Search

Abstract: Murine embryonic stem (mES) cells have been used to evaluate cytotoxicity and developmental injury following exposure to embryotoxic agents. However, maintaining a homogeneous population of undifferentiated mES cells for this purpose has been complicated by the need for continuous co-culture with murine embryonic fibroblast (mEF) cells or limited passaging on plastic surfaces coated with gelatin. Here, we compare the synthetic basement membrane Matrigel™ with 0.1% gelatin substratum for feeder-free propagation of undifferentiated mES cells. Biomarkers of pluripotentiality, chromosome number, caspase-3 expression, and cardiomyocyte differentiation were monitored for mES cells cultured on Matrigel™ or 0.1% gelatin up to passage 7 (P7). Our results suggest that choice of substratum had no significant effect on population doubling time, cell viability, stage-specific embryonic antigen-1 (SSEA-1) expression, or early passage formation of beating cardiomyocytes (all P ⩾0.09). In other comparisons, however, Matrigel™ supported significantly higher synthesis of alkaline phosphatase (7.7×10−3 ±0.8 vs 6.6×10−3 ±0.8 units/liter/cell, respectively, P =0.012), overall expression of activated caspase-3 following exposure to 5, 10, 50, 100 and 500 parts per billion (ppb) sodium arsenite (P <0.0001), and percent development to beating cardiomyocytes at P7 (P =0.01). Together, our findings suggest that Matrigel™ shows promise as a substrate for feeder-free propagation of undifferentiated mES cells for embryotoxicity endpoints. [Copyright &y& Elsevier]

HIV-1, like the other lentiviruses, has evolved the ability to infect nondividing cells including macrophages. HIV-1 replication in monocytes/macrophages entails peculiar features and differs in many respects from that in CD4 T lymphocytes. HIV-1 exhibits different tropism for CD4 T cells and macrophages. The virus can enter macrophages via several routes. Mitosis is not required for nuclear import of viral DNA or for its integration into the host cell genome. Specific cellular factors are required for HIV-1 transcription in macrophages. The assembly and budding of viral particles in macrophages take place in late endosomal compartments. Viral particles can use the exosome pathway to exit cells. Given their functions in host defence against pathogens and the regulation of the immune response plus their permissivity to HIV-1 infection, monocytes/macrophages exert a dual role in HIV infection. They contribute to the establishment and persistence of HIV-1 infection, and may activate surrounding T cells favouring their infection. Furthermore, monocytes/macrophages act as a Trojan horse to transmit HIV-1 to the central nervous system. They also exhibit antiviral activity and express many molecules that inhibit HIV-1 replication. Activated microglia and macrophages may also exert a neurotrophic and neuroprotective effect on infected brain regulating glutamate metabolism or by secretion of neurotrophins. This review will discuss specific aspects of viral replication in monocytes/macrophages and the role of their interactions with the cellular environment in HIV-1 infection swinging between protection and pathogenesis. [Copyright &y& Elsevier]

Peripheral nerve inflammation is a common clinical problem that accompanies nerve injury and several diseases including Guillain-Barré syndrome and acute and chronic inflammatory demyelinating polyneuropathy. To determine if neuropeptides are induced in motor neurons after inflammation and to study the mechanisms involved, a nerve cuff soaked in complete Freund''s adjuvant (CFA) was applied locally to the facial nerve of Balb/C mice. This procedure resulted in an influx of lymphocytes and macrophages to the affected area and a blockade of retrograde axonal transport distal, but not proximal, to the site of application. The same treatment resulted in a strong ipsilateral induction of pituitary adenylyl cyclase activating peptide (PACAP) gene expression in motor neurons in the facial motor nucleus. Because the changes could have occurred due to the loss of target-derived factors or to the production of new factors by immune cells, we studied the effect of the inflammatory stimulus on PACAP mRNA in mice with severe combined immunodeficiency (SCID). As expected, SCID mice showed a severely reduced influx of T-lymphocytes but not macrophages to the peripheral nerve. Moreover, although retrograde transport distal to the inflammation site was blocked similarly in control and SCID mice, the number of motor neurons expressing PACAP mRNA after CFA application was significantly reduced in SCID mice. The data indicate that the induction of PACAP mRNA during nerve inflammation requires the involvement of lymphocytes. However, because the induction of PACAP gene expression was only partially blocked in SCID mice, macrophages, loss of target-derived factors, or other mechanisms may also contribute to the upregulation of PACAP gene expression in motor neurons after nerve inflammation. [Copyright &y& Elsevier]

