Limited hair cell induction from human induced pluripotent stem cells using a simple stepwise method.

PubMed

Ohnishi, Hiroe; Skerleva, Desislava; Kitajiri, Shin-ichiro; Sakamoto, Tatsunori; Yamamoto, Norio; Ito, Juichi; Nakagawa, Takayuki

2015-07-10

Disease-specific induced pluripotent stem cells (iPS) cells are expected to contribute to exploring useful tools for studying the pathophysiology of inner ear diseases and to drug discovery for treating inner ear diseases. For this purpose, stable induction methods for the differentiation of human iPS cells into inner ear hair cells are required. In the present study, we examined the efficacy of a simple induction method for inducing the differentiation of human iPS cells into hair cells. The induction of inner ear hair cell-like cells was performed using a stepwise method mimicking inner ear development. Human iPS cells were sequentially transformed into the preplacodal ectoderm, otic placode, and hair cell-like cells. As a first step, preplacodal ectoderm induction, human iPS cells were seeded on a Matrigel-coated plate and cultured in a serum free N2/B27 medium for 8 days according to a previous study that demonstrated spontaneous differentiation of human ES cells into the preplacodal ectoderm. As the second step, the cells after preplacodal ectoderm induction were treated with basic fibroblast growth factor (bFGF) for induction of differentiation into otic-placode-like cells for 15 days. As the final step, cultured cells were incubated in a serum free medium containing Matrigel for 48 days. After preplacodal ectoderm induction, over 90% of cultured cells expressed the genes that express in preplacodal ectoderm. By culture with bFGF, otic placode marker-positive cells were obtained, although their number was limited. Further 48-day culture in serum free media resulted in the induction of hair cell-like cells, which expressed a hair cell marker and had stereocilia bundle-like constructions on their apical surface. Our results indicate that hair cell-like cells are induced from human iPS cells using a simple stepwise method with only bFGF, without the use of xenogeneic cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

In vivo programming of tumor antigen-specific T lymphocytes from pluripotent stem cells to promote cancer immunosurveillance.

PubMed

Lei, Fengyang; Zhao, Baohua; Haque, Rizwanul; Xiong, Xiaofang; Budgeon, Lynn; Christensen, Neil D; Wu, Yuzhang; Song, Jianxun

2011-07-15

Adoptive T-cell immunotherapy has garnered wide attention, but its effective use is limited by the need of multiple ex vivo manipulations and infusions that are complex and expensive. In this study, we show how highly reactive antigen (Ag)-specific CTLs can be generated from induced pluripotent stem (iPS) cells to provide an unlimited source of functional CTLs for adoptive immunotherapy. iPS cell-derived T cells can offer the advantages of avoiding possible immune rejection and circumventing ethical and practical issues associated with other stem cell types. iPS cells can be differentiated into progenitor T cells in vitro by stimulation with the Notch ligand Delta-like 1 (DL1) overexpressed on bone marrow stromal cells, with complete maturation occurring upon adoptive transfer into Rag1-deficient mice. Here, we report that these iPS cells can be differentiated in vivo into functional CTLs after overexpression of MHC I-restricted Ag-specific T-cell receptors (TCR). In this study, we generated murine iPS cells genetically modified with ovalbumin (OVA)-specific and MHC-I restricted TCR (OT-I) by retrovirus-mediated transduction. After their adoptive transfer into recipient mice, the majority of OT-I/iPS cells underwent differentiation into CD8+ CTLs. TCR-transduced iPS cells developed in vivo responded in vitro to peptide stimulation by secreting interleukin 2 and IFN-γ. Most importantly, adoptive transfer of TCR-transduced iPS cells triggered infiltration of OVA-reactive CTLs into tumor tissues and protected animals from tumor challenge. Taken together, our findings offer proof of concept for a potentially more efficient approach to generate Ag-specific T lymphocytes for adoptive immunotherapy. ©2011 AACR.

Potential of laryngeal muscle regeneration using induced pluripotent stem cell-derived skeletal muscle cells.

PubMed

Dirja, Bayu Tirta; Yoshie, Susumu; Ikeda, Masakazu; Imaizumi, Mitsuyoshi; Nakamura, Ryosuke; Otsuki, Koshi; Nomoto, Yukio; Wada, Ikuo; Hazama, Akihiro; Omori, Koichi

2016-01-01

Conclusion Induced pluripotent stem (iPS) cells may be a new potential cell source for laryngeal muscle regeneration in the treatment of vocal fold atrophy after recurrent laryngeal nerve paralysis. Objectives Unilateral vocal fold paralysis can lead to degeneration, atrophy, and loss of force of the thyroarytenoid muscle. At present, there are some treatments such as thyroplasty, arytenoid adduction, and vocal fold injection. However, such treatments cannot restore reduced mass of the thyroarytenoid muscle. iPS cells have been recognized as supplying a potential resource for cell transplantation. The aim of this study was to assess the effectiveness of the use of iPS cells for the regeneration of laryngeal muscle through the evaluation of both in vitro and in vivo experiments. Methods Skeletal muscle cells were generated from tdTomato-labeled iPS cells using embryoid body formation. Differentiation into skeletal muscle cells was analyzed by gene expression and immunocytochemistry. The tdTomato-labeled iPS cell-derived skeletal muscle cells were transplanted into the left atrophied thyroarytenoid muscle. To evaluate the engraftment of these cells after transplantation, immunohistochemistry was performed. Results The tdTomato-labeled iPS cells were successfully differentiated into skeletal muscle cells through an in vitro experiment. These cells survived in the atrophied thyroarytenoid muscle after transplantation.

Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines

PubMed Central

Bock, Christoph; Kiskinis, Evangelos; Verstappen, Griet; Gu, Hongcang; Boulting, Gabriella; Smith, Zachary D.; Ziller, Michael; Croft, Gist F.; Amoroso, Mackenzie W.; Oakley, Derek H.; Gnirke, Andreas; Eggan, Kevin; Meissner, Alexander

2011-01-01

SUMMARY The developmental potential of human pluripotent stem cells suggests that they can produce disease-relevant cell types for biomedical research. However, substantial variation has been reported among pluripotent cell lines, which could affect their utility and clinical safety. Such cell-line-specific differences must be better understood before one can confidently use embryonic stem (ES) or induced pluripotent stem (iPS) cells in translational research. Toward this goal we have established genome-wide reference maps of DNA methylation and gene expression for 20 previously derived human ES lines and 12 human iPS cell lines, and we have measured the in vitro differentiation propensity of these cell lines. This resource enabled us to assess the epigenetic and transcriptional similarity of ES and iPS cells and to predict the differentiation efficiency of individual cell lines. The combination of assays yields a scorecard for quick and comprehensive characterization of pluripotent cell lines. PMID:21295703

Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts

PubMed Central

Doi, Akiko; Park, In-Hyun; Wen, Bo; Murakami, Peter; Aryee, Martin J; Irizarry, Rafael; Herb, Brian; Ladd-Acosta, Christine; Rho, Junsung; Loewer, Sabine; Miller, Justine; Schlaeger, Thorsten; Daley, George Q; Feinberg, Andrew P

2010-01-01

Induced pluripotent stem (iPS) cells are derived by epigenetic reprogramming, but their DNA methylation patterns have not yet been analyzed on a genome-wide scale. Here, we find substantial hypermethylation and hypomethylation of cytosine-phosphate-guanine (CpG) island shores in nine human iPS cell lines as compared to their parental fibroblasts. The differentially methylated regions (DMRs) in the reprogrammed cells (denoted R-DMRs) were significantly enriched in tissue-specific (T-DMRs; 2.6-fold, P < 10−4) and cancer-specific DMRs (C-DMRs; 3.6-fold, P < 10−4). Notably, even though the iPS cells are derived from fibroblasts, their R-DMRs can distinguish between normal brain, liver and spleen cells and between colon cancer and normal colon cells. Thus, many DMRs are broadly involved in tissue differentiation, epigenetic reprogramming and cancer. We observed colocalization of hypomethylated R-DMRs with hypermethylated C-DMRs and bivalent chromatin marks, and colocalization of hypermethylated R-DMRs with hypomethylated C-DMRs and the absence of bivalent marks, suggesting two mechanisms for epigenetic reprogramming in iPS cells and cancer. PMID:19881528

Protection against cancer by dietary IP6 and inositol.

PubMed

Vucenik, Ivana; Shamsuddin, AbulKalam M

2006-01-01

Inositol hexaphosphate (IP(6)) is a naturally occurring polyphosphorylated carbohydrate, abundantly present in many plant sources and in certain high-fiber diets, such as cereals and legumes. In addition to being found in plants, IP(6) is contained in almost all mammalian cells, although in much smaller amounts, where it is important in regulating vital cellular functions such as signal transduction, cell proliferation, and differentiation. For a long time IP(6) has been recognized as a natural antioxidant. Recently IP(6) has received much attention for its role in cancer prevention and control of experimental tumor growth, progression, and metastasis. In addition, IP(6) possesses other significant benefits for human health, such as the ability to enhance immune system, prevent pathological calcification and kidney stone formation, lower elevated serum cholesterol, and reduce pathological platelet activity. In this review we show the efficacy and discuss some of the molecular mechanisms that govern the action of this dietary agent. Exogenously administered IP(6) is rapidly taken up into cells and dephosphorylated to lower inositol phosphates, which further affect signal transduction pathways resulting in cell cycle arrest. A striking anticancer action of IP(6) was demonstrated in different experimental models. In addition to reducing cell proliferation, IP(6) also induces differentiation of malignant cells. Enhanced immunity and antioxidant properties also contribute to tumor cell destruction. Preliminary studies in humans show that IP(6) and inositol, the precursor molecule of IP(6), appear to enhance the anticancer effect of conventional chemotherapy, control cancer metastases, and improve quality of life. Because it is abundantly present in regular diet, efficiently absorbed from the gastrointestinal tract, and safe, IP(6) + inositol holds great promise in our strategies for cancer prevention and therapy. There is clearly enough evidence to justify the initiation of full-scale clinical trials in humans.

Generation of male differentiated germ cells from various types of stem cells.

PubMed

Hou, Jingmei; Yang, Shi; Yang, Hao; Liu, Yang; Liu, Yun; Hai, Yanan; Chen, Zheng; Guo, Ying; Gong, Yuehua; Gao, Wei-Qiang; Li, Zheng; He, Zuping

2014-06-01

Infertility is a major and largely incurable disease caused by disruption and loss of germ cells. It affects 10-15% of couples, and male factor accounts for half of the cases. To obtain human male germ cells 'especially functional spermatids' is essential for treating male infertility. Currently, much progress has been made on generating male germ cells, including spermatogonia, spermatocytes, and spermatids, from various types of stem cells. These germ cells can also be used in investigation of the pathology of male infertility. In this review, we focused on advances on obtaining male differentiated germ cells from different kinds of stem cells, with an emphasis on the embryonic stem (ES) cells, the induced pluripotent stem (iPS) cells, and spermatogonial stem cells (SSCs). We illustrated the generation of male differentiated germ cells from ES cells, iPS cells and SSCs, and we summarized the phenotype for these stem cells, spermatocytes and spermatids. Moreover, we address the differentiation potentials of ES cells, iPS cells and SSCs. We also highlight the advantages, disadvantages and concerns on derivation of the differentiated male germ cells from several types of stem cells. The ability of generating mature and functional male gametes from stem cells could enable us to understand the precise etiology of male infertility and offer an invaluable source of autologous male gametes for treating male infertility of azoospermia patients. © 2014 Society for Reproduction and Fertility.

Marmoset induced pluripotent stem cells: Robust neural differentiation following pretreatment with dimethyl sulfoxide.

PubMed

Qiu, Zhifang; Mishra, Anuja; Li, Miao; Farnsworth, Steven L; Guerra, Bernadette; Lanford, Robert E; Hornsby, Peter J

2015-07-01

The marmoset is an important nonhuman primate model for regenerative medicine. For experimental autologous cell therapy based on induced pluripotent (iPS) cells in the marmoset, cells must be able to undergo robust and reliable directed differentiation that will not require customization for each specific iPS cell clone. When marmoset iPS cells were aggregated in a hanging drop format for 3 days, followed by exposure to dual SMAD inhibitors and retinoic acid in monolayer culture for 3 days, we found substantial variability in the response of different iPS cell clones. However, when clones were pretreated with 0.05-2% dimethyl sulfoxide (DMSO) for 24 hours, all clones showed a very similar maximal response to the directed differentiation scheme. Peak responses were observed at 0.5% DMSO in two clones and at 1% DMSO in a third clone. When patterns of gene expression were examined by microarray analysis, hierarchical clustering showed very similar responses in all 3 clones when they were pretreated with optimal DMSO concentrations. The change in phenotype following exposure to DMSO and the 6 day hanging drop/monolayer treatment was confirmed by immunocytochemistry. Analysis of DNA content in DMSO-exposed cells indicated that it is unlikely that DMSO acts by causing cells to exit from the cell cycle. This approach should be generally valuable in the directed neural differentiation of pluripotent cells for experimental cell therapy. Copyright © 2015. Published by Elsevier B.V.

Efficient biotechnological approach for lentiviral transduction of induced pluripotent stem cells.

PubMed

Zare, Mehrak; Soleimani, Masoud; Mohammadian, Mozhdeh; Akbarzadeh, Abolfazl; Havasi, Parvaneh; Zarghami, Nosratollah

2016-01-01

Induced pluripotent stem (iPS) cells are generated from differentiated adult somatic cells by reprogramming them. Unlimited self-renewal, and the potential to differentiate into any cell type, make iPS cells very promising candidates for basic and clinical research. Furthermore, iPS cells can be genetically manipulated for use as therapeutic tools. DNA can be introduced into iPS cells, using lentiviral vectors, which represent a helpful choice for efficient transduction and stable integration of transgenes. In this study, we compare two methods of lentiviral transduction of iPS cells, namely, the suspension method and the hanging drop method. In contrast to the conventional suspension method, in the hanging drop method, embryoid body (EB) formation and transduction occur concurrently. The iPS cells were cultured to form EBs, and then transduced with lentiviruses, using the conventional suspension method and the hanging drop method, to express miR-128 and green fluorescent protein (GFP). The number of transduced cells were assessed by fluorescent microscopy and flow cytometry. MTT assay and real-time PCR were performed to determine the cell viability and transgene expression, respectively. Morphologically, GFP+ cells were more detectable in the hanging drop method, and this finding was quantified by flow cytometric analysis. According to the results of the MTT assay, cell viability was considerably higher in the hanging drop method, and real-time PCR represented a higher relative expression of miR-128 in the iPS cells introduced with lentiviruses in drops. Altogether, it seems that lentiviral transduction of challenging iPS cells using the hanging drop method offers a suitable and sufficient strategy in their gene transfer, with less toxicity than the conventional suspension method.

Investigation of the pathogenesis of autoimmune diseases by iPS cells.

PubMed

Natsumoto, Bunki; Shoda, Hirofumi; Fujio, Keishi; Otsu, Makoto; Yamamoto, Kazuhiko

2017-01-01

The pluripotent stem cells have a self-renewal ability and can be differentiated into theoretically all of cell types. The induced pluripotent stem (iPS) cells overcame the ethical problems of the human embryonic stem (ES) cell, and enable pathologic analysis of intractable diseases and drug discovery. The in vitro disease model using disease-specific iPS cells enables repeated analyses of human cells without influence of environment factors. Even though autoimmune diseases are polygenic diseases, autoimmune disease-specific iPS cells are thought to be a promising tool for analyzing the pathogenesis of the diseases and drug discovery in future.

Human induced pluripotent stem cell differentiation and direct transdifferentiation into corneal epithelial-like cells

PubMed Central

Cieślar-Pobuda, Artur; Rafat, Mehrdad; Knoflach, Viktoria; Skonieczna, Magdalena; Hudecki, Andrzej; Małecki, Andrzej; Urasińska, Elżbieta; Ghavami, Seaid; Łos, Marek J.

2016-01-01

The corneal epithelium is maintained by a small pool of tissue stem cells located at the limbus. Through certain injuries or diseases this pool of stem cells may get depleted. This leads to visual impairment. Standard treatment options include autologous or allogeneic limbal stem cell (LSC) transplantation, however graft rejection and chronic inflammation lowers the success rate over long time. Induced pluripotent stem (iPS) cells have opened new possibilities for treating various diseases with patient specific cells, eliminating the risk of immune rejection. In recent years, several protocols have been developed, aimed at the differentiation of iPS cells into the corneal epithelial lineage by mimicking the environmental niche of limbal stem cells. However, the risk of teratoma formation associated with the use of iPS cells hinders most applications from lab into clinics. Here we show that the differentiation of iPS cells into corneal epithelial cells results in the expression of corneal epithelial markers showing a successful differentiation, but the process is long and the level of gene expression for the pluripotency markers does not vanish completely. Therefore we set out to determine a direct transdifferentiation approach to circumvent the intermediate state of pluripotency (iPS-stage). The resulting cells, obtained by direct transdifferentiation of fibroblasts into limbal cells, exhibited corneal epithelial cell morphology and expressed corneal epithelial markers. Hence we shows for the first time a direct transdifferentiation of human dermal fibroblasts into the corneal epithelial lineage that may serve as source for corneal epithelial cells for transplantation approaches. PMID:27275539

c-MYC independent nuclear reprogramming favors cardiogenic potential of induced pluripotent stem cells

PubMed Central

Martinez-Fernandez, Almudena; Nelson, Timothy J.; Ikeda, Yasuhiro; Terzic, Andre

2010-01-01

Induced pluripotent stem cell (iPS) technology has launched a new platform in regenerative medicine aimed at deriving unlimited replacement tissue from autologous sources through somatic cell reprogramming using stemness factor sets. In this way, authentic cardiomyocytes have been obtained from iPS and recently demonstrated in proof-of-principle studies to repair infarcted heart. Optimizing the cardiogenic potential of iPS progeny would ensure a maximized yield of bioengineered cardiac tissue. Here, we reprogrammed fibroblasts in the presence or absence of c-MYC to determine if the acquired cardiogenicity is sensitive to the method of nuclear reprogramming. Using lentiviral constructs that expressed stemness factors SOX2, OCT4, and KLF4 with or without c-MYC, iPS clones generated through fibroblast reprogramming demonstrated indistinguishable characteristics for 5 days of differentiation with similar cell morphology, growth rates, and chimeric embryo integration. However, 4-factor c-MYC dependent nuclear reprogramming produced iPS progeny that consistently prolonged the expression of pluripotent Oct-4 and Fgf4 genes and repressed cardiac differentiation. In contrast, 3-factor c-MYC-less iPS clones efficiently up-regulated pre-cardiac (CXCR4, Flk-1, and Mesp1/2) and cardiac (Nkx2.5, Mef2c, and Myocardin) gene expression patterns. In fact, 3-factor iPS progeny demonstrated early and robust cardiogenesis during in vitro differentiation with consistent beating activity, sarcomere maturation, and rhythmical intracellular calcium dynamics. Thus, nuclear reprogramming independent of c-MYC enhances production of pluripotent stem cells with innate cardiogenic potential. PMID:20221419

Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences

PubMed Central

Yu, Junying; Hu, Kejin; Smuga-Otto, Kim; Tian, Shulan; Stewart, Ron; Slukvin, Igor I.; Thomson, James A.

2009-01-01

Reprogramming differentiated human cells to induced pluripotent stem (iPS) cells has applications in basic biology, drug development, and transplantation. Human iPS cell derivation previously required vectors that integrate into the genome, which can create mutations and limit the utility of the cells in both research and clinical applications. Here we describe the derivation of human iPS cells using non-integrating episomal vectors. After removal of the episome, iPS cells completely free of vector and transgene sequences are derived that are similar to human embryonic stem (ES) cells in proliferative and developmental potential. These results demonstrate that reprogramming human somatic cells does not require genomic integration or the continued presence of exogenous reprogramming factors, and removes one obstacle to the clinical application of human iPS cells. PMID:19325077

Modeling human neurological disorders with induced pluripotent stem cells.

PubMed

Imaizumi, Yoichi; Okano, Hideyuki

2014-05-01

Human induced pluripotent stem (iPS) cells obtained by reprogramming technology are a source of great hope, not only in terms of applications in regenerative medicine, such as cell transplantation therapy, but also for modeling human diseases and new drug development. In particular, the production of iPS cells from the somatic cells of patients with intractable diseases and their subsequent differentiation into cells at affected sites (e.g., neurons, cardiomyocytes, hepatocytes, and myocytes) has permitted the in vitro construction of disease models that contain patient-specific genetic information. For example, disease-specific iPS cells have been established from patients with neuropsychiatric disorders, including schizophrenia and autism, as well as from those with neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. A multi-omics analysis of neural cells originating from patient-derived iPS cells may thus enable investigators to elucidate the pathogenic mechanisms of neurological diseases that have heretofore been unknown. In addition, large-scale screening of chemical libraries with disease-specific iPS cells is currently underway and is expected to lead to new drug discovery. Accordingly, this review outlines the progress made via the use of patient-derived iPS cells toward the modeling of neurological disorders, the testing of existing drugs, and the discovery of new drugs. The production of human induced pluripotent stem (iPS) cells from the patients' somatic cells and their subsequent differentiation into specific cells have permitted the in vitro construction of disease models that contain patient-specific genetic information. Furthermore, innovations of gene-editing technologies on iPS cells are enabling new approaches for illuminating the pathogenic mechanisms of human diseases. In this review article, we outlined the current status of neurological diseases-specific iPS cell research and described recently obtained knowledge in the form of actual examples. © 2013 International Society for Neurochemistry.

Non-Viral Generation of Marmoset Monkey iPS Cells by a Six-Factor-in-One-Vector Approach

PubMed Central

Debowski, Katharina; Warthemann, Rita; Lentes, Jana; Salinas-Riester, Gabriela; Dressel, Ralf; Langenstroth, Daniel; Gromoll, Jörg; Sasaki, Erika; Behr, Rüdiger

2015-01-01

Groundbreaking studies showed that differentiated somatic cells of mouse and human origin could be reverted to a stable pluripotent state by the ectopic expression of only four proteins. The resulting pluripotent cells, called induced pluripotent stem (iPS) cells, could be an alternative to embryonic stem cells, which are under continuous ethical debate. Hence, iPS cell-derived functional cells such as neurons may become the key for an effective treatment of currently incurable degenerative diseases. However, besides the requirement of efficacy testing of the therapy also its long-term safety needs to be carefully evaluated in settings mirroring the clinical situation in an optimal way. In this context, we chose the long-lived common marmoset monkey (Callithrix jacchus) as a non-human primate species to generate iPS cells. The marmoset monkey is frequently used in biomedical research and is gaining more and more preclinical relevance due to the increasing number of disease models. Here, we describe, to our knowledge, the first-time generation of marmoset monkey iPS cells from postnatal skin fibroblasts by non-viral means. We used the transposon-based, fully reversible piggyback system. We cloned the marmoset monkey reprogramming factors and established robust and reproducible reprogramming protocols with a six-factor-in-one-construct approach. We generated six individual iPS cell lines and characterized them in comparison with marmoset monkey embryonic stem cells. The generated iPS cells are morphologically indistinguishable from marmoset ES cells. The iPS cells are fully reprogrammed as demonstrated by differentiation assays, pluripotency marker expression and transcriptome analysis. They are stable for numerous passages (more than 80) and exhibit euploidy. In summary, we have established efficient non-viral reprogramming protocols for the derivation of stable marmoset monkey iPS cells, which can be used to develop and test cell replacement therapies in preclinical settings. PMID:25785453

Non-viral generation of marmoset monkey iPS cells by a six-factor-in-one-vector approach.

PubMed

Debowski, Katharina; Warthemann, Rita; Lentes, Jana; Salinas-Riester, Gabriela; Dressel, Ralf; Langenstroth, Daniel; Gromoll, Jörg; Sasaki, Erika; Behr, Rüdiger

2015-01-01

Groundbreaking studies showed that differentiated somatic cells of mouse and human origin could be reverted to a stable pluripotent state by the ectopic expression of only four proteins. The resulting pluripotent cells, called induced pluripotent stem (iPS) cells, could be an alternative to embryonic stem cells, which are under continuous ethical debate. Hence, iPS cell-derived functional cells such as neurons may become the key for an effective treatment of currently incurable degenerative diseases. However, besides the requirement of efficacy testing of the therapy also its long-term safety needs to be carefully evaluated in settings mirroring the clinical situation in an optimal way. In this context, we chose the long-lived common marmoset monkey (Callithrix jacchus) as a non-human primate species to generate iPS cells. The marmoset monkey is frequently used in biomedical research and is gaining more and more preclinical relevance due to the increasing number of disease models. Here, we describe, to our knowledge, the first-time generation of marmoset monkey iPS cells from postnatal skin fibroblasts by non-viral means. We used the transposon-based, fully reversible piggyback system. We cloned the marmoset monkey reprogramming factors and established robust and reproducible reprogramming protocols with a six-factor-in-one-construct approach. We generated six individual iPS cell lines and characterized them in comparison with marmoset monkey embryonic stem cells. The generated iPS cells are morphologically indistinguishable from marmoset ES cells. The iPS cells are fully reprogrammed as demonstrated by differentiation assays, pluripotency marker expression and transcriptome analysis. They are stable for numerous passages (more than 80) and exhibit euploidy. In summary, we have established efficient non-viral reprogramming protocols for the derivation of stable marmoset monkey iPS cells, which can be used to develop and test cell replacement therapies in preclinical settings.

Differential expression of three T lymphocyte-activating CXC chemokines by human atheroma-associated cells

PubMed Central

Mach, François; Sauty, Alain; Iarossi, Albert S.; Sukhova, Galina K.; Neote, Kuldeep; Libby, Peter; Luster, Andrew D.

