrogen could improve the intestinal environment. METHODS: SAMP8 and SAMR1 (control) mice were first fed a normal diet (ND) or high-fat diet (HFD) for 10 wk (n = 10 each group). Subsequently, HFD was supplemented with a placebo jelly or hydrogen-rich jelly (HRJ) for 4 wk. After treatment, isolated small intestinal tissues were used for hematoxylin and eosin staining, immunofluorescence staining, and thiobarbituric acid reactive substances (TBARS) assay. Furthermore, we analyzed alterations in the microbiota composition in cecal feces using 16S rRNA gene analysis for microbiota profiling. Statistical analyses were performed using unpaired Student's t tests or one-way analysis of variance and Tukey's post hoc test for multiple comparisons. RESULT: HFD feeding reduced the expression of caudal-related homeobox transcription factor 2 (CDX2) and 5-bromo-2'-deoxyuridine (BrdU) and enhanced malondialdehyde (MDA) levels in the small intestine of SAMP8. HRJ treatment improved the reduction in CDX2 and BrdU and enhanced MDA levels. We performed a sequence analysis of the gut microbiota at the genus level and identified 283 different bacterial genera from the 30 samples analyzed in the study. Among them, Parvibacter positively correlated with both HFD intake and aging, whereas 10 bacteria, including Anaerofustis, Anaerosporobacter, Butyricicoccus, and Ruminococcus were negatively correlated with both HFD and aging. HRJ treatment increased Lactinobactor and decreased Akkermansia, Gracilibacter, and Marvinbryantia abundance. CONCLUSION: Our findings suggest that treatment with molecular hydrogen may affect microbiota profiling and suppress intestinal injury in HFD-loaded SAMP8.

Two-carba cyclic phosphatidic acid treatment promotes phenotypic switch from M1 to M2 microglia and prevents behavioral abnormalities in a mouse model of neuropsychiatric systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease associated with the production of double-stranded DNA (dsDNA) antibodies and other antibodies that predominantly affects women with a wide range of lesions. Although neuropsychiatric lupus erythematosus (NPSLE), characterized by neuropsychiatric symptoms related to cerebrovascular diseases or depression, ranks high in severity, no specific treatment has been defined. Two-carba cyclic phosphatidic acid (2ccPA), a derivative of cyclic phosphatidic acid, was isolated from the true slime mold Physarum polycephalum in 1992. 2ccPA treatment suppresses neuroinflammation and promotes tissue repair in mouse multiple sclerosis and traumatic brain injury models. In this study, we performed behavioral tests on MRL/lpr mice as an NPSLE model. MRL/lpr mice showed increased depression-like behaviors compared with control mice, which were significantly suppressed by 2ccPA treatment. The expression of CD68, an M1 phenotypic marker of microglia, was significantly elevated in the prefrontal cortex and hippocampus of MRL/lpr mice, which was significantly suppressed by 2ccPA treatment. In contrast, the expression of Arginase1, an M2 phenotypic marker of microglia, was significantly increased by 2ccPA treatment. Compared to control mice, MRL/lpr mice showed higher plasma levels of anti-dsDNA antibodies, which are mainly involved in SLE pathogenesis. 2ccPA treatment decreased these levels in the MRL/lpr mice. These results suggest that 2ccPA treatment suppresses behavioral abnormalities by promoting a microglial phenotypic switch from M1 to M2 in MRL/lpr mice.

Evans Blue and Fluorescein Isothiocyanate-Dextran Double Labeling Reveals Precise Sequence of Vascular Leakage and Glial Responses After Exposure to Mild-Level Blast-Associated Shock Waves.