Abstract: Embryonic stem (ES) cells are genetically manipulable pluripotential cells that can be differentiated in vitro into neurons, oligodendrocytes, and astrocytes. Given their potential utility as a source of replacement cells for the injured nervous system and the likelihood that transplantation interventions might include co-application of growth factors, we examined the effects of neurotrophin and GDNF family ligands on the survival and excitotoxic vulnerability of ES cell-derived neurons (ES neurons) grown in vitro. ES cells were differentiated down a neural lineage in vitro using the 4−/4+ protocol (Bain et al., Dev Biol 168:342–57, 1995). RT-PCR demonstrated expression of receptors for neurotrophins and GDNF family ligands in ES neural lineage cells. Neuronal expression of GFRα1, GFRα2, and ret was confirmed by immunocytochemistry. Exposure to 30–100 ng/ml GDNF or neurturin (NRTN) resulted in activation of ret. Addition of NT-3 and GDNF did not increase cell division but did increase the number of neurons in the cultures 7 days after plating. Pretreatment with NT-3 enhanced the vulnerability of ES neurons to NMDA-induced death (100 μM NMDA for 10 min) and enhanced the NMDA-induced increase in neuronal [Ca2+]i, but did not alter expression of NMDA receptor subunits NR2A or NR2B. In contrast, pretreatment with GDNF reduced the vulnerability of ES neurons to NMDA-induced death while modestly enhancing the NMDA-induced increase in neuronal [Ca2+]i. These findings demonstrate that the response of ES-derived neurons to neurotrophins and GDNF family ligands is largely similar to that of other cultured central neurons. [Copyright &y& Elsevier]

To what extent are the pathways that regulate self-renewal conserved between stem cells at different stages of development and in different tissues? Some pathways play a strikingly conserved role in regulating the self-renewal of diverse stem cells, whereas other pathways are specific to stem cells in certain tissues or at certain stages of development. Recent studies have highlighted differences between the self-renewal of embryonic, fetal and adult stem cells. By understanding these similarities and differences we may come to a molecular understanding of how stem cells replicate themselves and why aspects of this process differ between stem cells. [Copyright &y& Elsevier]

Ciliary neurotrophic factor (CNTF), originally known for its involvement in the modulation of neuronal growth, has been discovered to exert anorexigenic effects and is currently being investigated in clinical studies for the treatment of obesity and insulin resistance. This neuropeptide acts on the central nervous system. However, we have recently demonstrated direct peripheral effects on adipocyte signalling and thermogenesis. Given the emerging endocrine role of adipose tissue in the regulation of energy homeostasis and insulin resistance, we investigated potential effects of CNTF on leptin expression and secretion. Our study demonstrates a direct inhibition of leptin expression and secretion by acute and chronic CNTF treatment. Furthermore, we demonstrate a differentiation- and Janus kinase 2 (JAK2)-independent, but phosphatidylinositol 3-kinase-dependent signalling pathway mediating this negative effect. These results provide novel evidence for a role of CNTF in the selective modulation of adipocyte endocrine function which may have important implications for the regulation of energy homeostasis. [Copyright &y& Elsevier]