1999-01-01

Activated T lymphocytes accumulate early in atheroma formation and persist at sites of lesion growth and rupture, suggesting that they may play an important role in the pathogenesis of atherosclerosis. Moreover, atherosclerotic lesions contain the Th1-type cytokine IFN-γ, a potentiator of atherosclerosis. The present study demonstrates the differential expression of the 3 IFN-γ–inducible CXC chemokines — IFN-inducible protein 10 (IP-10), monokine induced by IFN-γ (Mig), and IFN-inducible T-cell α chemoattractant (I-TAC) — by atheroma-associated cells, as well as the expression of their receptor, CXCR3, by all T lymphocytes within human atherosclerotic lesions in situ. Atheroma-associated endothelial cells (ECs), smooth muscle cells (SMCs), and macrophages (MØ) all expressed IP-10, whereas Mig and I-TAC were mainly expressed in ECs and MØ, as detected by double immunofluorescence staining. ECs of microvessels within lesions also expressed abundant I-TAC. In vitro experiments supported these results and showed that IL-1β, TNF-α, and CD40 ligand potentiated IP-10 expression from IFN-γ–stimulated ECs. In addition, nitric oxide (NO) treatment decreased IFN-γ induction of IP-10. Our findings suggest that the differential expression of IP-10, Mig, and I-TAC by atheroma-associated cells plays a role in the recruitment and retention of activated T lymphocytes observed within vascular wall lesions during atherogenesis. PMID:10525042

Effects of Inositol 1,4,5-triphosphate on Osteoclast Differentiation in RANKL-induced Osteoclastogenesis

PubMed Central

Son, Aran; Kim, Min Seuk; Jo, Hae; Byun, Hae Mi

2012-01-01

The receptor activator of NF-κB ligand (RANKL) signal is an activator of tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of NF-κB and other signal transduction pathways essential for osteoclastogenesis, such as Ca2+ signaling. However, the intracellular levels of inositol 1,4,5-trisphosphate (IP3) and IP3-mediated cellular function of RANKL during osteoclastogenesis are not known. In the present study, we determined the levels of IP3 and evaluated IP3-mediated osteoclast differentiation and osteoclast activity by RANKL treatment of mouse leukemic macrophage cells (RAW 264.7) and mouse bone marrow-derived monocyte/macrophage precursor cells (BMMs). During osteoclastogenesis, the expression levels of Ca2+ signaling proteins such as IP3 receptors (IP3Rs), plasma membrane Ca2+ ATPase, and sarco/endoplasmic reticulum Ca2+ ATPase type2 did not change by RANKL treatment for up to 6 days in both cell types. At 24 h after RANKL treatment, a higher steady-state level of IP3 was observed in RAW264.7 cells transfected with green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of phospholipase C (PLC) δ, a probe specifically detecting intracellular IP3 levels. In BMMs, the inhibition of PLC with U73122 [a specific inhibitor of phospholipase C (PLC)] and of IP3Rs with 2-aminoethoxydiphenyl borate (2APB; a non-specific inhibitor of IP3Rs) inhibited the generation of RANKL-induced multinucleated cells and decreased the bone-resorption rate in dentin slice, respectively. These results suggest that intracellular IP3 levels and the IP3-mediated signaling pathway play an important role in RANKL-induced osteoclastogenesis. PMID:22416217

Cell-Penetrating Peptide as a Means of Directing the Differentiation of Induced-Pluripotent Stem Cells.

PubMed

Kaitsuka, Taku; Tomizawa, Kazuhito

2015-11-06

Protein transduction using cell-penetrating peptides (CPPs) is useful for the delivery of large protein molecules, including some transcription factors. This method is safer than gene transfection methods with a viral vector because there is no risk of genomic integration of the exogenous DNA. Recently, this method was reported as a means for the induction of induced pluripotent stem (iPS) cells, directing the differentiation into specific cell types and supporting gene editing/correction. Furthermore, we developed a direct differentiation method to obtain a pancreatic lineage from mouse and human pluripotent stem cells via the protein transduction of three transcription factors, Pdx1, NeuroD, and MafA. Here, we discuss the possibility of using CPPs as a means of directing the differentiation of iPS cells and other stem cell technologies.

Rho-associated kinase inhibitors promote the cardiac differentiation of embryonic and induced pluripotent stem cells.

PubMed

Cheng, Ya-Ting; Yeih, Dong-Feng; Liang, Shu-Man; Chien, Chia-Ying; Yu, Yen-Ling; Ko, Bor-Sheng; Jan, Yee-Jee; Kuo, Cheng-Chin; Sung, Li-Ying; Shyue, Song-Kun; Chen, Ming-Fong; Yet, Shaw-Fang; Wu, Kenneth K; Liou, Jun-Yang

2015-12-15

Rho-associated kinase (ROCK) plays an important role in maintaining embryonic stem (ES) cell pluripotency. To determine whether ROCK is involved in ES cell differentiation into cardiac and hematopoietic lineages, we evaluated the effect of ROCK inhibitors, Y-27632 and fasudil on murine ES and induced pluripotent stem (iPS) cell differentiation. Gene expression levels were determined by real-time PCR, Western blot analysis and immunofluorescent confocal microscopy. Cell transplantation of induced differentiated cells were assessed in vivo in a mouse model (three groups, n=8/group) of acute myocardial infarction (MI). The cell engraftment was examined by immunohistochemical staining and the outcome was analyzed by echocardiography. Cells were cultured in hematopoietic differentiation medium in the presence or absence of ROCK inhibitor and colony formation as well as markers of ES, hematopoietic stem cells (HSC) and cells of cardiac lineages were analyzed. ROCK inhibition resulted in a drastic change in colony morphology accompanied by loss of hematopoietic markers (GATA-1, CD41 and β-Major) and expressed markers of cardiac lineages (GATA-4, Isl-1, Tbx-5, Tbx-20, MLC-2a, MLC-2v, α-MHC, cTnI and cTnT) in murine ES and iPS cells. Fasudil-induced cardiac progenitor (Mesp-1 expressing) cells were infused into a murine MI model. They engrafted into the peri-infarct and infarct regions and preserved left ventricular function. These findings provide new insights into the signaling required for ES cell differentiation into hematopoietic as well as cardiac lineages and suggest that ROCK inhibitors are useful in directing iPS cell differentiation into cardiac progenitor cells for cell therapy of cardiovascular diseases. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Modeling Alzheimer’s disease with human induced pluripotent stem (iPS) cells

PubMed Central

Mungenast, Alison E.; Siegert, Sandra; Tsai, Li-Huei

2018-01-01

In the last decade, induced pluripotent stem (iPS) cells have revolutionized the utility of human in vitro models of neurological disease. The iPS-derived and differentiated cells allow researchers to study the impact of a distinct cell type in health and disease as well as performing therapeutic drug screens on a human genetic background. In particular, clinical trials for Alzheimer’s disease (AD) have been often failing. Two of the potential reasons are first, the species gap involved in proceeding from initial discoveries in rodent models to human studies, and second, an unsatisfying patient stratification, meaning subgrouping patients based on the disease severity due to the lack of phenotypic and genetic markers. iPS cells overcome this obstacles and will improve our understanding of disease subtypes in AD. They allow researchers conducting in depth characterization of neural cells from both familial and sporadic AD patients as well as preclinical screens on human cells. In this review, we briefly outline the status quo of iPS cell research in neurological diseases along with the general advantages and pitfalls of these models. We summarize how genome-editing techniques such as CRISPR/Cas will allow researchers to reduce the problem of genomic variability inherent to human studies, followed by recent iPS cell studies relevant to AD. We then focus on current techniques for the differentiation of iPS cells into neural cell types that are relevant to AD research. Finally, we discuss how the generation of three-dimensional cell culture systems will be important for understanding AD phenotypes in a complex cellular milieu, and how both two- and three-dimensional iPS cell models can provide platforms for drug discovery and translational studies into the treatment of AD. PMID:26657644

Modeling Alzheimer's disease with human induced pluripotent stem (iPS) cells.

PubMed

Mungenast, Alison E; Siegert, Sandra; Tsai, Li-Huei

2016-06-01

In the last decade, induced pluripotent stem (iPS) cells have revolutionized the utility of human in vitro models of neurological disease. The iPS-derived and differentiated cells allow researchers to study the impact of a distinct cell type in health and disease as well as performing therapeutic drug screens on a human genetic background. In particular, clinical trials for Alzheimer's disease (AD) have been failing. Two of the potential reasons are first, the species gap involved in proceeding from initial discoveries in rodent models to human studies, and second, an unsatisfying patient stratification, meaning subgrouping patients based on the disease severity due to the lack of phenotypic and genetic markers. iPS cells overcome this obstacles and will improve our understanding of disease subtypes in AD. They allow researchers conducting in depth characterization of neural cells from both familial and sporadic AD patients as well as preclinical screens on human cells. In this review, we briefly outline the status quo of iPS cell research in neurological diseases along with the general advantages and pitfalls of these models. We summarize how genome-editing techniques such as CRISPR/Cas9 will allow researchers to reduce the problem of genomic variability inherent to human studies, followed by recent iPS cell studies relevant to AD. We then focus on current techniques for the differentiation of iPS cells into neural cell types that are relevant to AD research. Finally, we discuss how the generation of three-dimensional cell culture systems will be important for understanding AD phenotypes in a complex cellular milieu, and how both two- and three-dimensional iPS cell models can provide platforms for drug discovery and translational studies into the treatment of AD. Copyright © 2015 Elsevier Inc. All rights reserved.

Altered hematopoiesis in trisomy 21 as revealed through in vitro differentiation of isogenic human pluripotent cells

PubMed Central

MacLean, Glenn A.; Menne, Tobias F.; Guo, Guoji; Sanchez, Danielle J.; Park, In-Hyun; Daley, George Q.; Orkin, Stuart H.

2012-01-01

Trisomy 21 is associated with hematopoietic abnormalities in the fetal liver, a preleukemic condition termed transient myeloproliferative disorder, and increased incidence of acute megakaryoblastic leukemia. Human trisomy 21 pluripotent cells of various origins, human embryionic stem (hES), and induced pluripotent stem (iPS) cells, were differentiated in vitro as a model to recapitulate the effects of trisomy on hematopoiesis. To mitigate clonal variation, we isolated disomic and trisomic subclones from the same parental iPS line, thereby generating subclones isogenic except for chromosome 21. Under differentiation conditions favoring development of fetal liver-like, γ-globin expressing, definitive hematopoiesis, we found that trisomic cells of hES, iPS, or isogenic origins exhibited a two- to fivefold increase in a population of CD43+(Leukosialin)/CD235+(Glycophorin A) hematopoietic cells, accompanied by increased multilineage colony-forming potential in colony-forming assays. These findings establish an intrinsic disturbance of multilineage myeloid hematopoiesis in trisomy 21 at the fetal liver stage. PMID:23045682

Epigenetic rejuvenation.

PubMed

Manukyan, Maria; Singh, Prim B

2012-05-01

Induced pluripotent stem (iPS) cells have provided a rational means of obtaining histo-compatible tissues for 'patient-specific' regenerative therapies (Hanna et al. 2010; Yamanaka & Blau 2010). Despite the obvious potential of iPS cell-based therapies, there are certain problems that must be overcome before these therapies can become safe and routine (Ohi et al. 2011; Pera 2011). As an alternative, we have recently explored the possibility of using 'epigenetic rejuvenation', where the specialized functions of an old cell are rejuvenated in the absence of any change in its differentiated state (Singh & Zacouto 2010). The mechanism(s) that underpin 'epigenetic rejuvenation' are unknown and here we discuss model systems, using key epigenetic modifiers, which might shed light on the processes involved. Epigenetic rejuvenation has advantages over iPS cell techniques that are currently being pursued. First, the genetic and epigenetic abnormalities that arise through the cycle of dedifferentiation of somatic cells to iPS cells followed by redifferentiation of iPS cells into the desired cell type are avoided (Gore et al. 2011; Hussein et al. 2011; Pera 2011): epigenetic rejuvenation does not require passage through the de-/redifferentiation cycle. Second, because the aim of epigenetic rejuvenation is to ensure that the differentiated cell type retains its specialized function it makes redundant the question of transcriptional memory that is inimical to iPS cell-based therapies (Ohi et al. 2011). Third, to produce unrelated cell types using the iPS technology takes a long time, around three weeks, whereas epigenetic rejuvenation of old cells will take only a matter of days. Epigenetic rejuvenation provides the most safe, rapid and cheap route to successful regenerative medicine. © 2012 The Authors. Journal compilation © 2012 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

Common genetic variation drives molecular heterogeneity in human iPSCs.

PubMed

Kilpinen, Helena; Goncalves, Angela; Leha, Andreas; Afzal, Vackar; Alasoo, Kaur; Ashford, Sofie; Bala, Sendu; Bensaddek, Dalila; Casale, Francesco Paolo; Culley, Oliver J; Danecek, Petr; Faulconbridge, Adam; Harrison, Peter W; Kathuria, Annie; McCarthy, Davis; McCarthy, Shane A; Meleckyte, Ruta; Memari, Yasin; Moens, Nathalie; Soares, Filipa; Mann, Alice; Streeter, Ian; Agu, Chukwuma A; Alderton, Alex; Nelson, Rachel; Harper, Sarah; Patel, Minal; White, Alistair; Patel, Sharad R; Clarke, Laura; Halai, Reena; Kirton, Christopher M; Kolb-Kokocinski, Anja; Beales, Philip; Birney, Ewan; Danovi, Davide; Lamond, Angus I; Ouwehand, Willem H; Vallier, Ludovic; Watt, Fiona M; Durbin, Richard; Stegle, Oliver; Gaffney, Daniel J

2017-06-15

Technology utilizing human induced pluripotent stem cells (iPS cells) has enormous potential to provide improved cellular models of human disease. However, variable genetic and phenotypic characterization of many existing iPS cell lines limits their potential use for research and therapy. Here we describe the systematic generation, genotyping and phenotyping of 711 iPS cell lines derived from 301 healthy individuals by the Human Induced Pluripotent Stem Cells Initiative. Our study outlines the major sources of genetic and phenotypic variation in iPS cells and establishes their suitability as models of complex human traits and cancer. Through genome-wide profiling we find that 5-46% of the variation in different iPS cell phenotypes, including differentiation capacity and cellular morphology, arises from differences between individuals. Additionally, we assess the phenotypic consequences of genomic copy-number alterations that are repeatedly observed in iPS cells. In addition, we present a comprehensive map of common regulatory variants affecting the transcriptome of human pluripotent cells.

Dual Role of Act1 in Keratinocyte Differentiation and Host Defense: TRAF3IP2 Silencing Alters Keratinocyte Differentiation and Inhibits IL-17 Responses.

PubMed

Lambert, Sylviane; Swindell, William R; Tsoi, Lam C; Stoll, Stefan W; Elder, James T

2017-07-01

TRAF3IP2 is a candidate psoriasis susceptibility gene encoding Act1, an adaptor protein with ubiquitin ligase activity that couples the IL-17 receptor to downstream signaling pathways. We investigated the role of Act1 in keratinocyte responses to IL-17 using a tetracycline inducible short hairpin RNA targeting TRAF3IP2. Tetracycline exposure for 7 days effectively silenced TRAF3IP2 mRNA and Act1 protein, resulting in 761 genes with significant changes in expression (495 down, 266 up; >1.5-fold, P < 0.05). Gene ontology analysis showed that genes affected by TRAF3IP2 silencing are involved in epidermal differentiation, with early differentiation genes (KRT1, KRT10, DSC1, DSG1) being down-regulated and late differentiation genes (SPRR2, SPRR3, LCE3) being up-regulated. AP1 binding sites were enriched upstream of genes up-regulated by TRAF3IP2 silencing. Correspondingly, nuclear expression of FosB and Fra1 was increased in TRAF3IP2-silenced cells. Many genes involved in host defense were induced by IL-17 in a TRAF3IP2-dependent fashion. Inflammatory differentiation conditions (serum addition for 4 days postconfluence) markedly amplified these IL-17 responses and increased basal levels and TRAF3IP2 silencing-dependent up-regulation of multiple late differentiation genes. These findings suggest that TRAF3IP2 may alter both epidermal homeostasis and keratinocyte defense responses to influence psoriasis risk. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Ataxia-telangiectasia mutated (ATM) deficiency decreases reprogramming efficiency and leads to genomic instability in iPS cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kinoshita, Taisuke; Nagamatsu, Go, E-mail: gonag@sc.itc.keio.ac.jp; Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012

2011-04-08

Highlights: {yields} iPS cells were induced with a fluorescence monitoring system. {yields} ATM-deficient tail-tip fibroblasts exhibited quite a low reprogramming efficiency. {yields} iPS cells obtained from ATM-deficient cells had pluripotent cell characteristics. {yields} ATM-deficient iPS cells had abnormal chromosomes, which were accumulated in culture. -- Abstract: During cell division, one of the major features of somatic cell reprogramming by defined factors, cells are potentially exposed to DNA damage. Inactivation of the tumor suppressor gene p53 raised reprogramming efficiency but resulted in an increased number of abnormal chromosomes in established iPS cells. Ataxia-telangiectasia mutated (ATM), which is critical in the cellularmore » response to DNA double-strand breaks, may also play an important role during reprogramming. To clarify the function of ATM in somatic cell reprogramming, we investigated reprogramming in ATM-deficient (ATM-KO) tail-tip fibroblasts (TTFs). Although reprogramming efficiency was greatly reduced in ATM-KO TTFs, ATM-KO iPS cells were successfully generated and showed the same proliferation activity as WT iPS cells. ATM-KO iPS cells had a gene expression profile similar to ES cells and WT iPS cells, and had the capacity to differentiate into all three germ layers. On the other hand, ATM-KO iPS cells accumulated abnormal genome structures upon continuous passages. Even with the abnormal karyotype, ATM-KO iPS cells retained pluripotent cell characteristics for at least 20 passages. These data indicate that ATM does participate in the reprogramming process, although its role is not essential.« less

Podocalyxin Is a Glycoprotein Ligand of the Human Pluripotent Stem Cell-Specific Probe rBC2LCN

PubMed Central

Tateno, Hiroaki; Matsushima, Asako; Hiemori, Keiko; Onuma, Yasuko; Ito, Yuzuru; Hasehira, Kayo; Nishimura, Ken; Ohtaka, Manami; Takayasu, Satoko; Nakanishi, Mahito; Ikehara, Yuzuru; Nakanishi, Mio; Ohnuma, Kiyoshi; Chan, Techuan; Toyoda, Masashi; Akutsu, Hidenori; Umezawa, Akihiro; Asashima, Makoto

2013-01-01

In comprehensive glycome analysis with a high-density lectin microarray, we have previously shown that the recombinant N-terminal domain of the lectin BC2L-C from Burkholderia cenocepacia (rBC2LCN) binds exclusively to undifferentiated human induced pluripotent stem (iPS) cells and embryonic stem (ES) cells but not to differentiated somatic cells. Here we demonstrate that podocalyxin, a heavily glycosylated type 1 transmembrane protein, is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. When analyzed by DNA microarray, podocalyxin was found to be highly expressed in both iPS cells and ES cells. Western and lectin blotting revealed that rBC2LCN binds to podocalyxin with a high molecular weight of more than 240 kDa in undifferentiated iPS cells of six different origins and four ES cell lines, but no binding was observed in either differentiated mouse feeder cells or somatic cells. The specific binding of rBC2LCN to podocalyxin prepared from a large set of iPS cells (138 types) and ES cells (15 types) was also confirmed using a high-throughput antibody-overlay lectin microarray. Alkaline digestion greatly reduced the binding of rBC2LCN to podocalyxin, indicating that the major glycan ligands of rBC2LCN are presented on O-glycans. Furthermore, rBC2LCN was found to exhibit significant affinity to a branched O-glycan comprising an H type 3 structure (Ka, 2.5 × 104 M−1) prepared from human 201B7 iPS cells, indicating that H type 3 is a most probable potential pluripotency marker. We conclude that podocalyxin is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. PMID:23526252

Human induced pluripotent stem cells: a review of the US patent landscape.

PubMed

Georgieva, Bilyana P; Love, Jane M

2010-07-01

Human induced pluripotent stem (iPS) cells and human embryonic stem cells are cells that have the ability to differentiate into a variety of cell types. Embryonic stem cells are derived from human embryos; however, by contrast, human iPS cells can be obtained from somatic cells that have undergone a process of 'reprogramming' via genetic manipulation such that they develop pluripotency. Since iPS cells are not derived from human embryos, they are a less complicated source of human pluripotent cells and are considered valuable research tools and potentially useful in therapeutic applications in regenerative medicine. Worldwide, there are only three issued patents concerning iPS cells. Therefore, the patent landscape in this field is largely undefined. This article provides an overview of the issued patents as well as the pending published patent applications in the field.

Conference Scene: Induced pluripotent cells: a new path for regenerative medicine. 7 October 2010, BioPark, Welwyn Garden City, Hertfordshire, UK.

PubMed

Crutzen, Hélène S G

2011-01-01

Embryonic stem cells and induced pluripotent stem (iPS) cells, which are embryonic stem-like cells derived from adult tissues, have the broadest differentiation potential. These cells are unique in their ability to self-renew, to be maintained in an undifferentiated state for long periods of culturing and to give rise to many different cell lineages including germ-line cells. They therefore represent an invaluable tool for facilitating research towards the realization of regenerative medicine. The recent developments in embryonic stem cell and iPS cell technology have allowed human cell models to be developed that will hopefully provide novel platforms for disease analysis not only at the basic science level, but also for drug discovery and screening, and other clinical applications. This 1-day conference, chaired by Professor Peter Andrews from the University of Sheffield, UK, and Dr Chris Denning from the University of Nottingham, UK, focused on generation of iPS cells, their differentiation into specific fates and applications to disease modeling. It consisted of 11 talks by UK-based and international researchers, and three posters; Ms Azra Fatima from Cologne University, Germany, won the competition for her poster on the derivation of iPS cells from a patient with arrhythmogenic right ventricular cardiomyopathy.

TOPICAL REVIEW: Stem cell technology using bioceramics: hard tissue regeneration towards clinical application

NASA Astrophysics Data System (ADS)

Ohnishi, Hiroe; Oda, Yasuaki; Ohgushi, Hajime

2010-02-01

Mesenchymal stem cells (MSCs) are adult stem cells which show differentiation capabilities toward various cell lineages. We have already used MSCs for treatments of osteoarthritis, bone necrosis and bone tumor. For this purpose, culture expanded MSCs were combined with various ceramics and then implanted. Because of rejection response to allogeneic MSC implantation, we have utilized patients' own MSCs for the treatment. Bone marrow is a good cell source of MSCs, although the MSCs also exist in adipose tissue. When comparing osteogenic differentiation of these MSCs, bone marrow MSCs show more extensive bone forming capability than adipose MSCs. Thus, the bone marrow MSCs are useful for bone tissue regeneration. However, the MSCs show limited proliferation and differentiation capabilities that hindered clinical applications in some cases. Recent advances reveal that transduction of plural transcription factors into human adult cells results in generation of new type of stem cells called induced pluripotent stem cells (iPS cells). A drawback of the iPS cells for clinical applications is tumor formation after their in vivo implantation; therefore it is difficult to use iPS cells for the treatment. To circumvent the problem, we transduced a single factor of either SOX2 or NANOG into the MSCs and found high proliferation as well as osteogenic differentiation capabilities of the MSCs. The stem cells could be combined with bioceramics for clinical applications. Here, we summarize our recent technologies using adult stem cells in viewpoints of bone tissue regeneration.

Long-term maintenance of human induced pluripotent stem cells by automated cell culture system.

PubMed

Konagaya, Shuhei; Ando, Takeshi; Yamauchi, Toshiaki; Suemori, Hirofumi; Iwata, Hiroo

2015-11-17

Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem (iPS) cells, are regarded as new sources for cell replacement therapy. These cells can unlimitedly expand under undifferentiated conditions and be differentiated into multiple cell types. Automated culture systems enable the large-scale production of cells. In addition to reducing the time and effort of researchers, an automated culture system improves the reproducibility of cell cultures. In the present study, we newly designed a fully automated cell culture system for human iPS maintenance. Using an automated culture system, hiPS cells maintained their undifferentiated state for 60 days. Automatically prepared hiPS cells had a potency of differentiation into three germ layer cells including dopaminergic neurons and pancreatic cells.

Introduction of Exogenous HSV-TK Suicide Gene Increases Safety of Keratinocyte-Derived Induced Pluripotent Stem Cells by Providing Genetic "Emergency Exit" Switch.

PubMed

Sułkowski, Maciej; Konieczny, Paweł; Chlebanowska, Paula; Majka, Marcin

2018-01-09

Since their invention in 2006, induced Pluripotent Stem (iPS) cells remain a great promise for regenerative medicine circumventing the ethical issues linked to Embryonic Stem (ES) cell research. iPS cells can be generated in a patient-specific manner as an unlimited source of various cell types for in vitro drug screening, developmental biology studies and regenerative use. Having the capacity of differentiating into the cells of all three primary germ layers, iPS cells have high potential to form teratoma tumors. This remains their main disadvantage and hazard which, until resolved, prevents utilization of iPS cells in clinic. Here, we present an approach for increasing iPS cells safety by introducing genetic modification-exogenous suicide gene Herpes Simplex Virus Thymidine Kinase ( HSV-TK ). Its expression results in specific vulnerability of genetically modified cells to prodrug-ganciclovir (GCV). We show that HSV-TK expressing cells can be eradicated both in vitro and in vivo with high specificity and efficiency with low doses of GCV. Described strategy increases iPS cells safety for future clinical applications by generating "emergency exit" switch allowing eradication of transplanted cells in case of their malfunction.

Epigenetic modulation by TFII-I during embryonic stem cell differentiation.

PubMed

Bayarsaihan, Dashzeveg; Makeyev, Aleksandr V; Enkhmandakh, Badam

2012-10-01

TFII-I transcription factors play an essential role during early vertebrate embryogenesis. Genome-wide mapping studies by ChIP-seq and ChIP-chip revealed that TFII-I primes multiple genomic loci in mouse embryonic stem cells and embryonic tissues. Moreover, many TFII-I-bound regions co-localize with H3K4me3/K27me3 bivalent chromatin within the promoters of lineage-specific genes. This minireview provides a summary of current knowledge regarding the function of TFII-I in epigenetic control of stem cell differentiation. Copyright © 2012 Wiley Periodicals, Inc.

Induced Pluripotent Stem (iPS) Cells in Dentistry: A Review

PubMed Central

Malhotra, Neeraj

2016-01-01

iPS cells are derived from somatic cells via transduction and expression of selective transcription factors. Both viral-integrating (like retroviral) and non-integrating (like, mRNA or protein-based) techniques are available for the production of iPS cells. In the field of dentistry, iPS cells have been derived from stem cells of apical papilla, dental pulp stem cells, and stem cells from exfoliated deciduous teeth, gingival and periodontal ligament fibroblasts, and buccal mucosa fibroblasts. iPS cells have the potential to differentiate into all derivatives of the 3 primary germ layers i.e. ectoderm, endoderm, and mesoderm. They are autogeneically accessible, and can produce patient-specific or disease-specific cell lines without the issue of ethical controversy. They have been successfully tested to produce mesenchymal stem cells-like cells, neural crest-like cells, ameloblasts-like cells, odontoblasts-like cells, and osteoprogenitor cells. These cells can aid in regeneration of periodontal ligament, alveolar bone, cementum, dentin-pulp complex, as well as possible Biotooth formation. However certain key issues like, epigenetic memory of iPS cells, viral-transduction, tumorgenesis and teratoma formation need to be overcome, before they can be successfully used in clinical practice. The article discusses the sources, pros and cons, and current applications of iPS cells in dentistry with an emphasis on encountered challenges and their solutions. PMID:27572712

Chicken Induced Pluripotent Stem Cells: Establishment and Characterization.

PubMed

Fuet, Aurelie; Pain, Bertrand

2017-01-01

In mammals, the introduction of the OSKM (Oct4, Sox2, Klf4, and c-Myc) genes into somatic cells has allowed generating induced pluripotent stem (iPS) cells. So far, this process has been only clearly demonstrated in mammals. Here, using chicken as an avian model, we describe a set of protocols allowing the establishment, characterization, maintenance, differentiation, and injection of putative reprogrammed chicken Induced Pluripotent Stem (iPS) cells.

Differential gene expression profiles in neurons generated from lymphoblastoid B-cell line-derived iPS cells from monozygotic twin cases with treatment-resistant schizophrenia and discordant responses to clozapine.