Abstract Blast-induced shock waves (BSWs) are responsible for several aspects of psychiatric disorders that are collectively termed mild traumatic brain injury (mTBI). The pathophysiology of mTBI includes vascular leakage resulting from blood-brain barrier (BBB) disruption. In this study, the precise sequence of BBB breakdown was examined using an Evans blue and fluorescein isothiocyanate (FITC)-dextran double labeling technique. Evans blue solution was injected into the tail vein of male C57BL6/J mice just before and 4 h, 1 day, 3 days, and 7 days after a single BSW exposure at as low as 25-kPa peak overpressure. In contrast, the FITC-dextran solution was transcardially injected just before perfusion fixation. Differences in the labeling time-point revealed that BBB breakdown was initiated after approximately 3 h, with significant remodeling by 1 day, and continued until 7 days after BSW exposure. BBB breakdown was upregulated in three distinct regions, namely the brain surface and subsurface areas facing the skull, regions closely associated with capillaries, and the circumventricular organ and choroid plexus. These regions showed distinct responses to BSW; moreover, clusters of reactive astrocytes were closely associated with the sites of BBB breakdown. In severe cases, these reactive astrocytes recruited activated microglia. Our findings provide important insights into the pathogenesis underlying mTBI and indicate that even mild BSW exposure affects the whole brain.

Treatment with lysophosphatidic acid prevents microglial activation and depression-like behaviours in a murine model of neuropsychiatric systemic lupus erythematosus.

Neuropsychiatric systemic lupus erythematosus (NPSLE) is an incurable disease characterised by neuropsychiatric symptoms, particularly depression. Novel therapeutic options for NPSLE are urgently needed. Several previous reports have suggested that both microglial activation and impaired neurogenesis may be involved in the progression of depression. In contrast, the administration of lysophosphatidic acid (LPA) ameliorates depression and anxiety. Therefore, in the present study, we determined whether treatment with LPA affects microglial activation, impaired neurogenesis, and abnormal behaviour in MRL/lpr mice. In both tail suspension test and forced swim test, the MRL/lpr mice exhibited a significant increase in total immobility time compared with MRL/+ mice. Treatment with LPA significantly suppressed the prolonged immobility time in MRL/lpr mice. In contrast, pretreatment with ki16425 (a specific antagonist of LPA receptor 1 and 3) significantly reversed the effects of LPA. Furthermore, MRL/lpr mice exhibited impairments in spatial working memory and visual cognitive memory, which were suppressed by LPA treatment. The expression levels of TMEM119, CD68, GFAP, and caspase-3 in the hippocampus and prefrontal cortex of MRL/lpr mice were significantly higher than those in MRL/+ mice. Treatment with LPA inhibited these increases in MRL/lpr mice. Pretreatment with ki16425 reversed LPA-mediated inhibition of microglial activation. The quantity of sodium fluorescein that leaked into the brain tissues in MRL/lpr mice were significantly higher than that in MRL/+ mice. Treatment with LPA tended to decrease the sodium fluorescein leakage. These findings suggest that treatment with LPA may regulate microglial activation, which is important in the pathogenesis of NPSLE, as well as blood-brain-barrier weakening and abnormal behaviour.

Neonatal administration of a subanaesthetic dose of JM-1232(-) in mice results in no behavioural deficits in adulthood.

In animal models, neonatal exposure of general anaesthetics significantly increases apoptosis in the brain, resulting in persistent behavioural deficits later in adulthood. Consequently, there is growing concern about the use of general anaesthetics in obstetric and paediatric practice. JM-1232(-) has been developed as a novel intravenous anaesthetic, but the effects of JM-1232(-) on the developing brain are not understood. Here we show that neonatal administration of JM-1232(-) does not lead to detectable behavioural deficits in adulthood, contrarily to other widely-used intravenous anaesthetics. At postnatal day 6 (P6), mice were injected intraperitoneally with a sedative-equivalent dose of JM-1232(-), propofol, or midazolam. Western blot analysis of forebrain extracts using cleaved poly-(adenosine diphosphate-ribose) polymerase antibody showed that JM-1232(-) is accompanied by slight but measurable apoptosis 6 h after administration, but it was relatively small compared to those of propofol and midazolam. Behavioural studies were performed in adulthood, long after the neonatal anaesthesia, to evaluate the long-term effects on cognitive, social, and affective functions. P6 administration to JM-1232(-) was not accompanied by detectable long-term behavioural deficits in adulthood. However, animals receiving propofol or midazolam had impaired social and/or cognitive functions. These data suggest that JM-1232(-) has prospects for use in obstetric and paediatric practice.

The asymmetric learning rates of murine exploratory behavior in sparse reward environments.