Undifferentiated, murine embryonic stem (mES) cells have shown promise as a substrate for identifying embryotoxic chemicals and for studying mechanisms of early developmental injury. However, long-term maintenance of mES cells in an undifferentiated state is problematic. The present study evaluates the combination of MatrigelTM matrix and three growth factors for this purpose. Biomarkers of mES cell pluripotency, apoptosis, chromosome number and cardiomyocyte differentiation were monitored over 119 population doublings. D3 mES cells retained undifferentiated characteristics, including sustained expression of alkaline phosphatase and stage specific embryonic antigen-1 (SSEA-1) and continued transcription of Pou5f1 (Oct-4). Cell viability remained at ⩾95% and population-doubling times averaged 14.3 h over 10 weeks of observation. Caspase-3 activation, a marker of cellular death by apoptosis, was measured in early- and late-passage mES cells. Early-passage cells showed dose-responsive caspase-3 activation following exposure to sodium arsenite, whereas caspase-3 activation of late-passage cells dropped to background levels at toxicant dosages above 50 ppb. Aneuploidy and impaired differentiation into beating cardiomyocytes were noted for late-passage mES cells. MatrigelTM, combined with growth factors, may sustain undifferentiated mES cells. However, aneuploidy, reduced caspase-3 activation, and inability to differentiate suggests further modifications to the culture system may be needed for long-term propagation of cells for embryotoxicity endpoints. [Copyright &y& Elsevier]

The colony-stimulating factors (CSFs) are a group of cytokines central to the hematopoiesis of blood cells, the modulation of their functional responses, as well as the maintenance of homeostasis and overall immune competence. This group consists of the macrophage-CSF (M-CSF), granulocyte-CSF (G-CSF), granulocyte/macrophage-CSF (GM-CSF), and multi-CSF (IL-3). M-CSF and G-CSF are relatively lineage-specific, having a role in the proliferation, differentiation, and survival of macrophages, neutrophils, and their precursors. In contrast, GM-CSF and multi-CSF function at earlier stages of lineage commitment regulating the expansion and maturation of primitive hematopoietic progenitors. Colony stimulating factor production and degradation are strictly controlled, thus allowing for effective modulation of their biological functions in steady-state conditions as well as under periods of stress. Moreover, the mechanisms behind their expression and that of their cognate receptors ensures that their actions are tightly coordinated, within the context of a network of complex but finely tuned regulatory pathways derived from a variety of local and endocrine hematopoietic regulators.In this review we present some of the most salient information on CSF biology collected over the last three decades. We examine the gene and protein structure of each of the four CSFs and their corresponding receptors, and consider the main determinants behind their biological activities. The components responsible for their functional redundancy as well as the mechanisms that mediate their specificity are also discussed. Although most of available knowledge about CSFs is on human and mouse CSFs, an attempt was made to integrate recent findings in other systems in order to highlight a more widespread role for CSFs throughout evolution. [Copyright &y& Elsevier]

PIAS proteins, cytokine-dependent STAT-associated repressors, exhibit intrinsic E3-type SUMO ligase activities and form a family of transcriptional modulators. Three conserved domains have been identified so far in this protein family, the SAP box, the MIZ-Zn finger/RING module and the acidic C-terminal domain, which are essential for protein interactions, DNA binding or SUMO ligase activity. We have identified a novel conserved domain of 180 residues in PIAS proteins and shown that its ‘PINIT’ motif as well as other conserved motifs (in the SAP box and in the RING domain) are independently involved in nuclear retention of PIAS3L, the long form of PIAS3, that we have characterized in mouse embryonic stem cells. [Copyright &y& Elsevier]

The adipocyte-derived hormone leptin signals the status of body energy stores by activating its receptor in hypothalamic nuclei. In contrast to the initial expectations, leptin treatment of human obesity was largely unsuccessful. One explanation for this is the marked leptin resistance, which likely operates in part at the receptor level. The leptin receptor is a member of the class I cytokine receptor family, which uses the Janus kinase/signal transducer and activator of transcription pathway as a major signaling route. In this review, we focus on the molecular mechanisms underlying leptin receptor activation. Different modes of leptin-induced clustering of the ectodomains and the subsequent signaling events will be discussed. [Copyright &y& Elsevier]