PubMed

Nakazawa, Takanobu; Kikuchi, Masataka; Ishikawa, Mitsuru; Yamamori, Hidenaga; Nagayasu, Kazuki; Matsumoto, Takuya; Fujimoto, Michiko; Yasuda, Yuka; Fujiwara, Mikiya; Okada, Shota; Matsumura, Kensuke; Kasai, Atsushi; Hayata-Takano, Atsuko; Shintani, Norihito; Numata, Shusuke; Takuma, Kazuhiro; Akamatsu, Wado; Okano, Hideyuki; Nakaya, Akihiro; Hashimoto, Hitoshi; Hashimoto, Ryota

2017-03-01

Schizophrenia is a chronic psychiatric disorder with complex genetic and environmental origins. While many antipsychotics have been demonstrated as effective in the treatment of schizophrenia, a substantial number of schizophrenia patients are partially or fully unresponsive to the treatment. Clozapine is the most effective antipsychotic drug for treatment-resistant schizophrenia; however, clozapine has rare but serious side-effects. Furthermore, there is inter-individual variability in the drug response to clozapine treatment. Therefore, the identification of the molecular mechanisms underlying the action of clozapine and drug response predictors is imperative. In the present study, we focused on a pair of monozygotic twin cases with treatment-resistant schizophrenia, in which one twin responded well to clozapine treatment and the other twin did not. Using induced pluripotent stem (iPS) cell-based technology, we generated neurons from iPS cells derived from these patients and subsequently performed RNA-sequencing to compare the transcriptome profiles of the mock or clozapine-treated neurons. Although, these iPS cells similarly differentiated into neurons, several genes encoding homophilic cell adhesion molecules, such as protocadherin genes, showed differential expression patterns between these two patients. These results, which contribute to the current understanding of the molecular mechanisms of clozapine action, establish a new strategy for the use of monozygotic twin studies in schizophrenia research. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Accelerated generation of human induced pluripotent stem cells with retroviral transduction and chemical inhibitors under physiological hypoxia

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shimada, Hidenori; Hashimoto, Yoshiya; Nakada, Akira

2012-01-13

Highlights: Black-Right-Pointing-Pointer Very rapid generation of human iPS cells under optimized conditions. Black-Right-Pointing-Pointer Five chemical inhibitors under hypoxia boosted reprogramming. Black-Right-Pointing-Pointer We performed genome-wide DNA methylation analysis. -- Abstract: Induced pluripotent stem (iPS) cells are generated from somatic cells by the forced expression of a defined set of pluripotency-associated transcription factors. Human iPS cells can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for extra-embryonic tissues. This technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain largemore » amounts of disease-specific cells for biomedical research. Despite their great potential, the long reprogramming process (up to 1 month) remains one of the most significant challenges facing standard virus-mediated methodology. In this study, we report the accelerated generation of human iPS cells from adipose-derived stem (ADS) cells, using a new combination of chemical inhibitors under a setting of physiological hypoxia in conjunction with retroviral transduction of Oct4, Sox2, Klf4, and L-Myc. Under optimized conditions, we observed human embryonic stem (ES)-like cells as early as 6 days after the initial retroviral transduction. This was followed by the emergence of fully reprogrammed cells bearing Tra-1-81-positive and DsRed transgene-silencing properties on day 10. The resulting cell lines resembled human ES cells in many respects including proliferation rate, morphology, pluripotency-associated markers, global gene expression patterns, genome-wide DNA methylation states, and the ability to differentiate into all three of the germ layers, both in vitro and in vivo. Our method, when combined with chemical inhibitors under conditions of physiological hypoxia, offers a powerful tool for rapidly generating bona fide human iPS cells and facilitates the application of iPS cell technology to biomedical research.« less

3D spheroid culture of hESC/hiPSC-derived hepatocyte-like cells for drug toxicity testing.

PubMed

Takayama, Kazuo; Kawabata, Kenji; Nagamoto, Yasuhito; Kishimoto, Keisuke; Tashiro, Katsuhisa; Sakurai, Fuminori; Tachibana, Masashi; Kanda, Katsuhiro; Hayakawa, Takao; Furue, Miho Kusuda; Mizuguchi, Hiroyuki

2013-02-01

Although it is expected that hepatocyte-like cells differentiated from human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells will be utilized in drug toxicity testing, the actual applicability of hepatocyte-like cells in this context has not been well examined so far. To generate mature hepatocyte-like cells that would be applicable for drug toxicity testing, we established a hepatocyte differentiation method that employs not only stage-specific transient overexpression of hepatocyte-related transcription factors but also a three-dimensional spheroid culture system using a Nanopillar Plate. We succeeded in establishing protocol that could generate more matured hepatocyte-like cells than our previous protocol. In addition, our hepatocyte-like cells could sensitively predict drug-induced hepatotoxicity, including reactive metabolite-mediated toxicity. In conclusion, our hepatocyte-like cells differentiated from human ES cells or iPS cells have potential to be applied in drug toxicity testing. Copyright © 2012 Elsevier Ltd. All rights reserved.

β-Globin-Expressing Definitive Erythroid Progenitor Cells Generated from Embryonic and Induced Pluripotent Stem Cell-Derived Sacs.

PubMed

Fujita, Atsushi; Uchida, Naoya; Haro-Mora, Juan J; Winkler, Thomas; Tisdale, John

2016-06-01

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a potential alternative source for red blood cell transfusion. However, when using traditional methods with embryoid bodies, ES cell-derived erythroid cells predominantly express embryonic type ɛ-globin, with lesser fetal type γ-globin and very little adult type β-globin. Furthermore, no β-globin expression is detected in iPS cell-derived erythroid cells. ES cell-derived sacs (ES sacs) have been recently used to generate functional platelets. Due to its unique structure, we hypothesized that ES sacs serve as hemangioblast-like progenitors capable to generate definitive erythroid cells that express β-globin. With our ES sac-derived erythroid differentiation protocol, we obtained ∼120 erythroid cells per single ES cell. Both primitive (ɛ-globin expressing) and definitive (γ- and β-globin expressing) erythroid cells were generated from not only ES cells but also iPS cells. Primitive erythropoiesis is gradually switched to definitive erythropoiesis during prolonged ES sac maturation, concurrent with the emergence of hematopoietic progenitor cells. Primitive and definitive erythroid progenitor cells were selected on the basis of glycophorin A or CD34 expression from cells within the ES sacs before erythroid differentiation. This selection and differentiation strategy represents an important step toward the development of in vitro erythroid cell production systems from pluripotent stem cells. Further optimization to improve expansion should be required for clinical application. Stem Cells 2016;34:1541-1552. © 2016 AlphaMed Press.

The synergistic effect of micro/nano-structured and Cu2+-doped hydroxyapatite particles to promote osteoblast viability and antibacterial activity.

PubMed

Shi, Feng; Liu, Yumei; Zhi, Wei; Xiao, Dongqin; Li, Hongyu; Duan, Ke; Qu, Shuxin; Weng, Jie

2017-06-06

Microstructure and chemical constitution are important factors affecting the biological activity of biomaterials. This study aimed to fabricate hydroxyapatite (HAp) particles with both micro/nanohybrid structure and Cu 2+ doping to promote osteogenic differentiation and antibacterial property. In the presence of inositol hexakisphosphate (IP6), micro/nano-structured and Cu 2+ -doped HAp (HAp-IP6-Cu) microspheres were successfully fabricated via hydrothermal method. Morphological observation showed that HAp-IP6-Cu microspheres with a diameter of 3.1-4.1 μm were chrysanthemum-like and composed of nano-flakes approximately 50 nm in thickness. Compared with the HAp micro-rods or IP6 modified HAp (HAp-IP6) microspheres, HAp-IP6-Cu microspheres had a larger specific surface area, better hydrophilicity and stronger ability to adsorb bovine serum albumin. To evaluate the synergistic effects of micro/nanohybrid structure and Cu 2+ on cell behavior, rat calvarial osteoblasts (RCOs) were cultured on HAp-IP6-Cu, HAp-IP6 and HAp layers as well as their extracts, respectively. Results demonstrated that HAp-IP6-Cu layer promoted the adhesion, proliferation and osteogenic differentiation of RCOs. The cells grew on HAp-IP6-Cu and HAp-IP6 layers exhibited greater spreading than those on HAp layer. In addition, quantitative test by the agar disk diffusion technique found that HAp-IP6-Cu microspheres were effectively against S taphylococcus aureus and E scherichia coli. These results demonstrated that HAp-IP6-Cu microspheres may be a potential candidate as a bioactive and anti-infective biomaterial for bone regeneration.

Using Human Induced Pluripotent Stem Cells to Model Skeletal Diseases.

PubMed

Barruet, Emilie; Hsiao, Edward C

2016-01-01

Musculoskeletal disorders affecting the bones and joints are major health problems among children and adults. Major challenges such as the genetic origins or poor diagnostics of severe skeletal disease hinder our understanding of human skeletal diseases. The recent advent of human induced pluripotent stem cells (human iPS cells) provides an unparalleled opportunity to create human-specific models of human skeletal diseases. iPS cells have the ability to self-renew, allowing us to obtain large amounts of starting material, and have the potential to differentiate into any cell types in the body. In addition, they can carry one or more mutations responsible for the disease of interest or be genetically corrected to create isogenic controls. Our work has focused on modeling rare musculoskeletal disorders including fibrodysplasia ossificans progressive (FOP), a congenital disease of increased heterotopic ossification. In this review, we will discuss our experiences and protocols differentiating human iPS cells toward the osteogenic lineage and their application to model skeletal diseases. A number of critical challenges and exciting new approaches are also discussed, which will allow the skeletal biology field to harness the potential of human iPS cells as a critical model system for understanding diseases of abnormal skeletal formation and bone regeneration.

Elimination of remaining undifferentiated induced pluripotent stem cells in the process of human cardiac cell sheet fabrication using a methionine-free culture condition.

PubMed

Matsuura, Katsuhisa; Kodama, Fumiko; Sugiyama, Kasumi; Shimizu, Tatsuya; Hagiwara, Nobuhisa; Okano, Teruo

2015-03-01

Cardiac tissue engineering is a promising method for regenerative medicine. Although we have developed human cardiac cell sheets by integration of cell sheet-based tissue engineering and scalable bioreactor culture, the risk of contamination by induced pluripotent stem (iPS) cells in cardiac cell sheets remains unresolved. In the present study, we established a novel culture method to fabricate human cardiac cell sheets with a decreased risk of iPS cell contamination while maintaining viabilities of iPS cell-derived cells, including cardiomyocytes and fibroblasts, using a methionine-free culture condition. When cultured in the methionine-free condition, human iPS cells did not survive without feeder cells and could not proliferate or form colonies on feeder cells or in coculture with cells for cardiac cell sheet fabrication. When iPS cell-derived cells after the cardiac differentiation were transiently cultured in the methionine-free condition, gene expression of OCT3/4 and NANOG was downregulated significantly compared with that in the standard culture condition. Furthermore, in fabricated cardiac cell sheets, spontaneous and synchronous beating was observed in the whole area while maintaining or upregulating the expression of various cardiac and extracellular matrix genes. These findings suggest that human iPS cells are methionine dependent and a methionine-free culture condition for cardiac cell sheet fabrication might reduce the risk of iPS cell contamination.

Induced pluripotent stem (iPS) cells: a new source for cell-based therapeutics?

PubMed

de Lázaro, Irene; Yilmazer, Açelya; Kostarelos, Kostas

2014-07-10

The generation of induced pluripotent stem (iPS) cells from somatic cells by the ectopic expression of defined transcription factors has provided the regenerative medicine field with a new tool for cell replacement strategies. The advantages that these pluripotent cells can offer in comparison to other sources of stem cells include the generation of patient-derived cells and the lack of embryonic tissue while maintaining a versatile differentiation potential. The promise of iPS cell derivatives for therapeutic applications is encouraging albeit very early in development, with the first clinical study currently ongoing in Japan. Many challenges are yet to be circumvented before this technology can be clinically translated widely though. The delivery and expression of the reprogramming factors, the genomic instability, epigenetic memory and impact of cell propagation in culture are only some of the concerns. This article aims to critically discuss the potential of iPS cells as a new source of cell therapeutics. Copyright © 2014 Elsevier B.V. All rights reserved.

An In Vitro Expansion System for Generation of Human iPS Cell-Derived Hepatic Progenitor-Like Cells Exhibiting a Bipotent Differentiation Potential

PubMed Central

Yanagida, Ayaka; Ito, Keiichi; Chikada, Hiromi; Nakauchi, Hiromitsu; Kamiya, Akihide

2013-01-01

Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13highCD133+ cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development. PMID:23935837

An in vitro expansion system for generation of human iPS cell-derived hepatic progenitor-like cells exhibiting a bipotent differentiation potential.

PubMed

Yanagida, Ayaka; Ito, Keiichi; Chikada, Hiromi; Nakauchi, Hiromitsu; Kamiya, Akihide

2013-01-01

Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13(high)CD133(+) cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development.

Multiphoton autofluorescence lifetime imaging of induced pluripotent stem cells

NASA Astrophysics Data System (ADS)

Uchugonova, Aisada

2017-06-01

The multiphoton fluorescence lifetime imaging tomograph MPTflex with its flexible 360-deg scan head, articulated arm, and tunable femtosecond laser source was employed to study induced pluripotent stem cell (iPS) cultures. Autofluorescence (AF) lifetime imaging was performed with 250-ps temporal resolution and submicron spatial resolution using time-correlated single-photon counting. The two-photon excited AF was based on the metabolic coenzymes NAD(P)H and flavin adenine dinucleotide/flavoproteins. iPS cells generated from mouse embryonic fibroblasts (MEFs) and cocultured with growth-arrested MEFs as feeder cells have been studied. Significant differences on AF lifetime signatures were identified between iPS and feeder cells as well as between their differentiating counterparts.

Vector-Free and Transgene-Free Human iPS Cells Differentiate into Functional Neurons and Enhance Functional Recovery after Ischemic Stroke in Mice

PubMed Central

Mohamad, Osama; Faulkner, Ben; Chen, Dongdong; Yu, Shan Ping; Wei, Ling

2013-01-01

Stroke is a leading cause of human death and disability in the adult population in the United States and around the world. While stroke treatment is limited, stem cell transplantation has emerged as a promising regenerative therapy to replace or repair damaged tissues and enhance functional recovery after stroke. Recently, the creation of induced pluripotent stem (iPS) cells through reprogramming of somatic cells has revolutionized cell therapy by providing an unlimited source of autologous cells for transplantation. In addition, the creation of vector-free and transgene-free human iPS (hiPS) cells provides a new generation of stem cells with a reduced risk of tumor formation that was associated with the random integration of viral vectors seen with previous techniques. However, the potential use of these cells in the treatment of ischemic stroke has not been explored. In the present investigation, we examined the neuronal differentiation of vector-free and transgene-free hiPS cells and the transplantation of hiPS cell-derived neural progenitor cells (hiPS-NPCs) in an ischemic stroke model in mice. Vector-free hiPS cells were maintained in feeder-free and serum-free conditions and differentiated into functional neurons in vitro using a newly developed differentiation protocol. Twenty eight days after transplantation in stroke mice, hiPS-NPCs showed mature neuronal markers in vivo. No tumor formation was seen up to 12 months after transplantation. Transplantation of hiPS-NPCs restored neurovascular coupling, increased trophic support and promoted behavioral recovery after stroke. These data suggest that using vector-free and transgene-free hiPS cells in stem cell therapy are safe and efficacious in enhancing recovery after focal ischemic stroke in mice. PMID:23717557

Extracellular Matrix from Periodontal Ligament Cells Could Induce the Differentiation of Induced Pluripotent Stem Cells to Periodontal Ligament Stem Cell-Like Cells.

PubMed

Hamano, Sayuri; Tomokiyo, Atsushi; Hasegawa, Daigaku; Yoshida, Shinichiro; Sugii, Hideki; Mitarai, Hiromi; Fujino, Shoko; Wada, Naohisa; Maeda, Hidefumi

2018-01-15

The periodontal ligament (PDL) plays an important role in anchoring teeth in the bone socket. Damage to the PDL, such as after severe inflammation, can be treated with a therapeutic strategy that uses stem cells derived from PDL tissue (PDLSCs), a strategy that has received intense scrutiny over the past decade. However, there is an insufficient number of PDLSCs within the PDL for treating such damage. Therefore, we sought to induce the differentiation of induced pluripotent stem (iPS) cells into PDLSCs as an initial step toward PDL therapy. To this end, we first induced iPS cells into neural crest (NC)-like cells. We then captured the p75 neurotrophic receptor-positive cells (iPS-NC cells) and cultured them on an extracellular matrix (ECM) produced by human PDL cells (iPS-NC-PDL cells). These iPS-NC-PDL cells showed reduced expression of embryonic stem cell and NC cell markers as compared with iPS and iPS-NC cells, and enrichment of mesenchymal stem cell markers. The cells also had a higher proliferative capacity, multipotency, and elevated expression of PDL-related markers than iPS-NC cells cultured on fibronectin and laminin (iPS-NC-FL cells) or ECM produced by human skin fibroblast cells (iPS-NC-SF cells). Overall, we present a culture method to produce high number of PDLSC-like cells from iPS cells as a first step toward a strategy for PDL regeneration.

Reprogramming fibroblasts into induced pluripotent stem cells with Bmi1

PubMed Central

Moon, Jai-Hee; Heo, June Seok; Kim, Jun Sung; Jun, Eun Kyoung; Lee, Jung Han; Kim, Aeree; Kim, Jonggun; Whang, Kwang Youn; Kang, Yong-Kook; Yeo, Seungeun; Lim, Hee-Joung; Han, Dong Wook; Kim, Dong-Wook; Oh, Sejong; Yoon, Byung Sun; Schöler, Hans R; You, Seungkwon

2011-01-01

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by the transcription factors Oct4, Sox2, and Klf4 in combination with c-Myc. Recently, Sox2 plus Oct4 was shown to reprogram fibroblasts and Oct4 alone was able to reprogram mouse and human neural stem cells (NSCs) into iPS cells. Here, we report that Bmi1 leads to the transdifferentiation of mouse fibroblasts into NSC-like cells, and, in combination with Oct4, can replace Sox2, Klf4 and c-Myc during the reprogramming of fibroblasts into iPS cells. Furthermore, activation of sonic hedgehog signaling (by Shh, purmorphamine, or oxysterol) compensates for the effects of Bmi1, and, in combination with Oct4, reprograms mouse embryonic and adult fibroblasts into iPS cells. One- and two-factor iPS cells are similar to mouse embryonic stem cells in their global gene expression profile, epigenetic status, and in vitro and in vivo differentiation into all three germ layers, as well as teratoma formation and germline transmission in vivo. These data support that converting fibroblasts with Bmi1 or activation of the sonic hedgehog pathway to an intermediate cell type that expresses Sox2, Klf4, and N-Myc allows iPS generation via the addition of Oct4. PMID:21709693

Pancreatic β-Cell-Derived IP-10/CXCL10 Isletokine Mediates Early Loss of Graft Function in Islet Cell Transplantation.

PubMed

Yoshimatsu, Gumpei; Kunnathodi, Faisal; Saravanan, Prathab Balaji; Shahbazov, Rauf; Chang, Charles; Darden, Carly M; Zurawski, Sandra; Boyuk, Gulbahar; Kanak, Mazhar A; Levy, Marlon F; Naziruddin, Bashoo; Lawrence, Michael C

2017-11-01

Pancreatic islets produce and secrete cytokines and chemokines in response to inflammatory and metabolic stress. The physiological role of these "isletokines" in health and disease is largely unknown. We observed that islets release multiple inflammatory mediators in patients undergoing islet transplants within hours of infusion. The proinflammatory cytokine interferon-γ-induced protein 10 (IP-10/CXCL10) was among the highest released, and high levels correlated with poor islet transplant outcomes. Transgenic mouse studies confirmed that donor islet-specific expression of IP-10 contributed to islet inflammation and loss of β-cell function in islet grafts. The effects of islet-derived IP-10 could be blocked by treatment of donor islets and recipient mice with anti-IP-10 neutralizing monoclonal antibody. In vitro studies showed induction of the IP-10 gene was mediated by calcineurin-dependent NFAT signaling in pancreatic β-cells in response to oxidative or inflammatory stress. Sustained association of NFAT and p300 histone acetyltransferase with the IP-10 gene required p38 and c-Jun N-terminal kinase mitogen-activated protein kinase (MAPK) activity, which differentially regulated IP-10 expression and subsequent protein release. Overall, these findings elucidate an NFAT-MAPK signaling paradigm for induction of isletokine expression in β-cells and reveal IP-10 as a primary therapeutic target to prevent β-cell-induced inflammatory loss of graft function after islet cell transplantation. © 2017 by the American Diabetes Association.

Generation of hepatocyte-like cells from human induced pluripotent stem (iPS) cells by co-culturing embryoid body cells with liver non-parenchymal cell line TWNT-1.

PubMed

Javed, M Shahid; Yaqoob, Naeem; Iwamuro, Masaya; Kobayashi, Naoya; Fujiwara, Toshiyoshi

2014-02-01

To generate a homogeneous population of patient-specific hepatocyte-like cells (HLCs) from human iPS cells those show the morphologic and phenotypic properties of primary human hepatocytes. An experimental study. Department of Surgery, Okayama University, Graduate School of Medicine, Japan, from April to December 2011. Human iPS cells were generated and maintained on ES qualified matrigel coated plates supplemented with mTeSR medium or alternatively on mitotically inactivated MEF feeder layer in DMEM/F12 medium containing 20% KOSR, 4ng/ml bFGF-2, 1 x 10-4 M 2-mercaptoethanol, 1 mmol/L NEAA, 2mM L-glutamine and 1% penicillin-streptomycin. iPS cells were differentiated to HLCs by sequential culture using a four step differentiation protocol: (I) Generation of embryoid bodies (EBs) in suspension culture; (II) Induction of definitive endoderm (DE) from 2 days old EBs by growth in human activin-A (100 ng/ml) and basic fibroblasts growth factor (bFGF2) (100 ng/ml) on matrigel coated plates; (III) Induction of hepatic progenitors by co-culture with non-parenchymal human hepatic stellate cell line (TWNT-1); and (IV) Maturation by culture in dexamethasone. Characterization was performed by RT-PCR and functional assays. The generated HLCs showed microscopically morphological phenotype of human hepatocytes, expressed liver-specific genes (ASGPR, Albumin, AFP, Sox17, Fox A2), secreted human liver-specific proteins such as albumin, synthesized urea and metabolized ammonia. Functional HLCs were generated from human iPS cells, which could be used for autologus hepatocyte transplantation for liver failure and as in vitro model for determining the metabolic and toxicological properties of drug compounds.

CTCF-Mediated and Pax6-Associated Gene Expression in Corneal Epithelial Cell-Specific Differentiation

PubMed Central

Tsui, Shanli; Wang, Jie; Wang, Ling; Dai, Wei; Lu, Luo

2016-01-01

Background The purpose of the study is to elicit the epigenetic mechanism involving CCCTC binding factor (CTCF)-mediated chromatin remodeling that regulates PAX6 gene interaction with differentiation-associated genes to control corneal epithelial differentiation. Methods Cell cycle progression and specific keratin expressions were measured to monitor changes of differentiation-induced primary human limbal stem/progenitor (HLS/P), human corneal epithelial (HCE) and human telomerase-immortalized corneal epithelial (HTCE) cells. PAX6-interactive and differentiation-associated genes in chromatin remodeling mediated by the epigenetic factor CTCF were detected by circular chromosome conformation capture (4C) and ChIP (Chromatin immunoprecipitation)-on-chip approaches, and verified by FISH (Fluorescent in situ hybridization). Furthermore, CTCF activities were altered by CTCF-shRNA to study the effect of CTCF on mediating interaction of Pax6 and differentiation-associated genes in corneal epithelial cell fate. Results Our results demonstrated that differentiation-induced human corneal epithelial cells expressed typical corneal epithelial characteristics including morphological changes, increased keratin12 expression and G0/G1 accumulations. Expressions of CTCF and PAX6 were suppressed and elevated following the process of differentiation, respectively. During corneal epithelial cell differentiation, differentiation-induced RCN1 and ADAM17 were found interacting with PAX6 in the process of CTCF-mediated chromatin remodeling detected by 4C and verified by ChIP-on-chip and FISH. Diminished CTCF mRNA with CTCF-shRNA in HTCE cells weakened the interaction of PAX6 gene in controlling RCN1/ADAM17 and enhanced early onset of the genes in cell differentiation. Conclusion Our results explain how epigenetic factor CTCF-mediated chromatin remodeling regulates interactions between eye-specific PAX6 and those genes that are induced/associated with cell differentiation to modulate corneal epithelial cell-specific differentiation. PMID:27583466

In vitro modelling of familial amyloidotic polyneuropathy allows quantitative detection of transthyretin amyloid fibril-like structures in hepatic derivatives of patient-specific induced pluripotent stem cells.

PubMed

Hoepfner, Jeannine; Kleinsorge, Mandy; Papp, Oliver; Alfken, Susanne; Heiringhoff, Robin; Pich, Andreas; Sauer, Vanessa; Zibert, Andree; Göhring, Gudrun; Schmidt, Hartmut; Sgodda, Malte; Cantz, Tobias

2017-07-26

The transthyretin protein is thermodynamically destabilised by mutations in the transthyretin gene, promoting the formation of amyloid fibrils in various tissues. Consequently, impaired autonomic organ function is observed in patients suffering from transthyretin-related familial amyloidotic polyneuropathy (FAP). The influence of individual genetic backgrounds on fibril formation as a potential cause of genotype-phenotype variations needs to be investigated in order to ensure efficient patient-specific therapies. We reprogrammed FAP patient fibroblasts to induced pluripotent stem (iPS) cells and differentiated these cells into transthyretin-expressing hepatocyte-like cells (HLCs). HLCs differentiated from FAP iPS cells and healthy control iPS cells secreted the transthyretin protein in similar concentrations. Mass spectrometry revealed the presence of mutant transthyretin protein in FAP HLC supernatants. In comparison to healthy control iPS cells, we demonstrated the formation of transthyretin amyloid fibril-like structures in FAP HLC supernatants using the amyloid-specific dyes Congo red and thioflavin T. These dyes were also applicable for the quantitative determination of in vitro formed transthyretin fibril-like structures. Moreover, we confirmed the inhibition of fibril formation by the TTR kinetic stabiliser diclofenac. Thioflavin T fluorescence intensity measurements even allowed the quantification of amyloid fibril-like structures in 96-well plate formats as a prerequisite for patient-specific drug screening approaches.

Long-term Culture of Human iPS Cell-derived Telencephalic Neuron Aggregates on Collagen Gel.