Goal-oriented behaviors of animals can be modeled by reinforcement learning algorithms. Such algorithms predict future outcomes of selected actions utilizing action values and updating those values in response to the positive and negative outcomes. In many models of animal behavior, the action values are updated symmetrically based on a common learning rate, that is, in the same way for both positive and negative outcomes. However, animals in environments with scarce rewards may have uneven learning rates. To investigate the asymmetry in learning rates in reward and non-reward, we analyzed the exploration behavior of mice in five-armed bandit tasks using a Q-learning model with differential learning rates for positive and negative outcomes. The positive learning rate was significantly higher in a scarce reward environment than in a rich reward environment, and conversely, the negative learning rate was significantly lower in the scarce environment. The positive to negative learning rate ratio was about 10 in the scarce environment and about 2 in the rich environment. This result suggests that when the reward probability was low, the mice tend to ignore failures and exploit the rare rewards. Computational modeling analysis revealed that the increased learning rates ratio could cause an overestimation of and perseveration on rare-rewarding events, increasing total reward acquisition in the scarce environment but disadvantaging impartial exploration.

Extracellular signal-regulated kinases 2 (Erk2) and Erk5 in the central nervous system differentially contribute to central sensitization in male mice.

Nervous systems are designed to become extra sensitive to afferent nociceptive stimuli under certain circumstances such as inflammation and nerve injury. How pain hypersensitivity comes about is key issue in the field since it ultimately results in chronic pain. Central sensitization represents enhanced pain sensitivity due to increased neural signaling within the central nervous system (CNS). Particularly, much evidence indicates that underlying mechanism of central sensitization is associated with the change of spinal neurons. Extracellular signal-regulated kinases have received attention as key molecules in central sensitization. Previously, we revealed the isoform-specific function of extracellular signal-regulated kinase 2 (Erk2) in spinal neurons for central sensitization using mice with Cre-loxP-mediated deletion of Erk2 in the CNS. Still, how extracellular signal-regulated kinase 5 (Erk5) in spinal neurons contributes to central sensitization has not been directly tested, nor is the functional relevance of Erk5 and Erk2 known. Here, we show that Erk5 and Erk2 in the CNS play redundant and/or distinct roles in central sensitization, depending on the plasticity context (cell types, pain types, time, etc.). We used male mice with Erk5 deletion specifically in the CNS and found that Erk5 plays important roles in central sensitization in a formalin-induced inflammatory pain model. Deletion of both Erk2 and Erk5 leads to greater attenuation of central sensitization in this model, compared to deletion of either isoform alone. Conversely, Erk2 but not Erk5 plays important roles in central sensitization in neuropathic pain, a type of chronic pain caused by nerve damage. Our results suggest the elaborate mechanisms of Erk signaling in central sensitization.

Effects of oxidized low-density lipoprotein on differentiation of mouse neural progenitor cells into neural cells.

In Alzheimer's disease (AD), a decline in function of neural progenitor cells (NPCs) results in a reduced capacity for neural regeneration. It has been shown that plasma oxidized low-density lipoprotein (ox-LDL) levels are positively correlated with severity in patients with AD. However, the direct effects of ox-LDL on NPCs are unknown. Thus, we examined the effects of ox-LDL on the proliferation and differentiation of mouse NPCs into neural cells. Mouse induced pluripotent stem (iPS) cell-derived embryoid bodies were stimulated with Noggin and SB431542 for 4 days. Mouse NPCs were then collected using anti-polysialic acid-neural cell adhesion molecule antibodies in a magnetic separator. The proliferation of mouse NPCs was examined using the MTT assay. The differentiation of mouse NPCs into neural cells was examined by the expression of NeuN (a neuronal-specific nuclear protein) using immunofluorescence staining and Western blot analysis. Treatment with ox-LDL did not affect the proliferation of mouse NPCs. While treatment with all-trans retinoic acid (ATRA), epidermal growth factor (EGF), and basic fibroblast growth factor (FGF) significantly induced NeuN expression in the differentiated NPCs (P < 0.01), the addition of ox-LDL significantly inhibited the NeuN expression (P < 0.05). Pretreatment with SC-79 (an Akt activator) significantly reversed the inhibitory effect of ox-LDL on NeuN expression (P < 0.05). Treatment with ox-LDL significantly inhibited Akt phosphorylation (P < 0.05) and CREB phosphorylation induced by ATRA, EGF, and basic FGF (P < 0.05). The present study indicates that treatment with ox-LDL inhibits the differentiation of mouse NPCs into neural cells by inhibiting Akt and CREB activation.