Thrombopoietin (Tpo) and its receptor, c-mpl, are expressed in murine embryonic stem (ES) cells. ES cells are maintained in a pluripotent state by leukemia inhibitory factor (LIF) via activation of the Janus kinase (Jak)–STAT3 signaling pathway. Tpo, like LIF, activates STAT3. We report that Tpo increases the number of undifferentiated colonies derived from wild type or Shp-2 mutant (Shp-2Δ46–110) ES cells. Tpo plus LIF acted synergistically on the Shp-2Δ46–110 ES cells to maintain undifferentiated colonies but no evidence of synergism via Jak–STAT3 activation was detected. Collectively, these data suggest that Tpo can play a role in preventing ES cell differentiation via Jak–STAT3 activation and perhaps via novel pathways that are enhanced in the absence of functional Shp-2. [Copyright &y& Elsevier]

15N NMR relaxation parameters and amide 1H/2H-exchange rates have been used to characterize the structural flexibility of human growth hormone (rhGH) at neutral and acidic pH. Our results show that the rigidity of the molecule is strongly affected by the solution conditions. At pH 7.0 the backbone dynamics parameters of rhGH are uniform along the polypeptide chain and their values are similar to those of other folded proteins. In contrast, at pH 2.7 the overall backbone flexibility increases substantially compared to neutral pH and the average order parameter approaches the lower limit expected for a folded protein. However, a significant variation of the backbone dynamics through the molecule indicates that under acidic conditions the mobility of the residues becomes more dependent on their location within the secondary structure units. In particular, the order parameters of certain loop regions decrease dramatically and become comparable to those found in unfolded proteins. Furthermore, the HN-exchange rates at low pH reveal that the residues most protected from exchange are clustered at one end of the helical bundle, forming a stable nucleus. We suggest that this nucleus maintains the overall fold of the protein under destabilizing conditions. We therefore conclude that the acid state of rhGH consists of a structurally conserved, but dynamically more flexible helical core surrounded by an aura of highly mobile, unstructured loops. However, in spite of its prominent flexibility the acid state of rhGH cannot be considered a “molten globule” state because of its high stability. It appears from our work that under certain conditions, a protein can tolerate a considerable increase in flexibility of its backbone, along with an increased penetration of water into its core, while still maintaining a stable folded conformation. [Copyright &y& Elsevier]

Ciliary neurotrophic factor (CNTF) is a member of the gp130 family of cytokines. The functional receptor complex of CNTF is composed of the CNTF receptor α (CNTFR), gp130 and the leukemia inhibitory factor receptor (LIFR). Three regions on CNTF have been identified as binding sites for its receptors. The ligand–receptor interactions are mediated through the cytokine binding domains (CBDs) and/or the immunoglobulin-like domains of the receptors. However, in the case of CNTF, the precise nature of the protein–protein contacts in the signaling complex has not yet been resolved. In this study, we provide the first demonstration that the membrane distal CBD (CBD1) of LIFR associates in vitro with soluble CNTFR in the absence of CNTF. Moreover, purified CBD1 partially blocks CNTF signaling, but not that of interleukin-6 or LIF, in human embryonal carcinoma cell line Ntera/D1 cells. These data raise the possibility that LIFR has the capability to form a ligand-free complex with CNTFR. [Copyright &y& Elsevier]