PubMed

Oyama, Hiroshi; Takahashi, Koji; Tanaka, Yoshikazu; Takemoto, Hiroshi; Haga, Hisashi

2018-01-01

It takes several months to form the 3-dimensional morphology of the human embryonic brain. Therefore, establishing a long-term culture method for neuronal tissues derived from human induced pluripotent stem (iPS) cells is very important for studying human brain development. However, it is difficult to keep primary neurons alive for more than 3 weeks in culture. Moreover, long-term adherent culture to maintain the morphology of telencephalic neuron aggregates induced from human iPS cells is also difficult. Although collagen gel has been widely used to support long-term culture of cells, it is not clear whether human iPS cell-derived neuron aggregates can be cultured for long periods on this substrate. In the present study, we differentiated human iPS cells to telencephalic neuron aggregates and examined long-term culture of these aggregates on collagen gel. The results indicated that these aggregates could be cultured for over 3 months by adhering tightly onto collagen gel. Furthermore, telencephalic neuronal precursors within these aggregates matured over time and formed layered structures. Thus, long-term culture of telencephalic neuron aggregates derived from human iPS cells on collagen gel would be useful for studying human cerebral cortex development.Key words: Induced pluripotent stem cell, forebrain neuron, collagen gel, long-term culture.

Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy.

PubMed

Friedrichs, Stephanie; Malan, Daniela; Voss, Yvonne; Sasse, Philipp

2015-01-08

Disease-specific induced pluripotent stem (iPS) cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3)-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening.

A Paracrine Mechanism Accelerating Expansion of Human Induced Pluripotent Stem Cell-Derived Hepatic Progenitor-Like Cells

PubMed Central

Tsuruya, Kota; Chikada, Hiromi; Ida, Kinuyo; Anzai, Kazuya; Kagawa, Tatehiro; Inagaki, Yutaka; Mine, Tetsuya

2015-01-01

Hepatic stem/progenitor cells in liver development have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In this study, we focused on the cell surface molecules of human induced pluripotent stem (iPS) cell-derived hepatic progenitor-like cells (HPCs) and analyzed how these molecules modulate expansion of these cells. Human iPS cells were differentiated into immature hepatic lineage cells by cytokines. In addition to hepatic progenitor markers (CD13 and CD133), the cells were coimmunostained for various cell surface markers (116 types). The cells were analyzed by flow cytometry and in vitro colony formation culture with feeder cells. Twenty types of cell surface molecules were highly expressed in CD13+CD133+ cells derived from human iPS cells. Of these molecules, CD221 (insulin-like growth factor receptor), which was expressed in CD13+CD133+ cells, was quickly downregulated after in vitro expansion. The proliferative ability was suppressed by a neutralizing antibody and specific inhibitor of CD221. Overexpression of CD221 increased colony-forming ability. We also found that inhibition of CD340 (erbB2) and CD266 (fibroblast growth factor-inducible 14) signals suppressed proliferation. In addition, both insulin-like growth factor (a ligand of CD221) and tumor necrosis factor-like weak inducer of apoptosis (a ligand of CD266) were provided by feeder cells in our culture system. This study revealed the expression profiles of cell surface molecules in human iPS cell-derived HPCs and that the paracrine interactions between HPCs and other cells through specific receptors are important for proliferation. PMID:25808356

A Paracrine Mechanism Accelerating Expansion of Human Induced Pluripotent Stem Cell-Derived Hepatic Progenitor-Like Cells.

PubMed

Tsuruya, Kota; Chikada, Hiromi; Ida, Kinuyo; Anzai, Kazuya; Kagawa, Tatehiro; Inagaki, Yutaka; Mine, Tetsuya; Kamiya, Akihide

2015-07-15

Hepatic stem/progenitor cells in liver development have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In this study, we focused on the cell surface molecules of human induced pluripotent stem (iPS) cell-derived hepatic progenitor-like cells (HPCs) and analyzed how these molecules modulate expansion of these cells. Human iPS cells were differentiated into immature hepatic lineage cells by cytokines. In addition to hepatic progenitor markers (CD13 and CD133), the cells were coimmunostained for various cell surface markers (116 types). The cells were analyzed by flow cytometry and in vitro colony formation culture with feeder cells. Twenty types of cell surface molecules were highly expressed in CD13(+)CD133(+) cells derived from human iPS cells. Of these molecules, CD221 (insulin-like growth factor receptor), which was expressed in CD13(+)CD133(+) cells, was quickly downregulated after in vitro expansion. The proliferative ability was suppressed by a neutralizing antibody and specific inhibitor of CD221. Overexpression of CD221 increased colony-forming ability. We also found that inhibition of CD340 (erbB2) and CD266 (fibroblast growth factor-inducible 14) signals suppressed proliferation. In addition, both insulin-like growth factor (a ligand of CD221) and tumor necrosis factor-like weak inducer of apoptosis (a ligand of CD266) were provided by feeder cells in our culture system. This study revealed the expression profiles of cell surface molecules in human iPS cell-derived HPCs and that the paracrine interactions between HPCs and other cells through specific receptors are important for proliferation.

Epigenome analysis of pluripotent stem cells

PubMed Central

Ricupero, Christopher L.; Swerdel, Mavis R.; Hart, Ronald P.

2015-01-01

Summary Mis-regulation of gene expression due to epigenetic abnormalities has been linked with complex genetic disorders, psychiatric illness and cancer. In addition, the dynamic epigenetic changes that occur in pluripotent stem cells are believed to impact regulatory networks essential for proper lineage development. Chromatin immunoprecipitation (ChIP) is a technique used to isolate and enrich chromatin fragments using antibodies against specific chromatin modifications, such as DNA binding proteins or covalent histone modifications. Until recently, many ChIP protocols required millions of cells for each immunoprecipitation. This severely limited analysis of rare cell populations or post-mitotic, differentiated cell lines. Here, we describe a low cell number ChIP protocol with next generation sequencing and analysis, that has the potential to uncover novel epigenetic regulatory pathways that were previously difficult or impossible to obtain. PMID:23546758

Effects of peritoneal fluid from endometriosis patients on interferon-gamma-induced protein-10 (CXCL10) and interleukin-8 (CXCL8) released by neutrophils and CD4+ T cells.

PubMed

Kim, Ji-Yeon; Lee, Dong-Hyung; Joo, Jong-Kil; Jin, Jun-O; Wang, Ji-Won; Hong, Young-Seoub; Kwak, Jong-Young; Lee, Kyu-Sup

2009-09-01

Intraperitoneal immuno-inflammatory changes may be associated with the pathogenesis of endometriosis. We evaluated the effects of peritoneal fluid obtained from patients with endometriosis (ePF) on the release of interferon-gamma-induced protein-10 (IP-10/CXCL10) and interleukin-8 (IL-8/CXCL8) by neutrophils, CD4(+) T cells, and monocytes. Neutrophils, CD4(+) T cells, and monocytes were cultured with ePF and the chemokine levels in the supernatants were then measured using enzyme-linked immunosorbent assay. The addition of ePF to cultures of CD4(+) T cells led to a significant increase in the release of IP-10 when compared with control PF without endometriosis (cPF). There was a positive correlation between the levels of IL-8 and IP-10 in ePF (R = 0.89, P = 0.041), but not between the levels of IP-10 and IL-8 released by neutrophils, CD4(+) T cells, and monocytes. The levels of IP-10 in ePF were positively correlated with the release of IP-10 by ePF-treated neutrophils (R = 0.89, P < 0.001), CD4(+) T cells (R = 0.93, P < 0.001), and monocytes (R = 0.70, P = 0.01). Moreover, the addition of ePF significantly enhanced the interferon-gamma-induced release of IP-10 by nuetrophils and CD4(+) T cells. These findings suggest that neutrophils and T cells release differential levels of IP-10 and IL-8 in response to stimulation with ePF, and that these cells are a major source of IP-10 in the PF of endometriosis patients.

Information on Stem Cell Research

MedlinePlus

... of stem cells share similar properties there are differences as well. For example, ES cells and iPS cells are able to differentiate into any type of cell, whereas adult stem cells are more restricted in their potential. The promise of all stem cells for use ...

Investigation of tissue-specific expression and functions of MLF1-IP during development and in the immune system.

PubMed

Wang, Xuehai; Marcinkiewicz, Martin; Gatain, Yaned; Bouchard, Maxime; Mao, Jianning; Tremblay, Michel; Uetani, Noriko; Hanissian, Silva; Qi, Shijie; Wu, Jiangping; Luo, Hongyu

2013-01-01

Myeloid leukemia factor 1-interacting protein (MLF1-IP) has been found to exert functions in mitosis, although studies have been conducted only in cell lines up to now. To understand its roles during ontogeny and immunity, we analyzed its mRNA expression pattern by in situ hybridization and generated MLF1-IP gene knockout (KO) mice. MLF1-IP was expressed at elevated levels in most rudimentary tissues during the mid-gestation stage, between embryonic day 9.5 (e9.5) and e15.5. It declined afterwards in these tissues, but was very high in the testes and ovaries in adulthood. At post-natal day 10 (p10), the retina and cerebellum still expressed moderate MLF1-IP levels, although these tissues do not contain fast-proliferating cells at this stage. MLF1-IP expression in lymphoid organs, such as the thymus, lymph nodes, spleen and bone marrow, was high between e15.5 and p10, and decreased in adulthood. MLF1-IP KO embryos failed to develop beyond e6.5. On the other hand, MLF1-IP(+/-) mice were alive and fertile, with no obvious anomalies. Lymphoid organ size, weight, cellularity and cell sub-populations in MLF1-IP(+/-) mice were in the normal range. The functions of MLF1-IP(+/-) T cells and naïve CD4 cells, in terms of TCR-stimulated proliferation and Th1, Th17 and Treg cell differentiation in vitro, were comparable to those of wild type T cells. Our study demonstrates that MLF1-IP performs unique functions during mouse embryonic development, particularly around e6.5, when there was degeneration of epiblasts. However, the cells could proliferate dozens of rounds without MLF1-IP. MLF1-IP expression at about 50% of its normal level is sufficient to sustain mice life and the development of their immune system without apparent abnormalities. Our results also raise an intriguing question that MLF1-IP might have additional functions unrelated to cell proliferation.

Investigation of Tissue-Specific Expression and Functions of MLF1-IP during Development and in the Immune System

PubMed Central

Wang, Xuehai; Marcinkiewicz, Martin; Gatain, Yaned; Bouchard, Maxime; Mao, Jianning; Tremblay, Michel; Uetani, Noriko; Hanissian, Silva; Qi, Shijie; Wu, Jiangping; Luo, Hongyu

2013-01-01

Myeloid leukemia factor 1-interacting protein (MLF1-IP) has been found to exert functions in mitosis, although studies have been conducted only in cell lines up to now. To understand its roles during ontogeny and immunity, we analyzed its mRNA expression pattern by in situ hybridization and generated MLF1-IP gene knockout (KO) mice. MLF1-IP was expressed at elevated levels in most rudimentary tissues during the mid-gestation stage, between embryonic day 9.5 (e9.5) and e15.5. It declined afterwards in these tissues, but was very high in the testes and ovaries in adulthood. At post-natal day 10 (p10), the retina and cerebellum still expressed moderate MLF1-IP levels, although these tissues do not contain fast-proliferating cells at this stage. MLF1-IP expression in lymphoid organs, such as the thymus, lymph nodes, spleen and bone marrow, was high between e15.5 and p10, and decreased in adulthood. MLF1-IP KO embryos failed to develop beyond e6.5. On the other hand, MLF1-IP+/− mice were alive and fertile, with no obvious anomalies. Lymphoid organ size, weight, cellularity and cell sub-populations in MLF1-IP+/− mice were in the normal range. The functions of MLF1-IP+/− T cells and naïve CD4 cells, in terms of TCR-stimulated proliferation and Th1, Th17 and Treg cell differentiation in vitro, were comparable to those of wild type T cells. Our study demonstrates that MLF1-IP performs unique functions during mouse embryonic development, particularly around e6.5, when there was degeneration of epiblasts. However, the cells could proliferate dozens of rounds without MLF1-IP. MLF1-IP expression at about 50% of its normal level is sufficient to sustain mice life and the development of their immune system without apparent abnormalities. Our results also raise an intriguing question that MLF1-IP might have additional functions unrelated to cell proliferation. PMID:23724000

Human cord blood applications in cell therapy: looking back and look ahead.

PubMed

Zhou, Hongyan; Chang, Stephen; Rao, Mahendra

2012-08-01

Human umbilical cord blood (UCB) has been used as a reliable source of stem cells for blood-borne diseases and disorders. Recent advances in cell reprogramming technology to produce induced pluripotent stem (iPS) cells, which can be differentiated to multiple adult cell types, has further expanded the potential of cord blood cell therapy for treatment of non-blood-borne diseases. However, in order to harness this breakthrough technology and to provide clinical-grade cells for the patient, standardization of iPS production and differentiation, and good manufacturing practice (GMP) need to be employed. UCB is an ethical source of stem cells and has been used to treat diseases including leukemia, cancer and blood disorders. The development of iPS cell technology could potentially greatly increase the application of cord blood cells as a treatment for a broader range of diseases, UCB-iPS banks could, therefore, be a valuable complementary source of clinical-grade cells for cell therapy. The current applicability of GMP to UCB and UCB-iPS cell-based cell therapy will be discussed. Although cord blood stem cell therapies have been practiced for decades, UCB-iPS cell therapies are a new innovation currently in development. Successful clinical applications of such novel cell therapies will depend on the production of GMP-compliant cells and the establishment of cell banks.

Induced Pluripotent Stem Cell-Derived Red Blood Cells and Platelet Concentrates: From Bench to Bedside.

PubMed

Focosi, Daniele; Amabile, Giovanni

2017-12-27

Red blood cells and platelets are anucleate blood components indispensable for oxygen delivery and hemostasis, respectively. Derivation of these blood elements from induced pluripotent stem (iPS) cells has the potential to develop blood donor-independent and genetic manipulation-prone products to complement or replace current transfusion banking, also minimizing the risk of alloimmunization. While the production of erythrocytes from iPS cells has challenges to overcome, such as differentiation into adult-type phenotype that functions properly after transfusion, platelet products are qualitatively and quantitatively approaching a clinically-applicable level owing to advances in expandable megakaryocyte (MK) lines, platelet-producing bioreactors, and novel reagents. Guidelines that assure the quality of iPS cells-derived blood products for clinical application represent a novel challenge for regulatory agencies. Considering the minimal risk of tumorigenicity and the expected significant demand of such products, ex vivo production of iPS-derived blood components can pave the way for iPS translation into the clinic.

Type I intrinsically photosensitive retinal ganglion cells of early post-natal development correspond to the M4 subtype.

PubMed

Sexton, Timothy J; Bleckert, Adam; Turner, Maxwell H; Van Gelder, Russell N

2015-06-21

Intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate circadian light entrainment and the pupillary light response in adult mice. In early development these cells mediate different processes, including negative phototaxis and the timing of retinal vascular development. To determine if ipRGC physiologic properties also change with development, we measured ipRGC cell density and light responses in wild-type mouse retinas at post-natal days 8, 15 and 30. Melanopsin-positive cell density decreases by 17% between post-natal days 8 and 15 and by 25% between days 8 and 30. This decrease is due specifically to a decrease in cells co-labeled with a SMI-32, a marker for alpha-on ganglion cells (corresponding to adult morphologic type M4 ipRGCs). On multi-electrode array recordings, post-natal day 8 (P8) ipRGC light responses show more robust firing, reduced adaptation and more rapid recovery from short and extended light pulses than do the light responses of P15 and P30 ipRGCs. Three ipRGC subtypes - Types I-III - have been defined in early development based on sensitivity and latency on multielectrode array recordings. We find that Type I cells largely account for the unique physiologic properties of P8 ipRGCs. Type I cells have previously been shown to have relatively short latencies and high sensitivity. We now show that Type I cells show have rapid and robust recovery from long and short bright light exposures compared with Type II and III cells, suggesting differential light adaptation mechanisms between cell types. By P15, Type I ipRGCs are no longer detectable. Loose patch recordings of P8 M4 ipRGCs demonstrate Type I physiology. Type I ipRGCs are found only in early development. In addition to their previously described high sensitivity and rapid kinetics, these cells are uniquely resistant to adaptation and recover quickly and fully to short and prolonged light exposure. Type I ipRGCs correspond to the SMI-32 positive, M4 subtype and largely lose melanopsin expression in development. These cells constitute a unique morphologic and physiologic class of ipRGCs functioning early in postnatal development.

Induced Pluripotent Stem Cells from Nonhuman Primates.

PubMed

Mishra, Anuja; Qiu, Zhifang; Farnsworth, Steven L; Hemmi, Jacob J; Li, Miao; Pickering, Alexander V; Hornsby, Peter J

2016-01-01

Induced pluripotent stem cells from nonhuman primates (NHPs) have unique roles in cell biology and regenerative medicine. Because of the relatedness of NHPs to humans, NHP iPS cells can serve as a source of differentiated derivatives that can be used to address important questions in the comparative biology of primates. Additionally, when used as a source of cells for regenerative medicine, NHP iPS cells serve an invaluable role in translational experiments in cell therapy. Reprogramming of NHP somatic cells requires the same conditions as previously established for human cells. However, throughout the process, a variety of modifications to the human cell protocols must be made to accommodate significant species differences.

Induced pluripotent stem cells: Mechanisms, achievements and perspectives in farm animals

PubMed Central

Kumar, Dharmendra; Talluri, Thirumala R; Anand, Taruna; Kues, Wilfried A

2015-01-01

Pluripotent stem cells are unspecialized cells with unlimited self-renewal, and they can be triggered to differentiate into desired specialized cell types. These features provide the basis for an unlimited cell source for innovative cell therapies. Pluripotent cells also allow to study developmental pathways, and to employ them or their differentiated cell derivatives in pharmaceutical testing and biotechnological applications. Via blastocyst complementation, pluripotent cells are a favoured tool for the generation of genetically modified mice. The recently established technology to generate an induced pluripotency status by ectopic co-expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc allows to extending these applications to farm animal species, for which the derivation of genuine embryonic stem cells was not successful so far. Most induced pluripotent stem (iPS) cells are generated by retroviral or lentiviral transduction of reprogramming factors. Multiple viral integrations into the genome may cause insertional mutagenesis and may increase the risk of tumour formation. Non-integration methods have been reported to overcome the safety concerns associated with retro and lentiviral-derived iPS cells, such as transient expression of the reprogramming factors using episomal plasmids, and direct delivery of reprogramming mRNAs or proteins. In this review, we focus on the mechanisms of cellular reprogramming and current methods used to induce pluripotency. We also highlight problems associated with the generation of iPS cells. An increased understanding of the fundamental mechanisms underlying pluripotency and refining the methodology of iPS cell generation will have a profound impact on future development and application in regenerative medicine and reproductive biotechnology of farm animals. PMID:25815117

Preparation of pancreatic β-cells from human iPS cells with small molecules

PubMed Central

2012-01-01

Human induced pluripotent stem (iPS) cells obtained from patients are expected to be a useful source for cell transplantation therapy, because many patients (including those with type 1 diabetes and severe type 2 diabetes) are on waiting lists for transplantation for a long time due to the shortage of donors. At present, many concerns related to clinical application of human iPS cells have been raised, but rapid development of methods for the establishment, culture, and standardization of iPS cells will lead autologous cell therapy to be realistic sooner or later. However, establishment of a method for preparing some of desired cell types is still challenging. Regarding pancreatic β-cells, there have been many reports about differentiation of these cells from human embryonic stem (ES)/iPS cells, but a protocol for clinical application has still not been established. Since there is clear proof that cell transplantation therapy is effective for diabetes based on the results of clinical islet transplantation, pancreatic β-cells prepared from human iPS cells are considered likely to be effective for reducing the burden on patients. In this article, the current status of procedures for preparing pancreatic β-cells from human ES/iPS cells, including effective use of small molecules, is summarized, and some of the problems that still need to be overcome are discussed. PMID:22722666

Generation and validation of PAX7 reporter lines from human iPS cells using CRISPR/Cas9 technology.

PubMed

Wu, Jianbo; Hunt, Samuel D; Xue, Haipeng; Liu, Ying; Darabi, Radbod

2016-03-01

Directed differentiation of iPS cells toward various tissue progenitors has been the focus of recent research. Therefore, generation of tissue-specific reporter iPS cell lines provides better understanding of developmental stages in iPS cells. This technical report describes an efficient strategy for generation and validation of knock-in reporter lines in human iPS cells using the Cas9-nickase system. Here, we have generated a knock-in human iPS cell line for the early myogenic lineage specification gene of PAX7. By introduction of site-specific double-stranded breaks (DSB) in the genomic locus of PAX7 using CRISPR/Cas9 nickase pairs, a 2A-GFP reporter with selection markers has been incorporated before the stop codon of the PAX7 gene at the last exon. After positive and negative selection, single cell-derived human iPS clones have been isolated and sequenced for in-frame positioning of the reporter construct. Finally, by using a nuclease-dead Cas9 activator (dCas9-VP160) system, the promoter region of PAX7 has been targeted for transient gene induction to validate the GFP reporter activity. This was confirmed by flow cytometry analysis and immunostaining for PAX7 and GFP. This technical report provides a practical guideline for generation and validation of knock-in reporters using CRISPR/Cas9 system. Published by Elsevier B.V.

Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy

PubMed Central

Friedrichs, Stephanie; Malan, Daniela; Voss, Yvonne; Sasse, Philipp

2015-01-01

Disease-specific induced pluripotent stem (iPS) cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3)-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening. PMID:26237021

Enhanced Differentiation of Human Preosteoblasts on Electrospun Blend Fiber Mats of Polydioxanone and Anionic Sulfated Polysaccharides

PubMed Central

2017-01-01

The viability and differentiation of SaOS-2 preosteoblasts on fiber mats of blends comprising of the biodegradable poly(ester-ether) polydioxanone (PDX) and the sulfate-containing anionic polysaccharides kappa-carrageenan (KCG) and fucoidan (FUC) were investigated for a range of different blend compositions. The detailed analysis of the blend nanofiber properties revealed a different degree of miscibility of PDX and the polysaccharide leading to a different enrichment at the surface of the blend nanofibers, which were observed to be stable in phosphate buffer solution (PBS) for up to 5 weeks. The fibrous mats of PDX/FUC led to the highest osteogenic differentiation with very good cell viability. The electrospun blend fibers also supported human-induced pluripotent stem (iPS) cells and iPS cell-derived embryoid bodies with high cell viability, which underlines the potential of these novel blend fiber systems for optimized performance in bone tissue engineering applications. PMID:29285521

ChIPnorm: a statistical method for normalizing and identifying differential regions in histone modification ChIP-seq libraries.

PubMed

Nair, Nishanth Ulhas; Sahu, Avinash Das; Bucher, Philipp; Moret, Bernard M E

2012-01-01

The advent of high-throughput technologies such as ChIP-seq has made possible the study of histone modifications. A problem of particular interest is the identification of regions of the genome where different cell types from the same organism exhibit different patterns of histone enrichment. This problem turns out to be surprisingly difficult, even in simple pairwise comparisons, because of the significant level of noise in ChIP-seq data. In this paper we propose a two-stage statistical method, called ChIPnorm, to normalize ChIP-seq data, and to find differential regions in the genome, given two libraries of histone modifications of different cell types. We show that the ChIPnorm method removes most of the noise and bias in the data and outperforms other normalization methods. We correlate the histone marks with gene expression data and confirm that histone modifications H3K27me3 and H3K4me3 act as respectively a repressor and an activator of genes. Compared to what was previously reported in the literature, we find that a substantially higher fraction of bivalent marks in ES cells for H3K27me3 and H3K4me3 move into a K27-only state. We find that most of the promoter regions in protein-coding genes have differential histone-modification sites. The software for this work can be downloaded from http://lcbb.epfl.ch/software.html.

Unraveling the biology of bipolar disorder using induced pluripotent stem-derived neurons.

PubMed

Miller, Nathaniel D; Kelsoe, John R

2017-11-01

Bipolar disorder has been studied from numerous angles, from pathological studies to large-scale genomic studies, overall making moderate gains toward an understanding of the disorder. With the advancement of induced pluripotent stem (iPS) cell technology, in vitro models based on patient samples are now available that inherently incorporate the complex genetic variants that largely are the basis for this disorder. A number of groups are starting to apply iPS technology to the study of bipolar disorder. We selectively reviewed the literature related to understanding bipolar disorder based on using neurons derived from iPS cells. So far, most work has used the prototypical iPS cells. However, others have been able to transdifferentiate fibroblasts directly to neurons. Others still have utilized olfactory epithelium tissue as a source of neural-like cells that do not need reprogramming. In general, iPS and related cells can be used for studies of disease pathology, drug discovery, or stem cell therapy. Published studies have primarily focused on understanding bipolar disorder pathology, but initial work is also being done to use iPS technology for drug discovery. In terms of disease pathology, some evidence is pointing toward a differentiation defect with more ventral cell types being prominent. Additionally, there is evidence for a calcium signaling defect, a finding that builds on the genome-wide association study results. Continued work with iPS cells will certainly help us understand bipolar disorder and provide a way forward for improved treatments. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Microfluidic devices for stem-cell cultivation, differentiation and toxicity testing

NASA Astrophysics Data System (ADS)

Becker, Holger; Hansen-Hagge, Thomas; Kurtz, Andreas; Mrowka, Ralf; Wölfl, Stefan; Gärtner, Claudia

2017-02-01

The development of new drugs is time-consuming, extremely expensive and often promising drug candidates fail in late stages of the development process due to the lack of suitable tools to either predict toxicological effects or to test drug candidates in physiologically relevant environments prior to clinical tests. We therefore try to develop diagnostic multiorgan microfluidic chips based on patient specific induced pluripotent stem cell (iPS) technology to explore liver dependent toxic effects of drugs on individual human tissues such as liver or kidney cells. Based initially on standardized microfluidic modules for cell culture, we have developed integrated microfluidic devices which contain different chambers for cell/tissue cultivation. The devices are manufactured using injection molding of thermoplastic polymers such as polystyrene or cyclo-olefin polymer. In the project, suitable surface modification methods of the used materials had to be explored. We have been able to successfully demonstrate the seeding, cultivation and further differentiation of modified iPS, as shown by the use of differentiation markers, thus providing a suitable platform for toxicity testing and potential tissue-tissue interactions.

Recapitulating muscle disease phenotypes with myotonic dystrophy 1 iPS cells: a tool for disease modeling and drug discovery.

PubMed

Mondragon-Gonzalez, Ricardo; Perlingeiro, Rita C R

2018-06-13

Myotonic Dystrophy 1 (DM1) is a multi-system disorder primarily affecting the central nervous system, heart and skeletal muscle. It is caused by an expansion of the CTG trinucleotide repeats in the 3' untranslated region of the DMPK gene. Although patient myoblasts have been used for studying the disease in vitro , the invasiveness as well as the low accessibility to muscle biopsies motivate the development of alternative reliable myogenic models. Here, we established two DM1 iPS cell lines from patient-derived fibroblasts, and using the PAX7 conditional expression system, differentiated these into myogenic progenitors, and subsequently, terminally differentiated myotubes. Both DM1 myogenic progenitors and myotubes were found to express the intranuclear RNA foci exhibiting sequestration of MBNL1. Moreover, we found the DM1-related mis-splicing, namely BIN1 exon 11 in DM1 myotubes. We use this model to test a specific therapy, antisense oligonucleotide treatment, and find that this efficiently abolished RNA foci and rescued BIN1 mis-splicing in DM1 iPS cell-derived myotubes. Together, our results demonstrate that myotubes derived from DM1 iPS cells recapitulate the critical molecular features of DM1 and are sensitive to ASO treatment, confirming that these cells can be used for in vitro disease modeling and candidate drug testing or screening. © 2018. Published by The Company of Biologists Ltd.