Low body mass index and lymphocytopenia associate with Mycobacterium avium complex pulmonary disease in patients with rheumatoid arthritis.

OBJECTIVES: Patients with rheumatoid arthritis (RA) are at an increased risk of Mycobacterium avium complex pulmonary disease (MAC-PD). We aimed to identify factors associated with MAC-PD in RA patients, and investigate their clinical significance for diagnosis of this disease. METHODS: We examined 396 patients with RA for the presence of MAC-PD, using the criteria of the American Thoracic Society and conducted three years of follow-up on these patients. Multivariate logistic analyses were employed for selecting factors associated with MAC-PD. We developed a point system based on these factors which we call MAC-PD score to improve diagnosis of MAC-PD. RESULTS: During this study, 14 out of 396 patients were newly diagnosed with MAC-PD. Multivariate analyses revealed body mass index (BMI) <18.0 kg/m2 and lymphocyte count <1500/µl were associated with MAC-PD in RA patients. Points were assigned to them and totalled to provide the MAC-PD score. Among 20 patients with high-resolution computer tomography images consistent with MAC-PD, the scores were significantly higher in 14 patients with MAC-PD than those in six patients without MAC-PD. CONCLUSION: Using these data, in the forms of the MAC-PD score, could help to identify patients who should be considered for bronchoscopy more selectively.

Molecular Hydrogen Prevents Social Deficits and Depression-Like Behaviors Induced by Low-Intensity Blast in Mice.

Detonation of explosive devices creates blast waves, which can injure brains even in the absence of external injuries. Among these, blast-induced mild traumatic brain injury (bmTBI) is increasing in military populations, such as in the wars in Afghanistan, Iraq, and Syria. Although the clinical presentation of bmTBI is not precisely defined, it is frequently associated with psycho-neurological deficits and usually manifests in the form of poly-trauma including psychiatric morbidity and cognitive disruption. Although the underlying mechanisms of bmTBI are largely unknown, some studies suggested that bmTBI is associated with blood-brain barrier disruption, oxidative stress, and edema in the brain. The present study investigated the effects of novel antioxidant, molecular hydrogen gas, on bmTBI using a laboratory-scale shock tube model in mice. Hydrogen gas has a strong prospect for clinical use due to easy preparation, low-cost, and no side effects. The administration of hydrogen gas significantly attenuated the behavioral deficits observed in our bmTBI model, suggesting that hydrogen application might be a strong therapeutic method for treatment of bmTBI.

Nicotine treatment ameliorates DSS-induced colitis by suppressing MAdCAM-1 expression and leukocyte recruitment.

The enhanced recruitment of leukocytes to the inflamed colon is a key feature of ulcerative colitis (UC). The gut-specific adhesion molecules involved in leukocyte recruitment have emerged as recent therapeutic targets. Nicotine absorbed from smoking has been reported to work protectively in UC patients. Our hypothesis is that nicotine may suppress the aberrant leukocyte recruitment and colonic inflammation via the suppression of the overexpressed gut-specific adhesion molecules in the inflamed colon. To test this hypothesis, the severity of colitis and the degree of leukocyte recruitment induced by gut-specific adhesion molecules were assessed in dextran sulfate sodium (DSS) colitis mice (C57BL/6J mice treated with 3% DSS) with or without nicotine treatment. We also studied the in vitro changes in the expression of adhesion molecules by using a vascular endothelial cell line. DSS-induced colitis was accompanied by increases in disease activity index (DAI), histological score, recruitment of leukocytes, and the expression of adhesion molecules, mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) and VCAM-1. Nicotine treatment significantly attenuated MAdCAM-1 expression, leukocyte recruitment, DAI, and histological score. The expression of ß7-integrin, the ligand for MAdCAM-1, on leukocytes was not affected by nicotine treatment. In vitro study, the TNF-α-enhanced mRNA expression of MAdCAM-1 was reduced by the coadministration of nicotine in a dose-dependent manner, possibly via nicotinic receptor activation. These results supported our hypothesis that nicotine treatment ameliorated colitis through the suppression of MAdCAM-1 expression on the microvessels in the inflamed colon. Further investigation is warranted on the role of nicotine in the treatment of UC.