Ciliary neurotrophic factor (CNTF) is produced by astrocytes which have been implicated in regulating stress responses. We found that CNTF in the medial amygdala (MeA) promotes despair or passive coping, i.e., immobility in an acute forced swim stress, in female mice, while having no effect in males. Neutralizing CNTF antibody injected into the MeA of wildtype females reduced activation of downstream STAT3 (Y705) 24 and 48 h later. In concert, the antibody reduced immobility in the swim test in females and only after MeA injection, but not when injected in the central or basolateral amygdala. Antibody injected into the male MeA did not affect immobility. These data reveal a unique role of CNTF in female MeA in promoting despair or passive coping behavior. Moreover, 4 weeks of chronic unpredictable stress (CUS) increased immobility in the swim test and reduced sucrose preference in wildtype CNTF+/+, but not CNTF-/- littermate, females. Following CUS, 10 min of restraint stress increased plasma corticosterone levels only in CNTF+/+ females. In males, the CUS effects were present in both genotypes. Further, CUS increased CNTF expression in the MeA of female, but not male, mice. CUS did not alter CNTF in the female hippocampus, hypothalamus and bed nucleus of stria terminalis. This suggests that MeA CNTF has a female-specific role in promoting CUS-induced despair or passive coping, behavioral anhedonia and neuroendocrine responses. Compared to CNTF+/+ mice, CNTF-/- mice did not show differences in CUS-induced anxiety-like behavior and sensorimotor gating function as measured by elevated T-Maze, open field and pre-pulse inhibition of the acoustic startle response. Together, this study reveals a novel CNTF-mediated female-specific mechanism in stress responses and points to opportunities for developing treatments for stress-related disorders in women., Competing Interests: The authors declare no competing financial interests or potential conflicts of interest., (© 2022 The Authors.)

Introduction: Currently, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can be induced to differentiate at the cellular level but not to form mature tissues or organs suitable for transplantation. ESCs/iPSCs form immature teratomas after injection into immunodeficient mice. In humans, immature teratomas often transform into fully differentiated mature teratomas after administration of anticancer agents., Methods: We first investigated the ability of cisplatin to induce changes in mouse ESCs/iPSCs in vitro . Next, we designed experiments to analyze ESC/iPSC-derived immature teratoma tissue in vivo after treatment of cisplatin. Groups of six mice carrying ESC- or iPSC-derived teratomas were given either low or high dose intraperitoneal injection of cisplatin, while the control group received saline for 4 weeks., Results: Treatment of ESC/iPSC cultures with cisplatin for 3 days caused a dose-related decrease in cell numbers without inducing any morphological changes to the cells. ESC/iPSC-derived teratomas showed lower growth rates with a significantly higher mature components ratio in a concentration dependent manner after cisplatin treatment (P < 0.05); however, immunohistochemical analyses demonstrated a significantly reduced PCNA labelling index and an increase in an apoptosis marker on immature neural components (P < 0.05) along with emergence of h-Caldesmon + mature smooth muscle cells in treated mice. Moreover, newly differentiated components not found in the control group, such as mature adipose tissue, cartilage, and pancreas, as well as striated muscle, salivary glands, gastric mucosa with fundic glands, and hair follicles emerged. The identities of these components were confirmed by immunostaining for specific markers., Conclusions: Cisplatin has the ability to reduce immature components in ESC/iPSC-derived teratomas, presumably through apoptosis, and also to induce them to differentiate., Competing Interests: The authors declare no conflicts of interests in relation to this article., (© 2021 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

Fatigue is a persistent and debilitating symptom following radiation therapy for prostate cancer. However, it is not well-understood how radiation targeted to a small region of the body can lead to broad changes in behavior. In this study, we used targeted pelvic irradiation of healthy male mice to test whether inflammatory signaling mediates changes in voluntary physical activity levels. First, we tested the relationship between radiation dose, blood cell counts, and fatigue-like behavior measured as voluntary wheel-running activity. Next, we used oral minocycline treatments to reduce inflammation and found that minocycline reduces, but does not eliminate, the fatigue-like behavioral changes induced by radiation. We also used a strain of mice lacking the MyD88 adaptor protein and found that these mice also showed less fatigue-like behavior than the wild-type controls. Finally, using serum and brain tissue samples, we determined changes in inflammatory signaling induced by irradiation in wild-type, minocycline treated, and MyD88 knockout mice. We found that irradiation increased serum levels of IL-6, a change that was partially reversed in mice treated with minocycline or lacking MyD88. Overall, our results suggest that inflammation plays a causal role in radiation-induced fatigue and that IL-6 may be an important mediator., Competing Interests: There are no conflicts of interest to report.