Genome Therapy of Myotonic Dystrophy Type 1 iPS Cells for Development of Autologous Stem Cell Therapy.

PubMed

Gao, Yuanzheng; Guo, Xiuming; Santostefano, Katherine; Wang, Yanlin; Reid, Tammy; Zeng, Desmond; Terada, Naohiro; Ashizawa, Tetsuo; Xia, Guangbin

2016-08-01

Myotonic dystrophy type 1 (DM1) is caused by expanded Cytosine-Thymine-Guanine (CTG) repeats in the 3'-untranslated region (3' UTR) of the Dystrophia myotonica protein kinase (DMPK) gene, for which there is no effective therapy. The objective of this study is to develop genome therapy in human DM1 induced pluripotent stem (iPS) cells to eliminate mutant transcripts and reverse the phenotypes for developing autologous stem cell therapy. The general approach involves targeted insertion of polyA signals (PASs) upstream of DMPK CTG repeats, which will lead to premature termination of transcription and elimination of toxic mutant transcripts. Insertion of PASs was mediated by homologous recombination triggered by site-specific transcription activator-like effector nuclease (TALEN)-induced double-strand break. We found genome-treated DM1 iPS cells continue to maintain pluripotency. The insertion of PASs led to elimination of mutant transcripts and complete disappearance of nuclear RNA foci and reversal of aberrant splicing in linear-differentiated neural stem cells, cardiomyocytes, and teratoma tissues. In conclusion, genome therapy by insertion of PASs upstream of the expanded DMPK CTG repeats prevented the production of toxic mutant transcripts and reversal of phenotypes in DM1 iPS cells and their progeny. These genetically-treated iPS cells will have broad clinical application in developing autologous stem cell therapy for DM1.

Cerebral organoids derived from Sandhoff disease-induced pluripotent stem cells exhibit impaired neurodifferentiation[S

PubMed Central

Allende, Maria L.; Cook, Emily K.; Larman, Bridget C.; Nugent, Adrienne; Brady, Jacqueline M.; Golebiowski, Diane; Sena-Esteves, Miguel; Tifft, Cynthia J.

2018-01-01

Sandhoff disease, one of the GM2 gangliosidoses, is a lysosomal storage disorder characterized by the absence of β-hexosaminidase A and B activity and the concomitant lysosomal accumulation of its substrate, GM2 ganglioside. It features catastrophic neurodegeneration and death in early childhood. How the lysosomal accumulation of ganglioside might affect the early development of the nervous system is not understood. Recently, cerebral organoids derived from induced pluripotent stem (iPS) cells have illuminated early developmental events altered by disease processes. To develop an early neurodevelopmental model of Sandhoff disease, we first generated iPS cells from the fibroblasts of an infantile Sandhoff disease patient, then corrected one of the mutant HEXB alleles in those iPS cells using CRISPR/Cas9 genome-editing technology, thereby creating isogenic controls. Next, we used the parental Sandhoff disease iPS cells and isogenic HEXB-corrected iPS cell clones to generate cerebral organoids that modeled the first trimester of neurodevelopment. The Sandhoff disease organoids, but not the HEXB-corrected organoids, accumulated GM2 ganglioside and exhibited increased size and cellular proliferation compared with the HEXB-corrected organoids. Whole-transcriptome analysis demonstrated that development was impaired in the Sandhoff disease organoids, suggesting that alterations in neuronal differentiation may occur during early development in the GM2 gangliosidoses. PMID:29358305

Cerebral organoids derived from Sandhoff disease-induced pluripotent stem cells exhibit impaired neurodifferentiation.

PubMed

Allende, Maria L; Cook, Emily K; Larman, Bridget C; Nugent, Adrienne; Brady, Jacqueline M; Golebiowski, Diane; Sena-Esteves, Miguel; Tifft, Cynthia J; Proia, Richard L

2018-03-01

Sandhoff disease, one of the GM2 gangliosidoses, is a lysosomal storage disorder characterized by the absence of β-hexosaminidase A and B activity and the concomitant lysosomal accumulation of its substrate, GM2 ganglioside. It features catastrophic neurodegeneration and death in early childhood. How the lysosomal accumulation of ganglioside might affect the early development of the nervous system is not understood. Recently, cerebral organoids derived from induced pluripotent stem (iPS) cells have illuminated early developmental events altered by disease processes. To develop an early neurodevelopmental model of Sandhoff disease, we first generated iPS cells from the fibroblasts of an infantile Sandhoff disease patient, then corrected one of the mutant HEXB alleles in those iPS cells using CRISPR/Cas9 genome-editing technology, thereby creating isogenic controls. Next, we used the parental Sandhoff disease iPS cells and isogenic HEXB -corrected iPS cell clones to generate cerebral organoids that modeled the first trimester of neurodevelopment. The Sandhoff disease organoids, but not the HEXB -corrected organoids, accumulated GM2 ganglioside and exhibited increased size and cellular proliferation compared with the HEXB -corrected organoids. Whole-transcriptome analysis demonstrated that development was impaired in the Sandhoff disease organoids, suggesting that alterations in neuronal differentiation may occur during early development in the GM2 gangliosidoses.

Identification and characterization of novel IGFBP5 interacting protein: evidence IGFBP5-IP is a potential regulator of osteoblast cell proliferation

PubMed Central

Amaar, Yousef G.; Tapia, Blanca; Chen, Shin-Tai; Baylink, David J.; Mohan, Subburaman

2010-01-01

Insulin-like growth factor binding protein-5 (IGFBP5) is a multifunctional protein, which acts not only as a traditional binding protein, but also functions as a growth factor independent of IGFs to stimulate bone formation. It has been predicted that the intrinsic growth factor action of IGFBP5 involves binding of IGFBP5 to a putative receptor to induce downstream signaling pathways and/or nuclear translocation of IGFBP5 to influence transcription of genes involved in osteoblast cell proliferation/differentiation. Our study indentified proteins that bound to IGFBP5 using IGFBP5 as bait in a yeast two-hybrid screen of the U2 human osteosarcoma cell cDNA library. One of the clones that interacted strongly with the bait under high-stringency conditions corresponded to a novel IGFBP5 interacting protein (IGFBP5-IP) encoded by a gene that resides in mouse chromosome 10. The interaction between IGFBP5-IP and IGFBP5 is confirmed by in vitro coimmunoprecipitation studies that used pFlag and IGFBP5 polyclonal antibody, and cell lysates overexpressing both IGFBP5-IP and IGFBP5. Northern blot and RT-PCR analysis showed that the IGFBP-IP is expressed in both untransformed normal human osteoblasts and in osteosarcoma cell lines, which are known to produce IGFBP5. To determine the roles of IGFBP5-IP, we evaluated the effect of blocking the expression of IGFBP5-IP on osteoblast proliferation. We found that using a IGFBP5-IP-specific small interfering-hairpin plasmid resulted in a decrease in both basal and IGFBP5-induced osteoblast cell proliferation. On the basis of these findings, we predict that IGFBP5-IP may act as intracellular mediator of growth promoting actions of IGFBP5 and perhaps other osteoregulatory agents in bone cells. PMID:16269403

Electrical Stimulation Promotes Cardiac Differentiation of Human Induced Pluripotent Stem Cells

PubMed Central

Hernández, Damián; Millard, Rodney; Sivakumaran, Priyadharshini; Wong, Raymond C. B.; Crombie, Duncan E.; Hewitt, Alex W.; Liang, Helena; Hung, Sandy S. C.; Pébay, Alice; Shepherd, Robert K.; Dusting, Gregory J.; Lim, Shiang Y.

2016-01-01

Background. Human induced pluripotent stem cells (iPSCs) are an attractive source of cardiomyocytes for cardiac repair and regeneration. In this study, we aim to determine whether acute electrical stimulation of human iPSCs can promote their differentiation to cardiomyocytes. Methods. Human iPSCs were differentiated to cardiac cells by forming embryoid bodies (EBs) for 5 days. EBs were then subjected to brief electrical stimulation and plated down for 14 days. Results. In iPS(Foreskin)-2 cell line, brief electrical stimulation at 65 mV/mm or 200 mV/mm for 5 min significantly increased the percentage of beating EBs present by day 14 after plating. Acute electrical stimulation also significantly increased the cardiac gene expression of ACTC1, TNNT2, MYH7, and MYL7. However, the cardiogenic effect of electrical stimulation was not reproducible in another iPS cell line, CERA007c6. Beating EBs from control and electrically stimulated groups expressed various cardiac-specific transcription factors and contractile muscle markers. Beating EBs were also shown to cycle calcium and were responsive to the chronotropic agents, isoproterenol and carbamylcholine, in a concentration-dependent manner. Conclusions. Our results demonstrate that brief electrical stimulation can promote cardiac differentiation of human iPS cells. The cardiogenic effect of brief electrical stimulation is dependent on the cell line used. PMID:26788064

Chromatin immunoprecipitation assays: application of ChIP-on-chip for defining dynamic transcriptional mechanisms in bone cells.

PubMed

van der Deen, Margaretha; Hassan, Mohammad Q; Pratap, Jitesh; Teplyuk, Nadiya M; Young, Daniel W; Javed, Amjad; Zaidi, Sayyed K; Lian, Jane B; Montecino, Martin; Stein, Janet L; Stein, Gary S; van Wijnen, Andre J

2008-01-01

Normal cell growth and differentiation of bone cells requires the sequential expression of cell type specific genes to permit lineage specification and development of cellular phenotypes. Transcriptional activation and repression of distinct sets of genes support the anabolic functions of osteoblasts and the catabolic properties of osteoclasts. Furthermore, metastasis of tumors to the bone environment is controlled by transcriptional mechanisms. Insights into the transcriptional regulation of genes in bone cells may provide a conceptual basis for improved therapeutic approaches to treat bone fractures, genetic osteopathologies, and/or cancer metastases to bone. Chromatin immunoprecipitation (ChIP) is a powerful technique to establish in vivo binding of transcription factors to the promoters of genes that are either activated or repressed in bone cells. Combining ChIP with genomic microarray analysis, colloquially referred to as "ChIP-on-chip," has become a valuable method for analysis of endogenous protein/DNA interactions. This technique permits assessment of chromosomal binding sites for transcription factors or the location of histone modifications at a genomic scale. This chapter discusses protocols for performing chromatin immunoprecipitation experiments, with a focus on ChIP-on-chip analysis. The information presented is based on the authors' experience with defining interactions of Runt-related (RUNX) transcription factors with bone-related genes within the context of the native nucleosomal organization of intact osteoblastic cells.

Efficient generation of rat induced pluripotent stem cells using a non-viral inducible vector.

PubMed

Merkl, Claudia; Saalfrank, Anja; Riesen, Nathalie; Kühn, Ralf; Pertek, Anna; Eser, Stefan; Hardt, Markus Sebastian; Kind, Alexander; Saur, Dieter; Wurst, Wolfgang; Iglesias, Antonio; Schnieke, Angelika

2013-01-01

Current methods of generating rat induced pluripotent stem cells are based on viral transduction of pluripotency inducing genes (Oct4, Sox2, c-myc and Klf4) into somatic cells. These activate endogenous pluripotency genes and reprogram the identity of the cell to an undifferentiated state. Epigenetic silencing of exogenous genes has to occur to allow normal iPS cell differentiation. To gain more control over the expression of exogenous reprogramming factors, we used a novel doxycycline-inducible plasmid vector encoding Oct4, Sox2, c-Myc and Klf4. To ensure efficient and controlled generation of iPS cells by plasmid transfection we equipped the reprogramming vector with a bacteriophage φC31 attB site and used a φC31 integrase expression vector to enhance vector integration. A series of doxycycline-independent rat iPS cell lines were established. These were characterized by immunocytochemical detection of Oct4, SSEA1 and SSEA4, alkaline phosphatase staining, methylation analysis of the endogenous Oct4 promoter and RT-PCR analysis of endogenous rat pluripotency genes. We also determined the number of vector integrations and the extent to which reprogramming factor gene expression was controlled. Protocols were developed to generate embryoid bodies and rat iPS cells demonstrated as pluripotent by generating derivatives of all three embryonic germ layers in vitro, and teratoma formation in vivo. All data suggest that our rat iPS cells, generated by plasmid based reprogramming, are similar to rat ES cells. Methods of DNA transfection, protein transduction and feeder-free monolayer culture of rat iPS cells were established to enable future applications.

Retinoic acid-induced upregulation of the metalloendopeptidase nardilysin is accelerated by co-expression of the brain-specific protein p42(IP4) (centaurin α 1; ADAP1) in neuroblastoma cells.

PubMed

Borrmann, Claudia; Stricker, Rolf; Reiser, Georg

2011-11-01

The mainly neuronally expressed protein p42(IP4) (centaurin α1; ADAP1), which interacts with the metalloendopeptidase nardilysin (NRD) was found to be localized in neuritic plaques in Alzheimer disease (AD) brains. NRD was shown to enhance the cleavage of the amyloid precursor protein (APP) by α-secretases, thereby increasing the release of neuroprotective sAPPα. We here investigated in vitro the biochemical interaction of p42(IP4) and NRD and studied the physiological interaction in SH-SY5Y cells. NRD is a member of the M16 family of metalloendopeptidases. Some members of this M16 family act bi-functionally, as protease and as non-enzymatic scaffold protein. Here, we show that p42(IP4) enhances the enzymatic activity of NRD 3-4 times. However, p42(IP4) is not a substrate for NRD. Furthermore, we report that differentiation of SH-SY5Y cells by stimulation with 10μM retinoic acid (RA) results in upregulation of NRD protein levels, with a 6-fold rise after 15 days. NRD is expressed in the neurites of RA-stimulated SH-SY5Y cells, and localized in vesicular structures. Since p42(IP4) is not expressed in untreated SH-SY5Y cells, we could use this cell system as a model to find out, whether there is a functional interaction. Interestingly, SH-SY5Y cells, which we stably transfected with GFP-tagged-p42(IP4) showed an enhanced NRD protein expression already at an earlier time point after RA stimulation. Copyright © 2011 Elsevier B.V. All rights reserved.

Production of embryonic and fetal-like red blood cells from human induced pluripotent stem cells.

PubMed

Chang, Chan-Jung; Mitra, Koyel; Koya, Mariko; Velho, Michelle; Desprat, Romain; Lenz, Jack; Bouhassira, Eric E

2011-01-01

We have previously shown that human embryonic stem cells can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that regardless of the age of the donor cells, the iPS produced are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs by low and high-throughput expression and detailed analysis of globin expression patterns by HPLC. This suggests that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with erythroid differentiation regardless of the age or the tissue type of the donor cells, at least as detected in these assays. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications.

Induced pluripotent stem (iPS) cells from human fetal stem cells.

PubMed

Guillot, Pascale V

2016-02-01

Pluripotency defines the ability of stem cells to differentiate into all the lineages of the three germ layers and self-renew indefinitely. Somatic cells can regain the developmental potential of embryonic stem cells following ectopic expression of a set of transcription factors or, in certain circumstances, via modulation of culture conditions and supplementation with small molecule, that is, induced pluripotent stem (iPS) cells. Here, we discuss the use of fetal tissues for reprogramming, focusing in particular on stem cells derived from human amniotic fluid, and the development of chemical reprogramming. We next address the advantages and disadvantages of deriving pluripotent cells from fetal tissues and the potential clinical applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Generation and characterization of induced pluripotent stem cells from guinea pig fetal fibroblasts

PubMed Central

Wu, Yuehong; Li, Ouyang; He, Chengwen; Li, Yong; Li, Min; Liu, Xiaoming; Wang, Yujiong; He, Yulong

2017-01-01

Induced pluripotent stem cells (iPS) represent an important tool to develop disease-modeling assays, drug testing assays and cell-based replacement therapies. The application of iPS in these fields requires the development of suitable animal models. Of the suitable species, guinea pigs are particularly important and offer significant advantages. Successful iPS generation has been accomplished in a number of species; however, it has not been reported in the guinea pig. The present study successfully generated iPS from guinea pigs (giPS) using single polycistronic virus transduction with mouse octamer-binding transcription factor 4 (Oct4), sex determining region Y-box 2 (Sox2), Kruppel-like factor 4 and c-Myc. The giPS cell lines were cultured in media containing leukemia inhibitory factor and guinea pig fibroblast cells were used as feeder cells. These cultures were expanded under feeder-free culture conditions using ESGRO Complete Plus Clonal Grade medium containing 15% fetal bovine serum on gelatin-coated dishes. The resultant cells had a normal karyotype, exhibited alkaline phosphatase activity and expressed the pluripotency markers Oct4, Sox2 and Nanog. The cells differentiated in vivo to form teratomas that contained all three germ layers of the tissue cells. The generation of giPS may facilitate future studies investigating the mechanisms underlying innate immunity, particularly for tuberculosis. These experiments provide proof of principle that iPS technology may be adapted to use the guinea pig as a model of human diseases. PMID:28393187

Induced Pluripotent Stem Cell Derived Mesenchymal Stem Cells for Attenuating Age-Related Bone Loss

DTIC Science & Technology

2012-07-01

Mesenchymal stem cell (MSC) differentiation towards the bone forming osteoblastic lineage decreases as a function of age and may contribute to age-related...problem of age-related reduced availability of MSC we propose to examine the bone anabolic potential of induced pluripotent stem cell (iPS) derived MSC

Induction of pluripotent stem cells from fibroblast cultures.

PubMed

Takahashi, Kazutoshi; Okita, Keisuke; Nakagawa, Masato; Yamanaka, Shinya

2007-01-01

Clinical application of embryonic stem (ES) cells faces difficulties regarding use of embryos, as well as tissue rejection after implantation. One way to circumvent these issues is to generate pluripotent stem cells directly from somatic cells. Somatic cells can be reprogrammed to an embryonic-like state by the injection of a nucleus into an enucleated oocyte or by fusion with ES cells. However, little is known about the mechanisms underlying these processes. We have recently shown that the combination of four transcription factors can generate ES-like pluripotent stem cells directly from mouse fibroblast cultures. The cells, named induced pluripotent stem (iPS) cells, can be differentiated into three germ layers and committed to chimeric mice. Here we describe detailed methods and tips for the generation of iPS cells.

Generation and characterization of induced pluripotent stem cells from guinea pig fetal fibroblasts.

PubMed

Wu, Yuehong; Li, Ouyang; He, Chengwen; Li, Yong; Li, Min; Liu, Xiaoming Liu; Wang, Yujiong; He, Yulong

2017-06-01

Induced pluripotent stem cells (iPS) represent an important tool to develop disease‑modeling assays, drug testing assays and cell‑based replacement therapies. The application of iPS in these fields requires the development of suitable animal models. Of the suitable species, guinea pigs are particularly important and offer significant advantages. Successful iPS generation has been accomplished in a number of species; however, it has not been reported in the guinea pig. The present study successfully generated iPS from guinea pigs (giPS) using single polycistronic virus transduction with mouse octamer‑binding transcription factor 4 (Oct4), sex determining region Y‑box 2 (Sox2), Kruppel‑like factor 4 and c‑Myc. The giPS cell lines were cultured in media containing leukemia inhibitory factor and guinea pig fibroblast cells were used as feeder cells. These cultures were expanded under feeder‑free culture conditions using ESGRO Complete Plus Clonal Grade medium containing 15% fetal bovine serum on gelatin‑coated dishes. The resultant cells had a normal karyotype, exhibited alkaline phosphatase activity and expressed the pluripotency markers Oct4, Sox2 and Nanog. The cells differentiated in vivo to form teratomas that contained all three germ layers of the tissue cells. The generation of giPS may facilitate future studies investigating the mechanisms underlying innate immunity, particularly for tuberculosis. These experiments provide proof of principle that iPS technology may be adapted to use the guinea pig as a model of human diseases.

Buffer kinetics shape the spatiotemporal patterns of IP3-evoked Ca2+ signals

PubMed Central

Dargan, Sheila L; Parker, Ian

2003-01-01

Ca2+ liberation through inositol 1,4,5-trisphosphate receptors (IP3Rs) plays a universal role in cell regulation, and specificity of cell signalling is achieved through the spatiotemporal patterning of Ca2+ signals. IP3Rs display Ca2+-induced Ca2+ release (CICR), but are grouped in clusters so that regenerative Ca2+ signals may remain localized to individual clusters, or propagate globally between clusters by successive cycles of Ca2+ diffusion and CICR. We used confocal microscopy and photoreleased IP3 in Xenopus oocytes to study how these properties are modulated by mobile cytosolic Ca2+ buffers. EGTA (a buffer with slow ‘on-rate’) speeded Ca2+ signals and ‘balkanized’ Ca2+ waves by dissociating them into local signals. In contrast, BAPTA (a fast buffer with similar affinity) slowed Ca2+ responses and promoted ‘globalization’ of spatially uniform Ca2+ signals. These actions are likely to arise through differential effects on Ca2+ feedback within and between IP3R clusters, because Ca2+ signals evoked by influx through voltage-gated channels were little affected. We propose that cell-specific expression of Ca2+-binding proteins with distinct kinetics may shape the time course and spatial distribution of IP3-evoked Ca2+ signals for specific physiological roles. PMID:14555715

Epigenetic Plasticity Drives Adipogenic and Osteogenic Differentiation of Marrow-derived Mesenchymal Stem Cells*

PubMed Central

Meyer, Mark B.; Benkusky, Nancy A.; Sen, Buer; Rubin, Janet; Pike, J. Wesley

2016-01-01

Terminal differentiation of multipotent stem cells is achieved through a coordinated cascade of activated transcription factors and epigenetic modifications that drive gene transcription responsible for unique cell fate. Within the mesenchymal lineage, factors such as RUNX2 and PPARγ are indispensable for osteogenesis and adipogenesis, respectively. We therefore investigated genomic binding of transcription factors and accompanying epigenetic modifications that occur during osteogenic and adipogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (MSCs). As assessed by ChIP-sequencing and RNA-sequencing analyses, we found that genes vital for osteogenic identity were linked to RUNX2, C/EBPβ, retinoid X receptor, and vitamin D receptor binding sites, whereas adipocyte differentiation favored PPARγ, retinoid X receptor, C/EBPα, and C/EBPβ binding sites. Epigenetic marks were clear predictors of active differentiation loci as well as enhancer activities and selective gene expression. These marrow-derived MSCs displayed an epigenetic pattern that suggested a default preference for the osteogenic pathway; however, these patterns were rapidly altered near the Adipoq, Cidec, Fabp4, Lipe, Plin1, Pparg, and Cebpa genes during adipogenic differentiation. Surprisingly, we found that these cells also exhibited an epigenetic plasticity that enabled them to trans-differentiate from adipocytes to osteoblasts (and vice versa) after commitment, as assessed by staining, gene expression, and ChIP-quantitative PCR analysis. The osteogenic default pathway may be subverted during pathological conditions, leading to skeletal fragility and increased marrow adiposity during aging, estrogen deficiency, and skeletal unloading. Taken together, our data provide an increased mechanistic understanding of the epigenetic programs necessary for multipotent differentiation of MSCs that may prove beneficial in the development of therapeutic strategies. PMID:27402842

Excision of a viral reprogramming cassette by delivery of synthetic Cre mRNA

PubMed Central

Loh, Yuin-Han; Yang, Jimmy Chen; De Los Angeles, Alejandro; Guo, Chunguang; Cherry, Anne; Rossi, Derrick J.; Park, In-Hyun; Daley, George Q.

2012-01-01

The generation of patient-specific induced pluripotent stem (iPS) cells provides an invaluable resource for cell therapy, in vitro modeling of human disease, and drug screening. To date, most human iPS cells have been generated with integrating retro- and lenti-viruses and are limited in their potential utility because residual transgene expression may alter their differentiation potential or induce malignant transformation. Alternatively, transgene-free methods using adenovirus and protein transduction are limited by low efficiency. This report describes a protocol for the generation of transgene-free human induced pluripotent stem cells using retroviral transfection of a single vector, which includes the coding sequences of human OCT4, SOX2, KLF4, and cMYC linked with picornaviral 2A plasmids. Moreover, after reprogramming has been achieved, this cassette can be removed using mRNA transfection of Cre recombinase. The method described herein to excise reprogramming factors with ease and efficiency facilitates the experimental generation and use of transgene-free human iPS cells. PMID:22605648

Machine Learning Approach to Automated Quality Identification of Human Induced Pluripotent Stem Cell Colony Images.

PubMed

Joutsijoki, Henry; Haponen, Markus; Rasku, Jyrki; Aalto-Setälä, Katriina; Juhola, Martti

2016-01-01

The focus of this research is on automated identification of the quality of human induced pluripotent stem cell (iPSC) colony images. iPS cell technology is a contemporary method by which the patient's cells are reprogrammed back to stem cells and are differentiated to any cell type wanted. iPS cell technology will be used in future to patient specific drug screening, disease modeling, and tissue repairing, for instance. However, there are technical challenges before iPS cell technology can be used in practice and one of them is quality control of growing iPSC colonies which is currently done manually but is unfeasible solution in large-scale cultures. The monitoring problem returns to image analysis and classification problem. In this paper, we tackle this problem using machine learning methods such as multiclass Support Vector Machines and several baseline methods together with Scaled Invariant Feature Transformation based features. We perform over 80 test arrangements and do a thorough parameter value search. The best accuracy (62.4%) for classification was obtained by using a k-NN classifier showing improved accuracy compared to earlier studies.

Inducing pluripotency in somatic cells from the snow leopard (Panthera uncia), an endangered felid.

PubMed

Verma, R; Holland, M K; Temple-Smith, P; Verma, P J

2012-01-01

Induced pluripotency is a new approach to produce embryonic stem-like cells from somatic cells that provides a unique means to understand both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered species, where the limited availability of gametes makes production and research on embryonic stem cells difficult, we attempted generation of induced pluripotent stem (iPS) cells from snow leopard (Panthera uncia) fibroblasts by retroviral transfection with Moloney-based retroviral vectors (pMXs) encoding four factors (OCT4, SOX2, KLF4 and cMYC). This resulted in the formation of small colonies of cells, which could not be maintained beyond four passages (P4). However, addition of NANOG, to the transfection cocktail produced stable iPS cell colonies, which formed as early as D3. Colonies of cells were selected at D5 and expanded in vitro. The resulting cell line was positive for alkaline phosphatase (AP), OCT4, NANOG, and Stage-Specific embryonic Antigen-4 (SSEA-4) at P14. RT-PCR also confirmed that endogenous OCT4 and NANOG were expressed by snow leopard iPS cells from P4. All five human transgenes were transcribed at P4, but OCT4, SOX2 and NANOG transgenes were silenced as early as P14; therefore, reprogramming of the endogenous pluripotent genes had occurred. When injected into immune-deficient mice, snow leopard iPS cells formed teratomas containing tissues representative of the three germ layers. In conclusion, this was apparently the first derivation of iPS cells from the endangered snow leopard and the first report on induced pluripotency in felid species. Addition of NANOG to the reprogramming cocktail was essential for derivation of iPS lines in this felid. The iPS cells provided a unique source of pluripotent cells with utility in conservation through cryopreservation of genetics, as a source of reprogrammed donor cells for nuclear transfer or for directed differentiation to gametes in the future. Copyright © 2012 Elsevier Inc. All rights reserved.