Effects of muscarinic acetylcholine receptor stimulation on the differentiation of mouse induced pluripotent stem cells into neural progenitor cells.

Muscarinic acetylcholine receptors (mAchRs), which are expressed in various embryonic cells, may regulate neuronal differentiation. In the present study, we examined the effects of mAchR stimulation on the differentiation of mouse induced pluripotent stem (iPS) cells into neural progenitor cells (NPCs). Mouse iPS cells were cultured on ultra-low attachment dishes to induce embryoid body (EB) formation. All-trans retinoic acid (ATRA, 3 µmol/L) and/or pilocarpine (10 or 100 µmol/L), a mAchR agonist, were added to EB cultures for 4 days, following which the EBs were cultured on gelatin-coated plates for 7 days. Subtype-specific antibody staining revealed that mouse iPS cells predominantly express m2 - and m4 -AchR. Treatment with pilocarpine alone did not affect the expression of Nestin (a specific marker for neural progenitor cells). However, additional treatment with pilocarpine significantly suppressed ATRA-induced Nestin expression. Pretreating EBs with either AF-DX116 (an antagonist of both m2 - and m4 -AchR) or forskolin (an activator of adenylate cyclase) significantly reversed the pilocarpine-induced suppression of Nestin expression. In addition, treatment with pilocarpine significantly suppressed ATRA-induced phosphorylation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB). These findings suggest that the stimulation of m2 - or m4 -AchR suppresses ATRA-induced differentiation of mouse iPS cells into NPCs by inhibiting the cAMP/protein kinase A pathway and CREB activation.

Tumor growth limited to subcutaneous site vs tumor growth in pulmonary site exhibit differential effects on systemic immunities.

To evaluate systemic immunity associated with tumor growth limited to a subcutaneous site versus growth proceeding at multiple tumor sites, we established syngeneic mouse subcutaneous and pulmonary tumor models by local subcutaneous and intravenous injection of colon carcinoma CT26 cells. We found that splenic myeloid-derived suppressor cell (MDSC) levels were significantly increased in the subcutaneous tumor model but not in the pulmonary tumor model. Furthermore, both CD4+ and CD8+ T cells as well as CD4+ Foxp3+ T cells were significantly decreased in the subcutaneous tumor model and were largely unchanged in the pulmonary tumor model. In addition, the subcutaneous model, but not the pulmonary model, displayed a Th1 polarization bias. This bias was characterized by decreased IL-4, IL-9, and IL-10 production, whereas the pulmonary model displayed increased production of IL-10. These results suggest that the mode of tumor development has differential effects on systemic immunity that may, in turn, influence approaches to treatment of cancer patients.

Diagnostic performance of measuring antibodies to the glycopeptidolipid core antigen specific to Mycobacterium avium complex in patients with rheumatoid arthritis: results from a cross-sectional observational study.

INTRODUCTION: The aim of this study was to investigate the diagnostic performance of measuring antibodies to the glycopeptidolipid (GPL) core antigen specific to Mycobacterium avium complex (MAC) in patients with rheumatoid arthritis (RA). METHODS: We cross-sectionally investigated anti-GPL antibodies and radiographs of 396 patients with RA. A diagnosis of MAC pulmonary disease (MAC-PD) was made according to the criteria by the American Thoracic Society and the Infectious Diseases Society of America. Serum immunoglobulin A antibodies to MAC-specific GPL core antigen were measured by an enzyme immunoassay. All patients with RA with abnormal shadows on chest x-rays underwent chest computed tomography (CT). Bronchoscopy was performed on patients with negative cultures for MAC by expectorated sputum and positive CT findings compatible with MAC-PD. RESULTS: Ten patients were newly diagnosed with MAC-PD. Eight individuals who already had diagnoses of MAC-PD at the time of enrollment and nineteen who had negative expectorated sputum cultures for MAC and positive CT images compatible with MAC-PD and who refused bronchoscopy were excluded from the following analysis. Anti-GPL antibodies were detected in 12 of 369 patients. Eight of the ten patients with MAC-PD and 4 of 359 patients without MAC-PD tested positive for the anti-GPL antibodies. The specificity and sensitivity were 99 % and 80 %, respectively. Positive and negative predictive values were 67 %, and 97 %, respectively. When we analyzed diagnostic performance of the antibodies in 57 patients with RA who had abnormal shadows on chest x-rays, the positive and negative predictive values were 100 %, and 96 %, respectively. Twelve patients underwent bronchoscopy. Bronchoalveolar lavage fluid (BALF) samples from six patients were positive for MAC, and BALF samples from the remainder were negative. Anti-GPL antibodies were detected in the sera of all six patients with positive results for MAC by BALF sampling, whereas the antibodies were not detected in the sera from the remainder with negative results for MAC by BALF sampling. CONCLUSIONS: The measurement of anti-GPL antibodies is useful as a supplementary diagnostic tool for MAC-PD in patients with RA and may provide a new strategy, in combination with chest x-ray and CT, for differentiating MAC-PD from other pulmonary comorbidities in patients with RA.