Skeletal muscle injuries have bothered doctors and caused great burdens to the public medical insurance system for a long time. Once injured, skeletal muscles usually go through the processes of inflammation, repairing and remodeling. If repairing and remodeling stages are out of balance, scars will be formed to replace injured skeletal muscles. At present, clinicians usually use conventional methods to restore the injured skeletal muscles, such as flap transplantation. However, flap transplantation sometimes needs to sacrifice healthy autologous tissues and will bring extra harm to patients. In recent years, stem cells-based tissue engineering provides us new treatment ideas for skeletal muscle injuries. Stem cells are cells with multiple differentiation potential and have ability to differentiate into adult cells under special condition. Skeletal muscle tissues also have stem cells, called satellite cells, but they are in small amount and new muscle fibers that derived from them may not be enough to replace injured fibers. Bone marrow mesenchymal stem cells (BM-MSCs) could promote musculoskeletal tissue regeneration and activate the myogenic differentiation of satellite cells. Biomaterial is another important factor to promote tissue regeneration and greatly enhance physiological activities of stem cells in vivo . The combined use of stem cells and biomaterials will gradually become a mainstream to restore injured skeletal muscles in the future. This review article mainly focuses on the review of research about the application of BM-MSCs and several major biomaterials in skeletal muscle regeneration over the past decades., Competing Interests: The authors declare no conflict of interests., (© 2020 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

Background & Aims: Effective therapeutic approaches are urgently required to tackle the alarmingly poor survival outcomes in esophageal adenocarcinoma (EAC) patients. EAC originates from within the intestinal-type metaplasia, Barrett's esophagus, a condition arising on a background of gastroesophageal reflux disease and associated inflammation., Methods: This study used a druggable genome small interfering RNA (siRNA) screening library of 6022 siRNAs in conjunction with bioinformatics platforms, genomic studies of EAC tissues, somatic variation data of EAC from The Cancer Genome Atlas data of EAC, and pathologic and functional studies to define novel EAC-associated, and targetable, immune factors., Results: By using a druggable genome library we defined genes that sustain EAC cell growth, which included an unexpected immunologic signature. Integrating Cancer Genome Atlas data with druggable siRNA targets showed a striking concordance and an EAC-specific gene amplification event associated with 7 druggable targets co-encoded at Chr6p21.1. Over-representation of immune pathway-associated genes supporting EAC cell growth included leukemia inhibitory factor, complement component 1, q subcomponent A chain (C1QA), and triggering receptor expressed on myeloid cells 2 (TREM2), which were validated further as targets sharing downstream signaling pathways through genomic and pathologic studies. Finally, targeting the triggering receptor expressed on myeloid cells 2-, C1q-, and leukemia inhibitory factor-activated signaling pathways (TYROBP-spleen tyrosine kinase and JAK-STAT3) with spleen tyrosine kinase and Janus-activated kinase inhibitor fostamatinib R788 triggered EAC cell death, growth arrest, and reduced tumor burden in NOD scid gamma mice., Conclusions: These data highlight a subset of genes co-identified through siRNA targeting and genomic studies of expression and somatic variation, specifically highlighting the contribution that immune-related factors play in support of EAC development and suggesting their suitability as targets in the treatment of EAC.

Introduction: Formation of cell spheres is an important procedure in biomedical research. A large number of high-quality cell spheres of uniform size and shape are required for basic studies and therapeutic applications. Conventional approaches, including the hanging drop method and suspension culture, are used for cell sphere production. However, these methods are time consuming, cell spheres cannot be harvested easily, and it is difficult to control the size and geometry of cell spheres. To resolve these problems, a novel multiple-funnel cell culture insert was designed for size controlling, easy harvesting, and scale-up production of cell spheres., Methods: The culture substrate has 680 micro-funnels with a 1-mm width top, 0.89 mm depth, and 0.5 mm square bottom. Mouse embryonic stem cells were used to test the newly developed device. The seeded embryonic stem cells settled at the downward medium surface toward the bottom opening and aggregated as embryoid bodies (EBs). For cell sphere harvest, the bottom of the culture insert was put in contact with the medium surface in another culture dish, and the medium in the device flowed down with cell spheres by hydrostatic pressure., Results: Compact cell spheres with uniform size and shape were collected easily. The diameter of the spheres could be controlled by adjusting the seeding cell density. Spontaneous neural differentiation (nestin and Tju1) and retinoic acid-induced endodermal differentiation (Pdx-1 and insulin I) were improved in the EBs produced using the new insert compared to those in EBs produced by suspension culture., Conclusions: This novel cell culture insert shall improve future studies of cell spheres and benefit clinical applications of cell therapy.