Combined cistrome and transcriptome analysis of SKI in AML cells identifies SKI as a co-repressor for RUNX1

PubMed Central

Feld, Christine; Sahu, Peeyush; Frech, Miriam; Finkernagel, Florian; Nist, Andrea; Stiewe, Thorsten; Bauer, Uta-Maria; Neubauer, Andreas

2018-01-01

Abstract SKI is a transcriptional co-regulator and overexpressed in various human tumors, for example in acute myeloid leukemia (AML). SKI contributes to the origin and maintenance of the leukemic phenotype. Here, we use ChIP-seq and RNA-seq analysis to identify the epigenetic alterations induced by SKI overexpression in AML cells. We show that approximately two thirds of differentially expressed genes are up-regulated upon SKI deletion, of which >40% harbor SKI binding sites in their proximity, primarily in enhancer regions. Gene ontology analysis reveals that many of the differentially expressed genes are annotated to hematopoietic cell differentiation and inflammatory response, corroborating our finding that SKI contributes to a myeloid differentiation block in HL60 cells. We find that SKI peaks are enriched for RUNX1 consensus motifs, particularly in up-regulated SKI targets upon SKI deletion. RUNX1 ChIP-seq displays that nearly 70% of RUNX1 binding sites overlap with SKI peaks, mainly at enhancer regions. SKI and RUNX1 occupy the same genomic sites and cooperate in gene silencing. Our work demonstrates for the first time the predominant co-repressive function of SKI in AML cells on a genome-wide scale and uncovers the transcription factor RUNX1 as an important mediator of SKI-dependent transcriptional repression. PMID:29471413

Depletion of mRNA export regulator DBP5/DDX19, GLE1 or IPPK that is a key enzyme for the production of IP6, resulting in differentially altered cytoplasmic mRNA expression and specific cell defect

PubMed Central

Okamura, Masumi; Yamanaka, Yasutaka; Shigemoto, Maki; Kitadani, Yuya; Kobayashi, Yuhko; Kambe, Taiho; Nagao, Masaya; Kobayashi, Issei; Okumura, Katsuzumi

2018-01-01

DBP5, also known as DDX19, GLE1 and inositol hexakisphosphate (IP6) function in messenger RNA (mRNA) export at the cytoplasmic surface of the nuclear pore complex in eukaryotic cells. DBP5 is a DEAD-box RNA helicase, and its activity is stimulated by interactions with GLE1 and IP6. In addition, these three factors also have unique role(s). To investigate how these factors influenced the cytoplasmic mRNA expression and cell phenotype change, we performed RNA microarray analysis to detect the effect and function of DBP5, GLE1 and IP6 on the cytoplasmic mRNA expression. The expression of some cytoplasmic mRNA subsets (e.g. cell cycle, DNA replication) was commonly suppressed by the knock-down of DBP5, GLE1 and IPPK (IP6 synthetic enzyme). The GLE1 knock-down selectively reduced the cytoplasmic mRNA expression required for mitotic progression, results in an abnormal spindle phenotype and caused the delay of mitotic process. Meanwhile, G1/S cell cycle arrest was observed in DBP5 and IPPK knock-down cells. Several factors that function in immune response were also down-regulated in DBP5 or IPPK knock-down cells. Thereby, IFNβ-1 mRNA transcription evoked by poly(I:C) treatment was suppressed. These results imply that DBP5, GLE1 and IP6 have a conserved and individual function in the cytoplasmic mRNA expression. Variations in phenotype are due to the difference in each function of DBP5, GLE1 and IPPK in intracellular mRNA metabolism. PMID:29746542

Mechanical stimulation of cyclic tensile strain induces reduction of pluripotent related gene expressions via activation of Rho/ROCK and subsequent decreasing of AKT phosphorylation in human induced pluripotent stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Teramura, Takeshi, E-mail: teramura@med.kindai.ac.jp; Takehara, Toshiyuki; Onodera, Yuta

2012-01-13

Highlights: Black-Right-Pointing-Pointer Mechanical stimulation is an important factor for regulation of stem cell fate. Black-Right-Pointing-Pointer Cyclic stretch to human induced pluripotent stem cells activated small GTPase Rho. Black-Right-Pointing-Pointer Rho-kinase activation attenuated pluripotency via inhibition of AKT activation. Black-Right-Pointing-Pointer This reaction could be reproduced only by transfection of dominant active Rho. Black-Right-Pointing-Pointer Rho/ROCK are important molecules in mechanotransduction and control of stemness. -- Abstract: Mechanical stimulation has been shown to regulate the proliferation and differentiation of stem cells. However, the effects of the mechanical stress on the stemness or related molecular mechanisms have not been well determined. Pluripotent stem cells suchmore » as embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are used as good materials for cell transplantation therapy and research of mammalian development, since they can self-renew infinitely and differentiate into various cell lineages. Here we demonstrated that the mechanical stimulation to human iPS cells altered alignment of actin fibers and expressions of the pluripotent related genes Nanog, POU5f1 and Sox2. In the mechanically stimulated iPS cells, small GTPase Rho was activated and interestingly, AKT phosphorylation was decreased. Inhibition of Rho-associated kinase ROCK recovered the AKT phosphorylation and the gene expressions. These results clearly suggested that the Rho/ROCK is a potent primary effector of mechanical stress in the pluripotent stem cells and it participates to pluripotency-related signaling cascades as an upper stream regulator.« less

Cryopreservation of Human Stem Cells for Clinical Application: A Review

PubMed Central

Hunt, Charles J.

2011-01-01

Summary Stem cells have been used in a clinical setting for many years. Haematopoietic stem cells have been used for the treatment of both haematological and non-haematological disease; while more recently mesenchymal stem cells derived from bone marrow have been the subject of both laboratory and early clinical studies. Whilst these cells show both multipotency and expansion potential, they nonetheless do not form stable cell lines in culture which is likely to limit the breadth of their application in the field of regenerative medicine. Human embryonic stem cells are pluripotent cells, capable of forming stable cell lines which retain the capacity to differentiate into cells from all three germ layers. This makes them of special significance in both regenerative medicine and toxicology. Induced pluripotent stem (iPS) cells may also provide a similar breadth of utility without some of the confounding ethical issues surrounding embryonic stem cells. An essential pre-requisite to the commercial and clinical application of stem cells are suitable cryopreservation protocols for long-term storage. Whilst effective methods for cryopreservation and storage have been developed for haematopoietic and mesenchymal stem cells, embryonic cells and iPS cells have proved more refractory. This paper reviews the current state of cryopreservation as it pertains to stem cells and in particular the embryonic and iPS cell. PMID:21566712

Cryopreservation of Human Stem Cells for Clinical Application: A Review.

PubMed

Hunt, Charles J

2011-01-01

SUMMARY: Stem cells have been used in a clinical setting for many years. Haematopoietic stem cells have been used for the treatment of both haematological and non-haematological disease; while more recently mesenchymal stem cells derived from bone marrow have been the subject of both laboratory and early clinical studies. Whilst these cells show both multipotency and expansion potential, they nonetheless do not form stable cell lines in culture which is likely to limit the breadth of their application in the field of regenerative medicine. Human embryonic stem cells are pluripotent cells, capable of forming stable cell lines which retain the capacity to differentiate into cells from all three germ layers. This makes them of special significance in both regenerative medicine and toxicology. Induced pluripotent stem (iPS) cells may also provide a similar breadth of utility without some of the confounding ethical issues surrounding embryonic stem cells. An essential pre-requisite to the commercial and clinical application of stem cells are suitable cryopreservation protocols for long-term storage. Whilst effective methods for cryopreservation and storage have been developed for haematopoietic and mesenchymal stem cells, embryonic cells and iPS cells have proved more refractory. This paper reviews the current state of cryopreservation as it pertains to stem cells and in particular the embryonic and iPS cell.

Prospects for pluripotent stem cell therapies: into the clinic and back to the bench.

PubMed

Grabel, Laura

2012-02-01

Pluripotent stem cells, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, both hold great promise for the understanding and treatment of disease. They can be used for drug testing, as in vitro models for human disease progression, and for transplantation therapies. Research in this area has been influenced by the ever-changing political landscape, particularly in the United States. In this review, we discuss the prospects for clinical application using pluripotent cells, focusing on an evaluation of iPS cell potential, the continuing concern of tumor formation, and a summary of in vitro differentiation protocols and animal models used. We also describe the current clinical trials underway in the United States, as well as the ups and downs of funding for ES cell work. Copyright © 2011 Wiley Periodicals, Inc.

Implications of Parkinson's disease pathophysiology for the development of cell replacement strategies and drug discovery in neurodegenerative diseases.

PubMed

Pan-Montojo, Francisco; Funk, Richard H W

2012-11-01

Parkinson's disease (PD) is a progressive neurodegenerative disorder traditionally characterized by the loss of dopaminergic neurons in the substantia nigra (SN) at the midbrain. The potential use of adult or embryonic stem cells, induced pluriputent stem (iPS) cells and endogenous neurogenesis in cell replacement strategies has lead to numerous studies and clinical trials in this direction. It is now possible to differentiate stem cells into dopaminergic neurons in vitro and clinical trials have shown an improvement in PD-related symptoms after intra-striatal embryonic transplants and acceptable cell survival rates on the mid term. However, clinical improvement is transitory and associated with a strong placebo effect. Interestingly, recent pathological studies in PD patients who received embryonic stem cells show that in PD patients, grafted neurons show PD-related pathology. In this manuscript we review the latest findings regarding PD pathophysiology and give an outlook on the implications of these findings in how cell replacement strategies for PD treatment should be tested. These include changes in the type of animal models used, the preparation/conditioning of the cells before intracerebral injection, specially regarding backbone chronic diseases in iPS cells and determining the optimal proliferation, survival, differentiation and migration capacity of the grafted cells.

Synchrotron Radiation X-Ray Microfluorescence Reveals Polarized Distribution of Atomic Elements during Differentiation of Pluripotent Stem Cells

PubMed Central

Paulsen, Bruna S.; Rehen, Stevens K.

2011-01-01

The mechanisms underlying pluripotency and differentiation in embryonic and reprogrammed stem cells are unclear. In this work, we characterized the pluripotent state towards neural differentiated state through analysis of trace elements distribution using the Synchrotron Radiation X-ray Fluorescence Spectroscopy. Naive and neural-stimulated embryoid bodies (EB) derived from embryonic and induced pluripotent stem (ES and iPS) cells were irradiated with a spatial resolution of 20 µm to make elemental maps and qualitative chemical analyses. Results show that these embryo-like aggregates exhibit self-organization at the atomic level. Metallic elements content rises and consistent elemental polarization pattern of P and S in both mouse and human pluripotent stem cells were observed, indicating that neural differentiation and elemental polarization are strongly correlated. PMID:22195032

Vaccination with vascular progenitor cells derived from induced pluripotent stem cells elicits antitumor immunity targeting vascular and tumor cells.

PubMed

Koido, Shigeo; Ito, Masaki; Sagawa, Yukiko; Okamoto, Masato; Hayashi, Kazumi; Nagasaki, Eijiro; Kan, Shin; Komita, Hideo; Kamata, Yuko; Homma, Sadamu

2014-05-01

Vaccination of BALB/c mice with dendritic cells (DCs) loaded with the lysate of induced vascular progenitor (iVP) cells derived from murine-induced pluripotent stem (iPS) cells significantly suppressed the tumor of CMS-4 fibrosarcomas and prolonged the survival of CMS-4-inoculated mice. This prophylactic antitumor activity was more potent than that of immunization with DCs loaded with iPS cells or CMS-4 tumor cells. Tumors developed slowly in mice vaccinated with DCs loaded with iVP cells (DC/iVP) and exhibited a limited vascular bed. Immunohistochemistry and a tomato-lectin perfusion study demonstrated that the tumors that developed in the iVP-immunized mice showed a marked decrease in tumor vasculature. Immunization with DC/iVP induced a potent suppressive effect on vascular-rich CMS-4 tumors, a weaker effect on BNL tumors with moderate vasculature, and nearly no effect on C26 tumors with poor vasculature. Treatment of DC/iVP-immunized mice with a monoclonal antibody against CD4 or CD8, but not anti-asialo GM1, inhibited the antitumor activity. CD8(+) T cells from DC/iVP-vaccinated mice showed significant cytotoxic activity against murine endothelial cells and CMS-4 cells, whereas CD8(+) T cells from DC/iPS-vaccinated mice did not. DNA microarray analysis showed that the products of 29 vasculature-associated genes shared between genes upregulated by differentiation from iPS cells into iVP cells and genes shared by iVP cells and isolated Flk-1(+) vascular cells in CMS-4 tumor tissue might be possible targets in the immune response. These results suggest that iVP cells from iPS cells could be used as a cancer vaccine targeting tumor vascular cells and tumor cells.

Impact of non-thermal plasma surface modification on porous calcium hydroxyapatite ceramics for bone regeneration

PubMed Central

Moriguchi, Yu; Lee, Dae-Sung; Thamina, Khair; Masuda, Kazuto; Itsuki, Dai; Yoshikawa, Hideki; Hamaguchi, Satoshi; Myoui, Akira

2018-01-01

In the physiochemical sciences, plasma is used to describe an ionized gas. Previous studies have implicated plasma surface treatment in the enhancement of hydrophilicity of implanted musculoskeletal reconstructive materials. Hydroxyapatite (HA) ceramics, widely used in bone tissue regeneration, have made great advancements to skeletal surgery. In the present study, we investigate the impact of low-pressure plasma on the interconnected porous calcium hydroxyapatite (IP-CHA) both in vitro and in vivo. Our results indicate that dielectric barrier discharge (DBD) plasma, when used with oxygen, can augment the hydrophilicity of non-porous HA surfaces and the osteoconductivity of the IP-CHA disc via increased water penetration of inner porous structures, as demonstrated through microfocus computed tomography (μCT) assay. In vivo implantation of plasma-treated IP-CHA displayed superior bone ingrowth than untreated IP-CHA. Though plasma-treated IP-CHA did not alter osteoblast cell proliferation, it accelerated osteogenic differentiation of seeded marrow mesenchymal stem cells. In vitro X-ray photoelectron spectroscopy (XPS) revealed that this plasma treatment increases levels of oxygen, rather than nitrogen, on the plasma-treated IP-CHA surface. These findings suggest that plasma treatment, an easy and simple processing, can significantly improve the osteoconductive potential of commonly used artificial bones such as IP-CHA. Further optimization of plasma treatment and longer-term follow-up of in vivo application are required toward its clinical application. PMID:29538457

Chromatin immunoprecipitation in microfluidic droplets: towards fast and cheap analyses.

PubMed

Teste, Bruno; Champ, Jerome; Londono-Vallejo, Arturo; Descroix, Stéphanie; Malaquin, Laurent; Viovy, Jean-Louis; Draskovic, Irena; Mottet, Guillaume

2017-01-31

Genetic organization is governed by the interaction of DNA with histone proteins, and differential modifications of these proteins is a fundamental mechanism of gene regulation. Histone modifications are primarily studied through chromatin immunoprecipitation (ChIP) assays, however conventional ChIP procedures are time consuming, laborious and require a large number of cells. Here we report for the first time the development of ChIP in droplets based on a microfluidic platform combining nanoliter droplets, magnetic beads (MB) and magnetic tweezers (MT). The droplet approach enabled compartmentalization and improved mixing, while reducing the consumption of samples and reagents in an integrated workflow. Anti-histone antibodies grafted to MB were used as a solid support to capture and transfer the target chromatin from droplets to droplets in order to perform chromatin immunoprecipitation, washing, elution and purification of DNA. We designed a new ChIP protocol to investigate four different types of modified histones with known roles in gene activation or repression. We evaluated the performances of this new ChIP in droplet assay in comparison with conventional methods. The proposed technology dramatically reduces analytical time from a few days to 7 hours, simplifies the ChIP protocol and decreases the number of cells required by 100 fold while maintaining a high degree of sensitivity and specificity. Therefore this droplet-based ChIP assay represents a new, highly advantageous and convenient approach to epigenetic analyses.

Epigenetic Library Screen Identifies Abexinostat as Novel Regulator of Adipocytic and Osteoblastic Differentiation of Human Skeletal (Mesenchymal) Stem Cells

PubMed Central

Ali, Dalia; Hamam, Rimi; Alfayez, Musaed; Kassem, Moustapha; Aldahmash, Abdullah

2016-01-01

The epigenetic mechanisms promoting lineage-specific commitment of human skeletal (mesenchymal or stromal) stem cells (hMSCs) into adipocytes or osteoblasts are still not fully understood. Herein, we performed an epigenetic library functional screen and identified several novel compounds, including abexinostat, which promoted adipocytic and osteoblastic differentiation of hMSCs. Using gene expression microarrays, chromatin immunoprecipitation for H3K9Ac combined with high-throughput DNA sequencing (ChIP-seq), and bioinformatics, we identified several key genes involved in regulating stem cell proliferation and differentiation that were targeted by abexinostat. Concordantly, ChIP-quantitative polymerase chain reaction revealed marked increase in H3K9Ac epigenetic mark on the promoter region of AdipoQ, FABP4, PPARγ, KLF15, CEBPA, SP7, and ALPL in abexinostat-treated hMSCs. Pharmacological inhibition of focal adhesion kinase (PF-573228) or insulin-like growth factor-1R/insulin receptor (NVP-AEW51) signaling exhibited significant inhibition of abexinostat-mediated adipocytic differentiation, whereas inhibition of WNT (XAV939) or transforming growth factor-β (SB505124) signaling abrogated abexinostat-mediated osteogenic differentiation of hMSCs. Our findings provide insight into the understanding of the relationship between the epigenetic effect of histone deacetylase inhibitors, transcription factors, and differentiation pathways governing adipocyte and osteoblast differentiation. Manipulating such pathways allows a novel use for epigenetic compounds in hMSC-based therapies and tissue engineering. Significance This unbiased epigenetic library functional screen identified several novel compounds, including abexinostat, that promoted adipocytic and osteoblastic differentiation of human skeletal (mesenchymal or stromal) stem cells (hMSCs). These data provide new insight into the understanding of the relationship between the epigenetic effect of histone deacetylase inhibitors, transcription factors, and differentiation pathways controlling adipocyte and osteoblast differentiation of hMSCs. Manipulating such pathways allows a novel use for epigenetic compounds in hMSC-based therapies for tissue engineering, bone disease, obesity, and metabolic-disorders. PMID:27194745

Calcium Signaling enhancement during oocyte maturation

NASA Astrophysics Data System (ADS)

Jung, Peter; Ullah, Ghanim; Machaca, Khaled

2006-03-01

A Ca2+ signal with a special spatial and temporal characteristic universally removes cell-cycle arrest after fertilization of a mature egg cell. The Ca2+ signal is characterized by a fast rise of intracellular Ca2+ and a slow decay on the time scale of minutes. We use computational modeling of Ca2+ release on the microscale (Ca2+ puffs) and cell-scale in conjunction with experimental knowledge of the changes in the Ca2+ signaling apparatus during oocyte maturation and changing signaling patterns to explore the relationship between organization and sensitivity of IP3 receptors and SERCA pumps and the resulting signaling patterns. We hypothesize that potentiation of the IP3 receptors during oocyte maturation is the main cause for the differentiation in the signaling patterns.

Chromatin immunoprecipitation of mouse embryos.

PubMed

Voss, Anne K; Dixon, Mathew P; McLennan, Tamara; Kueh, Andrew J; Thomas, Tim

2012-01-01

During prenatal development, a large number of different cell types are formed, the vast majority of which contain identical genetic material. The basis of the great variety in cell phenotype and function is the differential expression of the approximately 25,000 genes in the mammalian genome. Transcriptional activity is regulated at many levels by proteins, including members of the basal transcriptional apparatus, DNA-binding transcription factors, and chromatin-binding proteins. Importantly, chromatin structure dictates the availability of a specific genomic locus for transcriptional activation as well as the efficiency, with which transcription can occur. Chromatin immunoprecipitation (ChIP) is a method to assess if chromatin modifications or proteins are present at a specific locus. ChIP involves the cross linking of DNA and associated proteins and immunoprecipitation using specific antibodies to DNA-associated proteins followed by examination of the co-precipitated DNA sequences or proteins. In the last few years, ChIP has become an essential technique for scientists studying transcriptional regulation and chromatin structure. Using ChIP on mouse embryos, we can document the presence or absence of specific proteins and chromatin modifications at genomic loci in vivo during mammalian development. Here, we describe a ChIP technique adapted for mouse embryos.

Screening of drugs to treat 8p11 myeloproliferative syndrome using patient-derived induced pluripotent stem cells with fusion gene CEP110-FGFR1.

PubMed

Yamamoto, Shohei; Otsu, Makoto; Matsuzaka, Emiko; Konishi, Chieko; Takagi, Haruna; Hanada, Sachiyo; Mochizuki, Shinji; Nakauchi, Hiromitsu; Imai, Kohzoh; Tsuji, Kohichiro; Ebihara, Yasuhiro

2015-01-01

Induced pluripotent stem (iPS) cells provide powerful tools for studying disease mechanisms and developing therapies for diseases. The 8p11 myeloproliferative syndrome (EMS) is an aggressive chronic myeloproliferative disorder (MPD) that is caused by constitutive activation of fibroblast growth factor receptor 1. EMS is rare and, consequently, effective treatment for this disease has not been established. Here, iPS cells were generated from an EMS patient (EMS-iPS cells) to assist the development of effective therapies for EMS. When iPS cells were co-cultured with murine embryonic stromal cells, EMS-iPS cells produced more hematopoietic progenitor and hematopoietic cells, and CD34+ cells derived from EMS-iPS cells exhibited 3.2-7.2-fold more macrophage and erythroid colony forming units (CFUs) than those derived from control iPS cells. These data indicate that EMS-iPS cells have an increased hematopoietic differentiation capacity, which is characteristic of MPDs. To determine whether a tyrosine kinase inhibitor (TKI) could suppress the increased number of CFUs formed by EMS-iPS-induced CD34+ cells, cells were treated with one of four TKIs (CHIR258, PKC 412, ponatinib, and imatinib). CHIR258, PKC 412, and ponatinib reduced the number of CFUs formed by EMS-iPS-induced CD34+ cells in a dose-dependent manner, whereas imatinib did not. Similar effects were observed on primary peripheral blood cells (more than 90% of which were blasts) isolated from the patient. This study provides evidence that the EMS-iPS cell line is a useful tool for the screening of drugs to treat EMS and to investigate the mechanism underlying this disease.

Ultrastructural demonstration of Cx43 gap junctions in induced pluripotent stem cells from human cord blood.

PubMed

Beckmann, Anja; Schubert, Madline; Hainz, Nadine; Haase, Alexandra; Martin, Ulrich; Tschernig, Thomas; Meier, Carola

2016-11-01

Gap junction proteins are essential for direct intercellular communication but also influence cellular differentiation and migration. The expression of various connexin gap junction proteins has been demonstrated in embryonic stem cells, with Cx43 being the most intensely studied. As Cx43 is the most prominent gap junction protein in the heart, cardiomyocyte-differentiated stem cells have been studied intensely. To date, however, little is known about the expression and the subcellular distribution of Cx43 in undifferentiated stem cells or about the structural arrangement of channels. We, therefore, here investigate expression of Cx43 in undifferentiated human cord-blood-derived induced pluripotent stem cells (hCBiPS2). For this purpose, we carried out quantitative real-time PCR and immunohistochemistry. For analysis of Cx43 ultrastructure and protein assembly, we performed freeze-fracture replica immunogold labeling (FRIL). Cx43 expression was detected at mRNA and protein level in hCBIPS2 cells. For the first time, ultrastructural data are presented on gap junction morphology in induced pluripotent stem (iPS) cells from cord blood: Our FRIL and electron microscopical analysis revealed the occurrence of gap junction plaques in undifferentiated iPS cells. In addition, these gap junctions were shown to contain the gap junction protein Cx43.

Application of Stem Cells in Oral Disease Therapy: Progresses and Perspectives

PubMed Central

Yang, Bo; Qiu, Yi; Zhou, Niu; Ouyang, Hong; Ding, Junjun; Cheng, Bin; Sun, Jianbo

2017-01-01

Stem cells are undifferentiated and pluripotent cells that can differentiate into specialized cells with a more specific function. Stem cell therapies become preferred methods for the treatment of multiple diseases. Oral and maxillofacial defect is one kind of the diseases that could be most possibly cured by stem cell therapies. Here we discussed oral diseases, oral adult stem cells, iPS cells, and the progresses/challenges/perspectives of application of stem cells for oral disease treatment. PMID:28421002

The Role of NG2 Glial Cells in ALS Pathogenesis

DTIC Science & Technology

2013-10-01

line of OPC differentiation from iPS cells. SHH, sonic hedgehog ; RA, retinoitic acid; bFGF, basic FGF; PDGF, platelet-derived growth factor; IGF...University School of Medicine, Baltimore, Maryland, USA. 3Department of Anatomy , Kitasato University School of Medicine, Sagamihara, Japan. 4Brain Science...6Present address: Shriners Hospital Pediatric Research Center, Department of Anatomy and Cell Biology, Temple University School of Medicine

Efficient programming of human eye conjunctiva-derived induced pluripotent stem (ECiPS) cells into definitive endoderm-like cells.

PubMed

Massumi, Mohammad; Hoveizi, Elham; Baktash, Parvaneh; Hooti, Abdollah; Ghazizadeh, Leili; Nadri, Samad; Pourasgari, Farzaneh; Hajarizadeh, Athena; Soleimani, Masoud; Nabiuni, Mohammad; Khorramizadeh, Mohammad R

2014-03-10

Due to pluripotency of induced pluripotent stem (iPS) cells, and the lack of immunological incompatibility and ethical issues, iPS cells have been considered as an invaluable cell source for future cell replacement therapy. This study was aimed first at establishment of novel iPS cells, ECiPS, which directly reprogrammed from human Eye Conjunctiva-derived Mesenchymal Stem Cells (EC-MSCs); second, comparing the inductive effects of Wnt3a/Activin A biomolecules to IDE1 small molecule in derivation of definitive endoderm (DE) from the ECiPS cells. To that end, first, the EC-MSCs were transduced by SOKM-expressing lentiviruses and characterized for endogenous expression of embryonic markers Then the established ECiPS cells were induced to DE formation by Wnt3a/Activin A or IDE1. Quantification of GSC, Sox17 and Foxa2 expression, as DE-specific markers, in both mRNA and protein levels revealed that induction of ECiPS cells by either Wnt3a/Activin A or IDE1 could enhance the expression level of the genes; however the levels of increase were higher in Wnt3a/Activin A induced ECiPS-EBs than IDE1 induced cells. Furthermore, the flow cytometry analyses showed no synergistic effect between Activin A and Wnt3a to derive DE-like cells from ECiPS cells. The comparative findings suggest that although both Wnt3a/Activin A signaling and IDE1 molecule could be used for differentiation of iPS into DE cells, the DE-inducing effect of Wnt3a/Activin A was statistically higher than IDE1. Copyright © 2014 Elsevier Inc. All rights reserved.