Involvement of ß-adrenoceptors in the differentiation of human induced pluripotent stem cells into mesodermal progenitor cells.

Previous studies suggest that ß-adrenoceptor stimulation may enhance the cardiac differentiation of mouse embryonic stem (ES) cells. It remains unclear whether the differentiations of ES cells and induced pluripotent stem (iPS) cells rely on similar molecular mechanisms. In addition, no previous studies have shown that human iPS cells express ß-adrenoceptors. Therefore, in the present study, we determined the involvement of ß-adrenoceptors in the differentiation of human iPS cells into mesodermal progenitor cells. The induction of differentiation of human iPS cells into kinase insert domain receptor (KDR)-positive mesodermal progenitor cells was performed on feeder cells in a differentiation medium with basic fibroblast growth factor (bFGF), bone morphogenetic protein-4 (BMP-4), and activin A. When the iPS cells that were exposed to bFGF, BMP-4, and activin A were treated with L-isoproterenol (a ß-adrenoceptor agonist) for 4 days, the expression of KDR was significantly increased compared to that in the cells that were not treated with L-isoproterenol. Pretreatment of the cells with either atenolol (a ß1-adrenoceptor antagonist) or ICI-118551 (a ß2-adrenoceptor antagonist) significantly inhibited the L-isoproterenol-induced increase in KDR expression. In addition, pretreatment with both H89 (a protein kinase A inhibitor) and SB203580 (a p38 MAPK inhibitor) significantly inhibited the L-isoproterenol-induced increase in KDR expression. Treatment with L-isoproterenol enhanced the phosphorylation of p38 MAPK in human iPS cells exposed to bFGF, BMP-4, and activin A. These results suggest that ß-adrenoceptor stimulation in human iPS cells may enhance their differentiation into mesodermal progenitor cells via the activation of either protein kinase A or p38 MAPK.

Stimulation of 5-HT4 receptor enhances differentiation of mouse induced pluripotent stem cells into neural progenitor cells.

Activation of serotonin (5-hydroxytryptamine; 5-HT) receptors plays a role in adult neurogenesis and differentiation of neural progenitor cells (NPC). Herein, we examined the involvement of 5-HT receptors in the differentiation of mouse induced pluripotent stem (iPS) cells into NPC. To induce embryoid body (EB) formation, mouse iPS cells were cultured on ultralow-attachment dishes. All-trans retinoic acid (ATRA; 1 µmol/L) and/or 5-HT (0.03 or 0.1 µmol/L) was added to the EB cultures for 4 days and then EB plated on gelatin-coated plates were cultured for 7 or 14 days. Immunofluorescence staining revealed that mouse iPS cells expressed both 5-HT2A and 5-HT4 receptors and, to a lesser extent, 5-HT1A receptors. Treatment with 5-HT significantly enhanced the ATRA-induced expression of nestin, a specific marker for NPC, and phosphorylation of cAMP response element-binding protein (CREB). Pretreatment of EB cultures with either 1 µmol/L GR113808 (a selective 5-HT4 receptor antagonist) or 1 µmol/L H89 (a protein kinase (PKA) inhibitor) significantly inhibited these effects of 5-HT. These findings suggest that stimulation of 5-HT4 receptors may enhance ATRA-induced neural differentiation of mouse iPS cells through activation of PKA and CREB.