The hypothalamic arcuate nucleus (ARC) is a major integration center for energy and glucose homeostasis that responds to leptin. Resistance to leptin in the ARC is an important component of the development of obesity and type 2 diabetes. Recently, we showed that Endospanin1 (Endo1) is a negative regulator of the leptin receptor (OBR) that interacts with OBR and retains the receptor inside the cell, leading to a decreased activation of the anorectic STAT3 pathway. Endo1 is up-regulated in the ARC of high fat diet (HFD)-fed mice, and its silencing in the ARC of lean and obese mice prevents and reverses the development of obesity., Objective: Herein we investigated whether decreased Endo1 expression in the hypothalamic ARC, associated with reduced obesity, could also ameliorate glucose homeostasis accordingly., Methods: We studied glucose homeostasis in lean or obese mice silenced for Endo1 in the ARC via stereotactic injection of shRNA-expressing lentiviral vectors., Results: We observed that despite being leaner, Endo1-silenced mice showed impaired glucose homeostasis on HFD. Mechanistically, we show that Endo1 interacts with p85, the regulatory subunit of PI3K, and mediates leptin-induced PI3K activation., Conclusions: Our results thus define Endo1 as an important hypothalamic integrator of leptin signaling, and its silencing differentially regulates the OBR-dependent functions.

Temozolomide (TMZ) is an oral alkylating agent used to treat glioblastoma multiforme (GBM) and astrocytomas. However, at least 50% of TMZ treated patients do not respond to TMZ. This is due primarily to the over-expression of O 6 -methylguanine methyltransferase (MGMT) and/or lack of a DNA repair pathway in GBM cells. Multiple GBM cell lines are known to contain TMZ resistant cells and several acquired TMZ resistant GBM cell lines have been developed for use in experiments designed to define the mechanism of TMZ resistance and the testing of potential therapeutics. However, the characteristics of intrinsic and adaptive TMZ resistant GBM cells have not been systemically compared. This article reviews the characteristics and mechanisms of TMZ resistance in natural and adapted TMZ resistant GBM cell lines. It also summarizes potential treatment options for TMZ resistant GBMs.

Bioengineered cardiac tissues represent a promising strategy for regenerative medicine. However, methods of vascularization and suitable cell sources for tissue engineering and regenerative medicine have not yet been established. In this study, we developed methods for the induction of vascular endothelial cells from mouse embryonic stem (ES) cells using three-dimensional (3D) suspension culture, and fabricated cardiac cell sheets with a pre-vascularized structure by co-culture of mouse ES cell-derived endothelial cells. After induction, isolated CD31+ cells expressed several endothelial cell marker genes and exhibited the ability to form vascular network structures similar to CD31+ cells from neonatal mouse heart. Co-culture of ES cell-derived CD31+ cells with ES cell-derived cardiomyocytes and dermal fibroblasts resulted in the formation of cardiac cell sheets with microvascular network formation. In contrast, microvascular network formation was reduced in co-cultures without cardiomyocytes, suggesting that cardiomyocytes within the cell sheet might enhance vascular endothelial cell sprouting. Polymerase chain reaction array analysis revealed that the expression levels of several angiogenesis-related genes, including fibroblast growth factor 1 (FGF1), were up-regulated in co-culture with cardiomyocytes compared with cultures without cardiomyocytes. The microvascular network in the cardiac sheets was attenuated by treatment with anti-FGF1 antibody. These results indicate that 3D suspension culture methods may be used to prepare functional vascular endothelial cells from mouse ES cells, and that cardiomyocyte-mediated paracrine effects might be important for fabricating pre-vascularized cardiac cell sheets.