Induced pluripotent stem cells with a pathological mitochondrial DNA deletion

PubMed Central

Cherry, Anne B. C.; Gagne, Katelyn E.; McLoughlin, Erin M.; Baccei, Anna; Gorman, Bryan; Hartung, Odelya; Miller, Justine D.; Zhang, Jin; Zon, Rebecca L.; Ince, Tan A.; Neufeld, Ellis J.; Lerou, Paul H.; Fleming, Mark D.; Daley, George Q.; Agarwal, Suneet

2013-01-01

In congenital mitochondrial DNA (mtDNA) disorders, a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues, which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown, and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders, as cytoplasmic genetic material is retained during direct reprogramming. Here we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage, we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth, mitochondrial function, and hematopoietic phenotype when differentiated in vitro, compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases. PMID:23400930

Premyogenic progenitors derived from human pluripotent stem cells expand in floating culture and differentiate into transplantable myogenic progenitors.

PubMed

Sakai-Takemura, Fusako; Narita, Asako; Masuda, Satoru; Wakamatsu, Toshifumi; Watanabe, Nobuharu; Nishiyama, Takashi; Nogami, Ken'ichiro; Blanc, Matthias; Takeda, Shin'ichi; Miyagoe-Suzuki, Yuko

2018-04-26

Human induced pluripotent stem cells (hiPSCs) are a potential source for cell therapy of Duchenne muscular dystrophy. To reliably obtain skeletal muscle progenitors from hiPSCs, we treated hiPS cells with a Wnt activator, CHIR-99021 and a BMP receptor inhibitor, LDN-193189, and then induced skeletal muscle cells using a previously reported sphere-based culture. This protocol greatly improved sphere formation efficiency and stably induced the differentiation of myogenic cells from hiPS cells generated from both healthy donors and a patient with congenital myasthenic syndrome. hiPSC-derived myogenic progenitors were enriched in the CD57(-) CD108(-) CD271(+) ERBB3(+) cell fraction, and their differentiation was greatly promoted by TGF-β inhibitors. TGF-β inhibitors down-regulated the NFIX transcription factor, and NFIX short hairpin RNA (shRNA) improved the differentiation of iPS cell-derived myogenic progenitors. These results suggest that NFIX inhibited differentiation of myogenic progenitors. hiPSC-derived myogenic cells differentiated into myofibers in muscles of NSG-mdx 4Cv mice after direct transplantation. Our results indicate that our new muscle induction protocol is useful for cell therapy of muscular dystrophies.

Inositol 1,4,5-trisphosphate receptor regulates replication, differentiation, infectivity and virulence of the parasitic protist Trypanosoma cruzi.

PubMed

Hashimoto, Muneaki; Enomoto, Masahiro; Morales, Jorge; Kurebayashi, Nagomi; Sakurai, Takashi; Hashimoto, Tetsuo; Nara, Takeshi; Mikoshiba, Katsuhiko

2013-03-01

In animals, inositol 1,4,5-trisphosphate receptors (IP3 Rs) are ion channels that play a pivotal role in many biological processes by mediating Ca(2+) release from the endoplasmic reticulum. Here, we report the identification and characterization of a novel IP3 R in the parasitic protist, Trypanosoma cruzi, the pathogen responsible for Chagas disease. DT40 cells lacking endogenous IP3 R genes expressing T. cruzi IP3 R (TcIP3 R) exhibited IP3 -mediated Ca(2+) release from the ER, and demonstrated receptor binding to IP3 . TcIP3 R was expressed throughout the parasite life cycle but the expression level was much lower in bloodstream trypomastigotes than in intracellular amastigotes or epimastigotes. Disruption of two of the three TcIP3 R gene loci led to the death of the parasite, suggesting that IP3 R is essential for T. cruzi. Parasites expressing reduced or increased levels of TcIP3 R displayed defects in growth, transformation and infectivity, indicating that TcIP3 R is an important regulator of the parasite's life cycle. Furthermore, mice infected with T. cruzi expressing reduced levels of TcIP3 R exhibited a reduction of disease symptoms, indicating that TcIP3 R is an important virulence factor. Combined with the fact that the primary structure of TcIP3 R has low similarity to that of mammalian IP3 Rs, TcIP3 R is a promising drug target for Chagas disease. © 2013 Blackwell Publishing Ltd.

A demonstration of the H3 trimethylation ChIP-seq analysis of galline follicular mesenchymal cells and male germ cells.

PubMed

Chokeshaiusaha, Kaj; Puthier, Denis; Nguyen, Catherine; Sananmuang, Thanida

2018-06-01

Trimethylation of histone 3 (H3) at 4th lysine N-termini (H3K4me3) in gene promoter region was the universal marker of active genes specific to cell lineage. On the contrary, coexistence of trimethylation at 27th lysine (H3K27me3) in the same loci-the bivalent H3K4m3/H3K27me3 was known to suspend the gene transcription in germ cells, and could also be inherited to the developed stem cell. In galline species, throughout example of H3K4m3 and H3K27me3 ChIP-seq analysis was still not provided. We therefore designed and demonstrated such procedures using ChIP-seq and mRNA-seq data of chicken follicular mesenchymal cells and male germ cells. Analytical workflow was designed and provided in this study. ChIP-seq and RNA-seq datasets of follicular mesenchymal cells and male germ cells were acquired and properly preprocessed. Peak calling by Model-based analysis of ChIP-seq 2 was performed to identify H3K4m3 or H3K27me3 enriched regions (Fold-change≥2, FDR≤0.01) in gene promoter regions. Integrative genomics viewer was utilized for cellular retinoic acid binding protein 1 ( CRABP1 ), growth differentiation factor 10 ( GDF10 ), and gremlin 1 ( GREM1 ) gene explorations. The acquired results indicated that follicular mesenchymal cells and germ cells shared several unique gene promoter regions enriched with H3K4me3 (5,704 peaks) and also unique regions of bivalent H3K4m3/H3K27me3 shared between all cell types and germ cells (1,909 peaks). Subsequent observation of follicular mesenchyme-specific genes- CRABP1 , GDF10 , and GREM1 correctly revealed vigorous transcriptions of these genes in follicular mesenchymal cells. As expected, bivalent H3K4m3/H3K27me3 pattern was manifested in gene promoter regions of germ cells, and thus suspended their transcriptions. According the results, an example of chicken H3K4m3/H3K27me3 ChIP-seq data analysis was successfully demonstrated in this study. Hopefully, the provided methodology should hereby be useful for galline ChIP-seq data analysis in the future.

Generation of induced pluripotent stem cells derived from an autosomal dominant polycystic kidney disease patient with a p.Ser1457fs mutation in PKD1.

PubMed

Huang, Ching-Ying; Ho, Ming-Ching; Lee, Jia-Jung; Hwang, Daw-Yang; Ko, Hui-Wen; Cheng, Yu-Che; Hsu, Yu-Hung; Lu, Huai-En; Chen, Hung-Chun; Hsieh, Patrick C H

2017-10-01

Autosomal dominant polycystic kidney disease is one of the most prevalent forms of inherited cystic kidney disease, and can be characterized by kidney cyst formation and enlargement. Here we report the generation of a Type 1 ADPKD disease iPS cell line, IBMS-iPSC-012-12, which retains the conserved deletion of PKD1, normal karyotype and exhibits the properties of pluripotent stem cells such as ES-like morphology, expression of pluripotent markers and capacity to differentiate into all three germ layers. Our results show that we have successfully generated a patient-specific iPS cell line with a mutation in PKD1 for study of renal disease pathophysiology. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

GluD1 is a common altered player in neuronal differentiation from both MECP2-mutated and CDKL5-mutated iPS cells.

PubMed

Livide, Gabriella; Patriarchi, Tommaso; Amenduni, Mariangela; Amabile, Sonia; Yasui, Dag; Calcagno, Eleonora; Lo Rizzo, Caterina; De Falco, Giulia; Ulivieri, Cristina; Ariani, Francesca; Mari, Francesca; Mencarelli, Maria Antonietta; Hell, Johannes Wilhelm; Renieri, Alessandra; Meloni, Ilaria

2015-02-01

Rett syndrome is a monogenic disease due to de novo mutations in either MECP2 or CDKL5 genes. In spite of their involvement in the same disease, a functional interaction between the two genes has not been proven. MeCP2 is a transcriptional regulator; CDKL5 encodes for a kinase protein that might be involved in the regulation of gene expression. Therefore, we hypothesized that mutations affecting the two genes may lead to similar phenotypes by dysregulating the expression of common genes. To test this hypothesis we used induced pluripotent stem (iPS) cells derived from fibroblasts of one Rett patient with a MECP2 mutation (p.Arg306Cys) and two patients with mutations in CDKL5 (p.Gln347Ter and p.Thr288Ile). Expression profiling was performed in CDKL5-mutated cells and genes of interest were confirmed by real-time RT-PCR in both CDKL5- and MECP2-mutated cells. The only major change in gene expression common to MECP2- and CDKL5-mutated cells was for GRID1, encoding for glutamate D1 receptor (GluD1), a member of the δ-family of ionotropic glutamate receptors. GluD1 does not form AMPA or NMDA glutamate receptors. It acts like an adhesion molecule by linking the postsynaptic and presynaptic compartments, preferentially inducing the inhibitory presynaptic differentiation of cortical neurons. Our results demonstrate that GRID1 expression is downregulated in both MECP2- and CDKL5-mutated iPS cells and upregulated in neuronal precursors and mature neurons. These data provide novel insights into disease pathophysiology and identify possible new targets for therapeutic treatment of Rett syndrome.

GluD1 is a common altered player in neuronal differentiation from both MECP2-mutated and CDKL5-mutated iPS cells

PubMed Central

Livide, Gabriella; Patriarchi, Tommaso; Amenduni, Mariangela; Amabile, Sonia; Yasui, Dag; Calcagno, Eleonora; Lo Rizzo, Caterina; De Falco, Giulia; Ulivieri, Cristina; Ariani, Francesca; Mari, Francesca; Mencarelli, Maria Antonietta; Hell, Johannes Wilhelm; Renieri, Alessandra; Meloni, Ilaria

2015-01-01

Rett syndrome is a monogenic disease due to de novo mutations in either MECP2 or CDKL5 genes. In spite of their involvement in the same disease, a functional interaction between the two genes has not been proven. MeCP2 is a transcriptional regulator; CDKL5 encodes for a kinase protein that might be involved in the regulation of gene expression. Therefore, we hypothesized that mutations affecting the two genes may lead to similar phenotypes by dysregulating the expression of common genes. To test this hypothesis we used induced pluripotent stem (iPS) cells derived from fibroblasts of one Rett patient with a MECP2 mutation (p.Arg306Cys) and two patients with mutations in CDKL5 (p.Gln347Ter and p.Thr288Ile). Expression profiling was performed in CDKL5-mutated cells and genes of interest were confirmed by real-time RT-PCR in both CDKL5- and MECP2-mutated cells. The only major change in gene expression common to MECP2- and CDKL5-mutated cells was for GRID1, encoding for glutamate D1 receptor (GluD1), a member of the δ-family of ionotropic glutamate receptors. GluD1 does not form AMPA or NMDA glutamate receptors. It acts like an adhesion molecule by linking the postsynaptic and presynaptic compartments, preferentially inducing the inhibitory presynaptic differentiation of cortical neurons. Our results demonstrate that GRID1 expression is downregulated in both MECP2- and CDKL5-mutated iPS cells and upregulated in neuronal precursors and mature neurons. These data provide novel insights into disease pathophysiology and identify possible new targets for therapeutic treatment of Rett syndrome. PMID:24916645

Downstream targets of HOXB4 in a cell line model of primitive hematopoietic progenitor cells.

PubMed

Lee, Han M; Zhang, Hui; Schulz, Vincent; Tuck, David P; Forget, Bernard G

2010-08-05

Enforced expression of the homeobox transcription factor HOXB4 has been shown to enhance hematopoietic stem cell self-renewal and expansion ex vivo and in vivo. To investigate the downstream targets of HOXB4 in hematopoietic progenitor cells, HOXB4 was constitutively overexpressed in the primitive hematopoietic progenitor cell line EML. Two genome-wide analytical techniques were used: RNA expression profiling using microarrays and chromatin immunoprecipitation (ChIP)-chip. RNA expression profiling revealed that 465 gene transcripts were differentially expressed in KLS (c-Kit(+), Lin(-), Sca-1(+))-EML cells that overexpressed HOXB4 (KLS-EML-HOXB4) compared with control KLS-EML cells that were transduced with vector alone. In particular, erythroid-specific gene transcripts were observed to be highly down-regulated in KLS-EML-HOXB4 cells. ChIP-chip analysis revealed that the promoter region for 1910 genes, such as CD34, Sox4, and B220, were occupied by HOXB4 in KLS-EML-HOXB4 cells. Side-by-side comparison of the ChIP-chip and RNA expression profiling datasets provided correlative information and identified Gp49a and Laptm4b as candidate "stemness-related" genes. Both genes were highly ranked in both dataset lists and have been previously shown to be preferentially expressed in hematopoietic stem cells and down-regulated in mature hematopoietic cells, thus making them attractive candidates for future functional studies in hematopoietic cells.

Mitochondrial DNA copy number is regulated in a tissue specific manner by DNA methylation of the nuclear-encoded DNA polymerase gamma A

PubMed Central

Kelly, Richard D. W.; Mahmud, Arsalan; McKenzie, Matthew; Trounce, Ian A.; St John, Justin C.

2012-01-01

DNA methylation is an essential mechanism controlling gene expression during differentiation and development. We investigated the epigenetic regulation of the nuclear-encoded, mitochondrial DNA (mtDNA) polymerase γ catalytic subunit (PolgA) by examining the methylation status of a CpG island within exon 2 of PolgA. Bisulphite sequencing identified low methylation levels (<10%) within exon 2 of mouse oocytes, blastocysts and embryonic stem cells (ESCs), while somatic tissues contained significantly higher levels (>40%). In contrast, induced pluripotent stem (iPS) cells and somatic nuclear transfer ESCs were hypermethylated (>20%), indicating abnormal epigenetic reprogramming. Real time PCR analysis of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) immunoprecipitated DNA suggests active DNA methylation and demethylation within exon 2 of PolgA. Moreover, neural differentiation of ESCs promoted de novo methylation and demethylation at the exon 2 locus. Regression analysis demonstrates that cell-specific PolgA expression levels were negatively correlated with DNA methylation within exon 2 and mtDNA copy number. Finally, using chromatin immunoprecipitation (ChIP) against RNA polymerase II (RNApII) phosphorylated on serine 2, we show increased DNA methylation levels are associated with reduced RNApII transcriptional elongation. This is the first study linking nuclear DNA epigenetic regulation with mtDNA regulation during differentiation and cell specialization. PMID:22941637

Loss of heme oxygenase-1 accelerates mesodermal gene expressions during embryoid body development from mouse embryonic stem cells.

PubMed

Lai, Yan-Liang; Lin, Chen-Yu; Jiang, Wei-Cheng; Ho, Yen-Chun; Chen, Chung-Huang; Yet, Shaw-Fang

2018-05-01

Heme oxygenase (HO)-1 is an inducible stress response protein and well known to protect cells and tissues against injury. Despite its important function in cytoprotection against physiological stress, the role of HO-1 in embryonic stem cell (ESC) differentiation remains largely unknown. We showed previously that induced pluripotent stem (iPS) cells that lack HO-1 are more sensitive to oxidant stress-induced cell death and more prone to lose pluripotent markers upon LIF withdrawal. To elucidate the role of HO-1 in ESC differentiation and to rule out the controversy of potential gene flaws in iPS cells, we derived and established mouse HO-1 knockout ESC lines from HO-1 knockout blastocysts. Using wild type D3 and HO-1 knockout ESCs in the 3-dimensional embryoid body (EB) differentiation model, we showed that at an early time point during EB development, an absence of HO-1 led to enhanced ROS level, concomitant with increased expressions of master mesodermal regulator brachyury and endodermal marker GATA6. In addition, critical smooth muscle cell (SMC) transcription factor serum response factor and its coactivator myocardin were enhanced. Furthermore, HO-1 deficiency increased Smad2 in ESCs and EBs, revealing a role of HO-1 in controlling Smad2 level. Smad2 not only mediates mesendoderm differentiation of mouse ESCs but also SMC development. Collectively, loss of HO-1 resulted in higher level of mesodermal and SMC regulators, leading to accelerated and enhanced SMC marker SM α-actin expression. Our results reveal a previously unrecognized function of HO-1 in regulating SMC gene expressions during ESC-EB development. More importantly, our findings may provide a novel strategy in enhancing ESC differentiation toward SMC lineage. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Regulation of H3K4me3 at Transcriptional Enhancers Characterizes Acquisition of Virus-Specific CD8+ T Cell-Lineage-Specific Function.

PubMed

Russ, Brendan E; Olshansky, Moshe; Li, Jasmine; Nguyen, Michelle L T; Gearing, Linden J; Nguyen, Thi H O; Olson, Matthew R; McQuilton, Hayley A; Nüssing, Simone; Khoury, Georges; Purcell, Damian F J; Hertzog, Paul J; Rao, Sudha; Turner, Stephen J

2017-12-19

Infection triggers large-scale changes in the phenotype and function of T cells that are critical for immune clearance, yet the gene regulatory mechanisms that control these changes are largely unknown. Using ChIP-seq for specific histone post-translational modifications (PTMs), we mapped the dynamics of ∼25,000 putative CD8 + T cell transcriptional enhancers (TEs) differentially utilized during virus-specific T cell differentiation. Interestingly, we identified a subset of dynamically regulated TEs that exhibited acquisition of a non-canonical (H3K4me3 + ) chromatin signature upon differentiation. This unique TE subset exhibited characteristics of poised enhancers in the naive CD8 + T cell subset and demonstrated enrichment for transcription factor binding motifs known to be important for virus-specific CD8 + T cell differentiation. These data provide insights into the establishment and maintenance of the gene transcription profiles that define each stage of virus-specific T cell differentiation. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Demethylation of induced pluripotent stem cells from type 1 diabetic patients enhances differentiation into functional pancreatic β cells.

PubMed

Manzar, Gohar S; Kim, Eun-Mi; Zavazava, Nicholas

2017-08-25

Type 1 diabetes (T1D) can be managed by transplanting either the whole pancreas or isolated pancreatic islets. However, cadaveric pancreas is scarcely available for clinical use, limiting this approach. As such, there is a great need to identify alternative sources of clinically usable pancreatic tissues. Here, we used induced pluripotent stem (iPS) cells derived from patients with T1D to generate glucose-responsive, insulin-producing cells (IPCs) via 3D culture. Initially, T1D iPS cells were resistant to differentiation, but transient demethylation treatment significantly enhanced IPC yield. The cells responded to high-glucose stimulation by secreting insulin in vitro The shape, size, and number of their granules, as observed by transmission electron microscopy, were identical to those found in cadaveric β cells. When the IPCs were transplanted into immunodeficient mice that had developed streptozotocin-induced diabetes, they promoted a dramatic decrease in hyperglycemia, causing the mice to become normoglycemic within 28 days. None of the mice died or developed teratomas. Because the cells are derived from "self," immunosuppression is not required, providing a much safer and reliable treatment option for T1D patients. Moreover, these cells can be used for drug screening, thereby accelerating drug discovery. In conclusion, our approach eliminates the need for cadaveric pancreatic tissue.

New Epigenetic Therapeutic Intervention for Metastatic Breast Cancer

DTIC Science & Technology

2017-04-01

papers as listed in 3.4B. Article Distinct Roles of Brd2 and Brd4 in Potentiating the Transcriptional Program for Th17 Cell Differentiation Graphical...this paper and include the following:d KEY RESOURCES TABLE d CONTACT FOR REAGENT AND RESOURCE SHARING d EXPERIMENTAL MODEL AND SUBJECT DETAILS d METHOD...Brd4, Smc1, Smc3, Nipbl, H3K27ac, H3K4me1 ChIP-seq data This paper GEO: GSE90788 and GSE63778 Other ChIP-seq data Ciofani et al., 2012 GEO: GSE40918

Haploinsufficiency of BAZ1B contributes to Williams syndrome through transcriptional dysregulation of neurodevelopmental pathways.

PubMed

Lalli, Matthew A; Jang, Jiwon; Park, Joo-Hye C; Wang, Yidi; Guzman, Elmer; Zhou, Hongjun; Audouard, Morgane; Bridges, Daniel; Tovar, Kenneth R; Papuc, Sorina M; Tutulan-Cunita, Andreea C; Huang, Yadong; Budisteanu, Magdalena; Arghir, Aurora; Kosik, Kenneth S

2016-04-01

Williams syndrome (WS) is a neurodevelopmental disorder caused by a genomic deletion of ∼28 genes that results in a cognitive and behavioral profile marked by overall intellectual impairment with relative strength in expressive language and hypersocial behavior. Advancements in protocols for neuron differentiation from induced pluripotent stem cells allowed us to elucidate the molecular circuitry underpinning the ontogeny of WS. In patient-derived stem cells and neurons, we determined the expression profile of the Williams-Beuren syndrome critical region-deleted genes and the genome-wide transcriptional consequences of the hemizygous genomic microdeletion at chromosome 7q11.23. Derived neurons displayed disease-relevant hallmarks and indicated novel aberrant pathways in WS neurons including over-activated Wnt signaling accompanying an incomplete neurogenic commitment. We show that haploinsufficiency of the ATP-dependent chromatin remodeler, BAZ1B, which is deleted in WS, significantly contributes to this differentiation defect. Chromatin-immunoprecipitation (ChIP-seq) revealed BAZ1B target gene functions are enriched for neurogenesis, neuron differentiation and disease-relevant phenotypes. BAZ1B haploinsufficiency caused widespread gene expression changes in neural progenitor cells, and together with BAZ1B ChIP-seq target genes, explained 42% of the transcriptional dysregulation in WS neurons. BAZ1B contributes to regulating the balance between neural precursor self-renewal and differentiation and the differentiation defect caused by BAZ1B haploinsufficiency can be rescued by mitigating over-active Wnt signaling in neural stem cells. Altogether, these results reveal a pivotal role for BAZ1B in neurodevelopment and implicate its haploinsufficiency as a likely contributor to the neurological phenotypes in WS. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Disruption of dopamine D1/D2 receptor complex is involved in the function of haloperidol in cardiac H9c2 cells.

PubMed

Lencesova, L; Szadvari, I; Babula, P; Kubickova, J; Chovancova, B; Lopusna, K; Rezuchova, I; Novakova, Z; Krizanova, O; Novakova, M

2017-12-15

Haloperidol is an antipsychotic agent and acts as dopamine D2 receptor (D2R) antagonist, as a prototypical ligand of sigma1 receptors (Sig1R) and it increases expression of type 1 IP 3 receptors (IP 3 R1). However, precise mechanism of haloperidol action on cardiomyocytes through dopaminergic signaling was not described yet. This study investigated a role of dopamine receptors in haloperidol-induced increase in IP 3 R1 and Sig1R, and compared physiological effect of melperone and haloperidol on basic heart parameters in rats. We used differentiated NG-108 cells and H9c2 cells. Gene expression, Western blot and immunofluorescence were used to evaluate haloperidol-induced differences; proximity ligation assay (PLA) and immunoprecipitation to determine interactions of D1/D2 receptors. To evaluate cardiac parameters, Wistar albino male rats were used. We have shown that antagonism of D2R with either haloperidol or melperone results in upregulation of both, IP 3 R1 and Sig1R, which is associated with increased D2R, but reduced D1R expression. Immunofluorescence, immunoprecipitation and PLA support formation of heteromeric D1/D2 complexes in H9c2 cells. Treatment with haloperidol (but not melperone) caused decrease in systolic and diastolic blood pressure and significant increase in heart rate. Because D1R/D2R complexes can engage Gq-like signaling in other experimental systems, these results are consistent with the possibility that disruption of D1R/D2R complex in H9c2 cells might cause a decrease in IP 3 R1 activity, which in turn may account for the increase expression of IP 3 R and Sig1R. D2R is probably not responsible for changes in cardiac parameters, since melperone did not have any effect. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

BloodChIP: a database of comparative genome-wide transcription factor binding profiles in human blood cells.

PubMed

Chacon, Diego; Beck, Dominik; Perera, Dilmi; Wong, Jason W H; Pimanda, John E

2014-01-01

The BloodChIP database (http://www.med.unsw.edu.au/CRCWeb.nsf/page/BloodChIP) supports exploration and visualization of combinatorial transcription factor (TF) binding at a particular locus in human CD34-positive and other normal and leukaemic cells or retrieval of target gene sets for user-defined combinations of TFs across one or more cell types. Increasing numbers of genome-wide TF binding profiles are being added to public repositories, and this trend is likely to continue. For the power of these data sets to be fully harnessed by experimental scientists, there is a need for these data to be placed in context and easily accessible for downstream applications. To this end, we have built a user-friendly database that has at its core the genome-wide binding profiles of seven key haematopoietic TFs in human stem/progenitor cells. These binding profiles are compared with binding profiles in normal differentiated and leukaemic cells. We have integrated these TF binding profiles with chromatin marks and expression data in normal and leukaemic cell fractions. All queries can be exported into external sites to construct TF-gene and protein-protein networks and to evaluate the association of genes with cellular processes and tissue expression.

Reprogramming of the MHC-I and its regulation by NFκB in human-induced pluripotent stem cells.

PubMed

Pick, Marjorie; Ronen, Daniel; Yanuka, Ofra; Benvenisty, Nissim

2012-12-01

The immunogenicity of human pluripotent stem cells plays a major role in their potential use in the clinic. We show that, during their reprogramming, human-induced pluripotent stem (iPS) cells downregulate expression of human leukocyte antigen (HLA)-A/B/C and β2 microglobulin (β2M), the two components of major histocompatibility complex-I (MHC-I). MHC-I expression in iPS cells can be restored by differentiation or treatment with interferon-gamma (IFNγ). To analyze the molecular mechanisms that regulate the expression of the MHC-I molecules in human iPS cells, we searched for correlation between the expression of HLA-A/B/C and β2M and the expression of transcription factors that bind to the promoter of these genes. Our results show a significant positive correlation between MHC-I expression and expression of the nuclear factors, nuclear factor kappa B 1 (NFκB1) and RelA, at the levels of RNA, protein and was confirmed by chromatin binding. Concordantly, we detected robust levels of NFκB1 and RelA proteins in the nucleus of somatic cells but not in the iPS cell derived from them. Overexpression of NFκB1 and RelA in undifferentiated pluripotent stem cells led to induction in expression of MHC-I, whereas silencing NFκB1 and RelA by small hairpin RNA decreased the expression of β2M after IFNγ treatment. Our data point to the critical role of NFκB proteins in regulating the MHC-I expression in human pluripotent stem cells. Copyright © 2012 AlphaMed Press.