Human trophoblast invasion and differentiation are essential for successful pregnancy outcome. The molecular mechanisms, however, are poorly understood. Interleukin (IL)-11, a cytokine, regulates endometrial epithelial cell adhesion. Leukemia inhibitory factor (LIF) is one of the key cytokines in the embryo implantation regulation. The present study aimed to assess the levels of LIF, IL-11, and IL-11 α receptor gene expression in the endometrium of women undergoing IVF and correlate their levels with the IVF pregnancy outcome. Also, the study aimed to detect any mutation in these three genes among IVF pregnant and non-pregnant women versus control menstrual blood of fertile women. Endometrial tissue biopsies were taken from 15 women undergoing IVF on the day of oocyte retrieval. The quantitative expression of IL-11, IL-11Rα, and LIF genes was assessed by real-time PCR and PCR products were sequenced. Menstrual blood from 10 fertile women was used as control to compare the DNA sequence versus DNA sequence of the studied genes in endometrial biopsies. LH, FSH, and E2 were assessed for enrolled patients by ELISA. Endometrial thickness was also assessed by pelvic ultrasonography. No significant difference was detected between quantitative expression of the three studied genes and pregnancy IVF outcome. Although DNA sequence changes were found in IL-11 and LIF genes of women with negative pregnancy IVF outcome compared to women with positive pregnancy IVF outcome, no DNA sequence changes were detected for IL-11Rα. Other studied parameters (e.g., age, LH, FSH, E2, and endometrial thickness) showed no significant differences or correlation of quantitative expression of the three studied involved genes. Data suggested that there were no significant differences between quantitative expression of IL-11, IL-11Rα, and LIF genes and the IVF pregnancy outcome. The present study may reveal that changes in IL-11 and LIF genes sequence may contribute in pregnancy IVF outcome.

Common marmoset (CM) is widely recognized as a useful non-human primate for disease modeling and preclinical studies. Thus, embryonic stem cells (ESCs) derived from CM have potential as an appropriate cell source to test human regenerative medicine using human ESCs. CM ESCs have been established by us and other groups, and can be cultured in vitro. However, the growth factors and downstream pathways for self-renewal of CM ESCs are largely unknown. In this study, we found that basic fibroblast growth factor (bFGF) rather than leukemia inhibitory factor (LIF) promoted CM ESC self-renewal via the activation of phosphatidylinositol-3-kinase (PI3K)-protein kinase B (AKT) pathway on mouse embryonic fibroblast (MEF) feeders. Moreover, bFGF and transforming growth factor β (TGFβ) signaling pathways cooperatively maintained the undifferentiated state of CM ESCs under feeder-free condition. Our findings may improve the culture techniques of CM ESCs and facilitate their use as a preclinical experimental resource for human regenerative medicine.

Homing and engraftment, a determining factor in hematopoietic stem cell transplantation success is defined as a process through which hematopoietic stem/progenitor cells (HSPCs) lodge recipient bone marrow. SDF-1/CXCR4 axis acts as a principle regulator in homing and engraftment, however, CXCR4 signaling is dependent upon expression of CXCR4 and its ligand SDF-1, which is highly dynamic. Hence, present investigation was aimed to explore the potential of CXCR4 constitutive active mutants (CXCR4-CAMs) in overcoming the limitation of CXCR4 signaling and up-modulate its efficiency in homing and engraftment. Regulated transgene expression study of these mutants revealed their significantly enhanced cell adhesion efficiency to endothelium and extracellular matrix protein. This altogether indicates promising prospects of CXCR4-CAMs in research aimed to improve HSPCs engraftment efficiency.