Derivation of Skeletal Myogenic Precursors from Human Pluripotent Stem Cells Using Conditional Expression of PAX7.

PubMed

Darabi, Radbod; Perlingeiro, Rita C R

2016-01-01

Cell-based therapies are considered as one of the most promising approaches for the treatment of degenerating pathologies including muscle disorders and dystrophies. Advances in the approach of reprogramming somatic cells into induced pluripotent stem (iPS) cells allow for the possibility of using the patient's own pluripotent cells to generate specific tissues for autologous transplantation. In addition, patient-specific tissue derivatives have been shown to represent valuable material for disease modeling and drug discovery. Nevertheless, directed differentiation of pluripotent stem cells into a specific lineage is not a trivial task especially in the case of skeletal myogenesis, which is generally poorly recapitulated during the in vitro differentiation of pluripotent stem cells.Here, we describe a practical and efficient method for the derivation of skeletal myogenic precursors from differentiating human pluripotent stem cells using controlled expression of PAX7. Flow cytometry (FACS) purified myogenic precursors can be expanded exponentially and differentiated in vitro into myotubes, enabling researchers to use these cells for disease modeling as well as therapeutic purposes.

Phosphatidylcholine-specific phospholipase C inhibition reduces HER2-overexpression, cell proliferation and in vivo tumor growth in a highly tumorigenic ovarian cancer model

PubMed Central

Spadaro, Francesca; Abalsamo, Laura; Pisanu, Maria Elena; Ricci, Alessandro; Cecchetti, Serena; Altabella, Luisa; Buoncervello, Maria; Lozneanu, Ludmila; Bagnoli, Marina; Ramoni, Carlo; Canevari, Silvana; Mezzanzanica, Delia

2017-01-01

Antagonizing the oncogenic effects of human epidermal growth factor receptor 2 (HER2) with current anti-HER2 agents has not yet yielded major progress in the treatment of advanced HER2-positive epithelial ovarian cancer (EOC). Using preclinical models to explore alternative molecular mechanisms affecting HER2 overexpression and oncogenicity may lead to new strategies for EOC patient treatment. We previously reported that phosphatidylcholine-specific phospholipase C (PC-PLC) exerts a pivotal role in regulating HER2 overexpression in breast cancer cells. The present study, conducted on two human HER2-overexpressing EOC cell lines - SKOV3 and its in vivo-passaged SKOV3.ip cell variant characterized by enhanced in vivo tumorigenicity - and on SKOV3.ip xenografts implanted in SCID mice, showed: a) about 2-fold higher PC-PLC and HER2 protein expression levels in SKOV3.ip compared to SKOV3 cells; b) physical association of PC-PLC with HER2 in non-raft domains; c) HER2 internalization and ca. 50% reduction of HER2 mRNA and protein expression levels in SKOV3.ip cells exposed to the PC-PLC inhibitor tricyclodecan-9-yl-potassium xanthate (D609); d) differential effects of D609 and trastuzumab on HER2 protein expression and cell proliferation; e) decreased in vivo tumor growth in SKOV3.ip xenografts during in vivo treatment with D609; f) potential use of in vivo magnetic resonance spectroscopy (MRS) and imaging (MRI) parameters as biomarkers of EOC response to PC-PLC inhibition. Overall, these findings support the view that PC-PLC inhibition may represent an effective means to target the tumorigenic effects of HER2 overexpression in EOC and that in vivo MR approaches can efficiently monitor its effects. PMID:28903399

Functional Significance of VEGFR-2 on Ovarian Cancer Cells

PubMed Central

Spannuth, Whitney A.; Nick, Alpa M.; Jennings, Nicholas B.; Armaiz-Pena, Guillermo N.; Mangala, Lingegowda S.; Danes, Christopher G.; Lin, Yvonne G.; Merritt, William M.; Thaker, Premal H.; Kamat, Aparna A.; Han, Liz Y.; Tonra, James R.; Coleman, Robert L.; Ellis, Lee M.; Sood, Anil K.

2009-01-01

Vascular endothelial growth factor receptor (VEGFR) has recently been discovered on ovarian cancer cells, but its functional significance is unknown and is the focus of the current study. By protein analysis, A2780-par and HeyA8 ovarian cancer cell lines expressed VEGFR-1 and HeyA8 and SKOV3ip1 expressed VEGFR-2. By in situ hybridization (ISH), 85% of human ovarian cancer specimens showed moderate to high VEGFR-2 expression while only 15% showed moderate to high VEGFR-1 expression. By immunofluorescence, little or no VEGFR-2 was detected in normal ovarian surface epithelial cells, whereas expression was detected in 75% of invasive ovarian cancer specimens. To differentiate between the effects of tumor versus host expression of VEGFR, nude mice were injected with SKOV3ip1 cells and treated with either human VEGFR-2 specific antibody (1121B), murine VEGFR-2 specific antibody (DC101), or the combination. Treatment with 1121B reduced SKOV3ip1 cell migration by 68% (p < 0.01) and invasion by 72% (p < 0.01), but exposure to VEGFR-1 antibody had no effect. Treatment with 1121B effectively blocked VEGF-induced phosphorylation of p130Cas. In vivo, treatment with either DC101 or 1121B significantly reduced tumor growth alone and in combination in the SKOV3ip1 and A2774 models. Decreased tumor burden after treatment with DC101 or 1121B correlated with increased tumor cell apoptosis, decreased proliferative index, and decreased microvessel density. These effects were significantly greater in the combination group (p<0.001). We show functionally active VEGFR-2 is present on most ovarian cancer cells. The observed anti-tumor activity of VEGF-targeted therapies may be mediated by both anti-angiogenic and direct anti-tumor effects. PMID:19058181

Constitutive Proteasomal Degradation of TWIST-1 in Epithelial Ovarian Cancer Stem Cells Impacts Differentiation and Metastatic Potential

PubMed Central

Yin, Gang; Alvero, Ayesha B.; Craveiro, Vinicius; Holmberg, Jennie C.; Fu, Han-Hsuan; Montagna, Michele K.; Yang, Yang; Chefetz-Menaker, Ilana; Nuti, Sudhakar; Rossi, Michael; Silasi, Dan-Arin; Rutherford, Thomas; Mor, Gil

2013-01-01

Epithelial-mesenchymal transition (EMT) is a critical process for embryogenesis but is abnormally activated during cancer metastasis and recurrence. This process enables epithelial cancer cells to acquire mobility and traits associated with stemness. It is unknown whether epithelial stem cells or epithelial cancer stem cells are able to undergo EMT, and what molecular mechanism regulates this process in these specific cell types. We found that Epithelial Ovarian Cancer Stem cells (EOC stem cells) are the source of metastatic progenitor cells through a differentiation process involving EMT and Mesenchymal-Epithelial Transition (MET). We demonstrate both in vivo and in vitro the differentiation of EOC stem cells into mesenchymal spheroid-forming cells (MSFCs) and their capacity to initiate an active carcinomatosis. Furthermore, we demonstrate that human EOC stem cells injected i.p in mice are able to form ovarian tumors, suggesting that the EOC stem cells have the ability to “home” to the ovaries and establish tumors. Most interestingly, we found that TWIST1 is constitutively degraded in EOC stem cells, and that the acquisition of TWIST1 requires additional signals that will trigger the differentiation process. These findings are relevant for understanding the differentiation and metastasis process in EOC stem cells. PMID:22349827

Importance of the stem cell microenvironment for ophthalmological cell-based therapy

PubMed Central

Wan, Peng-Xia; Wang, Bo-Wen; Wang, Zhi-Chong

2015-01-01

Cell therapy is a promising treatment for diseases that are caused by cell degeneration or death. The cells for clinical transplantation are usually obtained by culturing healthy allogeneic or exogenous tissue in vitro. However, for diseases of the eye, obtaining the adequate number of cells for clinical transplantation is difficult due to the small size of tissue donors and the frequent needs of long-term amplification of cells in vitro, which results in low cell viability after transplantation. In addition, the transplanted cells often develop fibrosis or degrade and have very low survival. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS) are also promising candidates for cell therapy. Unfortunately, the differentiation of ESCs can bring immune rejection, tumorigenicity and undesired differentiated cells, limiting its clinical application. Although iPS cells can avoid the risk of immune rejection caused by ES cell differentiation post-transplantation, the low conversion rate, the risk of tumor formation and the potentially unpredictable biological changes that could occur through genetic manipulation hinder its clinical application. Thus, the desired clinical effect of cell therapy is impaired by these factors. Recent research findings recognize that the reason for low survival of the implanted cells not only depends on the seeded cells, but also on the cell microenvironment, which determines the cell survival, proliferation and even reverse differentiation. When used for cell therapy, the transplanted cells need a specific three-dimensional structure to anchor and specific extra cellular matrix components in addition to relevant cytokine signaling to transfer the required information to support their growth. These structures present in the matrix in which the stem cells reside are known as the stem cell microenvironment. The microenvironment interaction with the stem cells provides the necessary homeostasis for cell maintenance and growth. A large number of studies suggest that to explore how to reconstruct the stem cell microenvironment and strengthen its combination with the transplanted cells are key steps to successful cell therapy. In this review, we will describe the interactions of the stem cell microenvironment with the stem cells, discuss the importance of the stem cell microenvironment for cell-based therapy in ocular diseases, and introduce the progress of stem cell-based therapy for ocular diseases. PMID:25815128

Integrated transcriptome analysis of human iPS cells derived from a fragile X syndrome patient during neuronal differentiation.

PubMed

Lu, Ping; Chen, Xiaolong; Feng, Yun; Zeng, Qiao; Jiang, Cizhong; Zhu, Xianmin; Fan, Guoping; Xue, Zhigang

2016-11-01

Fragile X syndrome (FXS) patients carry the expansion of over 200 CGG repeats at the promoter of fragile X mental retardation 1 (FMR1), leading to decreased or absent expression of its encoded fragile X mental retardation protein (FMRP). However, the global transcriptional alteration by FMRP deficiency has not been well characterized at single nucleotide resolution, i.e., RNA-seq. Here, we performed in-vitro neuronal differentiation of human induced pluripotent stem (iPS) cells that were derived from fibroblasts of a FXS patient (FXS-iPSC). We then performed RNA-seq and examined the transcriptional misregulation at each intermediate stage during in-vitro differentiation of FXS-iPSC into neurons. After thoroughly analyzing the transcriptomic data and integrating them with those from other platforms, we found up-regulation of many genes encoding TFs for neuronal differentiation (WNT1, BMP4, POU3F4, TFAP2C, and PAX3), down-regulation of potassium channels (KCNA1, KCNC3, KCNG2, KCNIP4, KCNJ3, KCNK9, and KCNT1) and altered temporal regulation of SHANK1 and NNAT in FXS-iPSC derived neurons, indicating impaired neuronal differentiation and function in FXS patients. In conclusion, we demonstrated that the FMRP deficiency in FXS patients has significant impact on the gene expression patterns during development, which will help to discover potential targeting candidates for the cure of FXS symptoms.

Long interspersed element-1 is differentially regulated by food-borne carcinogens via the aryl hydrocarbon receptor

PubMed Central

Okudaira, N; Okamura, T; Tamura, M; Iijma, K; Goto, M; Matsunaga, A; Ochiai, M; Nakagama, H; Kano, S; Fujii-Kuriyama, Y; Ishizaka, Y

2013-01-01

A single human cell contains more than 5.0 × 105 copies of long interspersed element-1 (L1), 80–100 of which are competent for retrotransposition (L1-RTP). Recent observations have revealed the presence of de novo L1 insertions in various tumors, but little is known about its mechanism. Here, we found that 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx), food-borne carcinogens that are present in broiled meats, induced L1-RTP. This induction was dependent on a cellular cascade comprising the aryl hydrocarbon receptor (AhR), a mitogen-activated protein kinase, and CCAAT/enhancer-binding protein β. Notably, these compounds exhibited differential induction of L1-RTP. MeIQx-induced L1-RTP was dependent on AhR nuclear translocator 1 (ARNT1), a counterpart of AhR required for gene expression in response to environmental pollutants. By contrast, PhIP-induced L1-RTP did not require ARNT1 but was dependent on estrogen receptor α (ERα) and AhR repressor. In vivo studies using transgenic mice harboring the human L1 gene indicated that PhIP-induced L1-RTP was reproducibly detected in the mammary gland, which is a target organ of PhIP-induced carcinoma. Moreover, picomolar levels of each compound induced L1-RTP, which is comparable to the PhIP concentration detected in human breast milk. Data suggest that somatic cells possess machineries that induce L1-RTP in response to the carcinogenic compounds. Together with data showing that micromolar levels of heterocyclic amines (HCAs) were non-genotoxic, our observations indicate that L1-RTP by environmental compounds is a novel type of genomic instability, further suggesting that analysis of L1-RTP by HCAs is a novel approach to clarification of modes of carcinogenesis. PMID:23208499

Long interspersed element-1 is differentially regulated by food-borne carcinogens via the aryl hydrocarbon receptor.

PubMed

Okudaira, N; Okamura, T; Tamura, M; Iijma, K; Goto, M; Matsunaga, A; Ochiai, M; Nakagama, H; Kano, S; Fujii-Kuriyama, Y; Ishizaka, Y

2013-10-10

A single human cell contains more than 5.0 × 10(5) copies of long interspersed element-1 (L1), 80-100 of which are competent for retrotransposition (L1-RTP). Recent observations have revealed the presence of de novo L1 insertions in various tumors, but little is known about its mechanism. Here, we found that 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx), food-borne carcinogens that are present in broiled meats, induced L1-RTP. This induction was dependent on a cellular cascade comprising the aryl hydrocarbon receptor (AhR), a mitogen-activated protein kinase, and CCAAT/enhancer-binding protein β. Notably, these compounds exhibited differential induction of L1-RTP. MeIQx-induced L1-RTP was dependent on AhR nuclear translocator 1 (ARNT1), a counterpart of AhR required for gene expression in response to environmental pollutants. By contrast, PhIP-induced L1-RTP did not require ARNT1 but was dependent on estrogen receptor α (ERα) and AhR repressor. In vivo studies using transgenic mice harboring the human L1 gene indicated that PhIP-induced L1-RTP was reproducibly detected in the mammary gland, which is a target organ of PhIP-induced carcinoma. Moreover, picomolar levels of each compound induced L1-RTP, which is comparable to the PhIP concentration detected in human breast milk. Data suggest that somatic cells possess machineries that induce L1-RTP in response to the carcinogenic compounds. Together with data showing that micromolar levels of heterocyclic amines (HCAs) were non-genotoxic, our observations indicate that L1-RTP by environmental compounds is a novel type of genomic instability, further suggesting that analysis of L1-RTP by HCAs is a novel approach to clarification of modes of carcinogenesis.

Genome-wide analysis of endogenously expressed ZEB2 binding sites reveals inverse correlations between ZEB2 and GalNAc-transferase GALNT3 in human tumors.

PubMed

Balcik-Ercin, Pelin; Cetin, Metin; Yalim-Camci, Irem; Odabas, Gorkem; Tokay, Nurettin; Sayan, A Emre; Yagci, Tamer

2018-03-07

ZEB2 is a transcriptional repressor that regulates epithelial-to-mesenchymal transition (EMT) through binding to bipartite E-box motifs in gene regulatory regions. Despite the abundant presence of E-boxes within the human genome and the multiplicity of pathophysiological processes regulated during ZEB2-induced EMT, only a small fraction of ZEB2 targets has been identified so far. Hence, we explored genome-wide ZEB2 binding by chromatin immunoprecipitation-sequencing (ChIP-seq) under endogenous ZEB2 expression conditions. For ChIP-Seq we used an anti-ZEB2 monoclonal antibody, clone 6E5, in SNU398 hepatocellular carcinoma cells exhibiting a high endogenous ZEB2 expression. The ChIP-Seq targets were validated using ChIP-qPCR, whereas ZEB2-dependent expression of target genes was assessed by RT-qPCR and Western blotting in shRNA-mediated ZEB2 silenced SNU398 cells and doxycycline-induced ZEB2 overexpressing colorectal carcinoma DLD1 cells. Changes in target gene expression were also assessed using primary human tumor cDNA arrays in conjunction with RT-qPCR. Additional differential expression and correlation analyses were performed using expO and Human Protein Atlas datasets. Over 500 ChIP-Seq positive genes were annotated, and intervals related to these genes were found to include the ZEB2 binding motif CACCTG according to TOMTOM motif analysis in the MEME Suite database. Assessment of ZEB2-dependent expression of target genes in ZEB2-silenced SNU398 cells and ZEB2-induced DLD1 cells revealed that the GALNT3 gene serves as a ZEB2 target with the highest, but inversely correlated, expression level. Remarkably, GALNT3 also exhibited the highest enrichment in the ChIP-qPCR validation assays. Through the analyses of primary tumor cDNA arrays and expO datasets a significant differential expression and a significant inverse correlation between ZEB2 and GALNT3 expression were detected in most of the tumors. We also explored ZEB2 and GALNT3 protein expression using the Human Protein Atlas dataset and, again, observed an inverse correlation in all analyzed tumor types, except malignant melanoma. In contrast to a generally negative or weak ZEB2 expression, we found that most tumor tissues exhibited a strong or moderate GALNT3 expression. Our observation that ZEB2 negatively regulates a GalNAc-transferase (GALNT3) that is involved in O-glycosylation adds another layer of complexity to the role of ZEB2 in cancer progression and metastasis. Proteins glycosylated by GALNT3 may be exploited as novel diagnostics and/or therapeutic targets.

Human embryonic stem cell therapies for neurodegenerative diseases.

PubMed

Tomaskovic-Crook, Eva; Crook, Jeremy M

2011-06-01

There is a renewed enthusiasm for the clinical translation of human embryonic stem (hES) cells. This is abetted by putative clinically-compliant strategies for hES cell maintenance and directed differentiation, greater understanding of and accessibility to cells through formal cell registries and centralized cell banking for distribution, the revised US government policy on funding hES cell research, and paradoxically the discovery of induced pluripotent stem (iPS) cells. Additionally, as we consider the constraints (practical and fiscal) of delivering cell therapies for global healthcare, the more efficient and economical application of allogeneic vs autologous treatments will bolster the clinical entry of hES cell derivatives. Neurodegenerative disorders such as Parkinson's disease are primary candidates for hES cell therapy, although there are significant hurdles to be overcome. The present review considers key advances and challenges to translating hES cells into novel therapies for neurodegenerative diseases, with special consideration given to Parkinson's disease and Alzheimer's disease. Importantly, despite the focus on degenerative brain disorders and hES cells, many of the issues canvassed by this review are relevant to systemic application of hES cells and other pluripotent stem cells such as iPS cells.

Genome-wide Analysis of Simultaneous GATA1/2, RUNX1, FLI1, and SCL Binding in Megakaryocytes Identifies Hematopoietic Regulators

PubMed Central

Tijssen, Marloes R.; Cvejic, Ana; Joshi, Anagha; Hannah, Rebecca L.; Ferreira, Rita; Forrai, Ariel; Bellissimo, Dana C.; Oram, S. Helen; Smethurst, Peter A.; Wilson, Nicola K.; Wang, Xiaonan; Ottersbach, Katrin; Stemple, Derek L.; Green, Anthony R.; Ouwehand, Willem H.; Göttgens, Berthold

2011-01-01

Summary Hematopoietic differentiation critically depends on combinations of transcriptional regulators controlling the development of individual lineages. Here, we report the genome-wide binding sites for the five key hematopoietic transcription factors—GATA1, GATA2, RUNX1, FLI1, and TAL1/SCL—in primary human megakaryocytes. Statistical analysis of the 17,263 regions bound by at least one factor demonstrated that simultaneous binding by all five factors was the most enriched pattern and often occurred near known hematopoietic regulators. Eight genes not previously appreciated to function in hematopoiesis that were bound by all five factors were shown to be essential for thrombocyte and/or erythroid development in zebrafish. Moreover, one of these genes encoding the PDZK1IP1 protein shared transcriptional enhancer elements with the blood stem cell regulator TAL1/SCL. Multifactor ChIP-Seq analysis in primary human cells coupled with a high-throughput in vivo perturbation screen therefore offers a powerful strategy to identify essential regulators of complex mammalian differentiation processes. PMID:21571218

Miz1, a Novel Target of ING4, Can Drive Prostate Luminal Epithelial Cell Differentiation.

PubMed

Berger, Penny L; Winn, Mary E; Miranti, Cindy K

2017-01-01

How prostate epithelial cells differentiate and how dysregulation of this process contributes to prostate tumorigenesis remain unclear. We recently identified a Myc target and chromatin reader protein, ING4, as a necessary component of human prostate luminal epithelial cell differentiation, which is often lost in primary prostate tumors. Furthermore, loss of ING4 in the context of oncogenic mutations is required for prostate tumorigenesis. Identifying the gene targets of ING4 can provide insight into how its loss disrupts differentiation and leads to prostate cancer. Using a combination of RNA-Seq, a best candidate approach, and chromatin immunoprecipitation (ChIP), we identified Miz1 as a new ING4 target. ING4 or Miz1 overexpression, shRNA knock-down, and a Myc-binding mutant were used in a human in vitro differentiation assay to assess the role of Miz1 in luminal cell differentiation. ING4 directly binds the Miz1 promoter and is required to induce Miz1 mRNA and protein expression during luminal cell differentiation. Miz1 mRNA was not induced in shING4 expressing cells or tumorigenic cells in which ING4 is not expressed. Miz1 dependency on ING4 was unique to differentiating luminal cells; Miz1 mRNA expression was not induced in basal cells. Although Miz1 is a direct target of ING4, and its overexpression can drive luminal cell differentiation, Miz1 was not required for differentiation. Miz1 is a newly identified ING4-induced target gene which can drive prostate luminal epithelial cell differentiation although it is not absolutely required. Prostate 77:49-59, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Histone deacetylase inhibitors suppress ABO transcription in vitro, leading to reduced expression of the antigens.

PubMed

Takahashi, Yoichiro; Kubo, Rieko; Sano, Rie; Nakajima, Tamiko; Takahashi, Keiko; Kobayashi, Momoko; Handa, Hiroshi; Tsukada, Junichi; Kominato, Yoshihiko

2017-03-01

The ABO system is of fundamental importance in the fields of transfusion and transplantation and has apparent associations with certain diseases, including cardiovascular disorders. ABO expression is reduced in the late phase of erythroid differentiation in vitro, whereas histone deacetylase inhibitors (HDACIs) are known to promote cell differentiation. Therefore, whether or not HDACIs could reduce the amount of ABO transcripts and A or B antigens is an intriguing issue. Quantitative polymerase chain reactions were carried out for the ABO transcripts in erythroid-lineage K562 and epithelial-lineage KATOIII cells after incubation with HDACIs, such as sodium butyrate, panobinostat, vorinostat, and sodium valproate. Flow cytometric analysis was conducted to evaluate the amounts of antigen in KATOIII cells treated with panobinostat. Quantitative chromatin immunoprecipitation (ChIP) assays and luciferase assays were performed on both cell types to examine the mechanisms of ABO suppression. HDACIs reduced the ABO transcripts in both K562 and KATOIII cells, with panobinostat exerting the most significant effect. Flow cytometric analysis demonstrated a decrease in B-antigen expression on panobinostat-treated KATOIII cells. ChIP assays indicated that panobinostat altered the modification of histones in the transcriptional regulatory regions of ABO, and luciferase assays demonstrated reduced activity of these elements. ABO transcription seems to be regulated by an epigenetic mechanism. Panobinostat appears to suppress ABO transcription, reducing the amount of antigens on the surface of cultured cells. © 2016 AABB.

Coordination of Myeloid Differentiation with Reduced Cell Cycle Progression by PU.1 Induction of MicroRNAs Targeting Cell Cycle Regulators and Lipid Anabolism.

PubMed

Solomon, Lauren A; Podder, Shreya; He, Jessica; Jackson-Chornenki, Nicholas L; Gibson, Kristen; Ziliotto, Rachel G; Rhee, Jess; DeKoter, Rodney P

2017-05-15

During macrophage development, myeloid progenitor cells undergo terminal differentiation coordinated with reduced cell cycle progression. Differentiation of macrophages from myeloid progenitors is accompanied by increased expression of the E26 transformation-specific transcription factor PU.1. Reduced PU.1 expression leads to increased proliferation and impaired differentiation of myeloid progenitor cells. It is not understood how PU.1 coordinates macrophage differentiation with reduced cell cycle progression. In this study, we utilized cultured PU.1-inducible myeloid cells to perform genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis coupled with gene expression analysis to determine targets of PU.1 that may be involved in regulating cell cycle progression. We found that genes encoding cell cycle regulators and enzymes involved in lipid anabolism were directly and inducibly bound by PU.1 although their steady-state mRNA transcript levels were reduced. Inhibition of lipid anabolism was sufficient to reduce cell cycle progression in these cells. Induction of PU.1 reduced expression of E2f1 , an important activator of genes involved in cell cycle and lipid anabolism, indirectly through microRNA 223. Next-generation sequencing identified microRNAs validated as targeting cell cycle and lipid anabolism for downregulation. These results suggest that PU.1 coordinates cell cycle progression with differentiation through induction of microRNAs targeting cell cycle regulators and lipid anabolism. Copyright © 2017 American Society for Microbiology.

Enhancer connectome in primary human cells identifies target genes of disease-associated DNA elements

PubMed Central

Mumbach, Maxwell R; Satpathy, Ansuman T; Boyle, Evan A; Dai, Chao; Gowen, Benjamin G; Cho, Seung Woo; Nguyen, Michelle L; Rubin, Adam J; Granja, Jeffrey M; Kazane, Katelynn R; Wei, Yuning; Nguyen, Trieu; Greenside, Peyton G; Corces, M Ryan; Tycko, Josh; Simeonov, Dimitre R; Suliman, Nabeela; Li, Rui; Xu, Jin; Flynn, Ryan A; Kundaje, Anshul; Khavari, Paul A; Marson, Alexander; Corn, Jacob E; Quertermous, Thomas; Greenleaf, William J; Chang, Howard Y

2018-01-01

The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer–promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases. PMID:28945252

Determination of the optimum number of cardiac cycles to differentiate intra-pulmonary shunt and patent foramen ovale by saline contrast two- and three-dimensional echocardiography.

PubMed

Bhatia, Nirmanmoh; Abushora, Mohannad Y; Donneyong, Macarius M; Stoddard, Marcus F

2014-03-01

Patent foramen ovale (PFO) and intra-pulmonary shunt (IPS) are potential causes of stroke. The most optimum cardiac cycle cutoff for bubbles to appear in the left heart on saline contrast transthoracic echocardiography (TTE) as criteria to differentiate the 2 entities is unknown. Ninety-five adult patients had saline contrast transesophageal echocardiography (TEE), two-dimensional (2D) and 3DTTE. Sensitivity and specificity of each cardiac cycle as cutoff to differentiate a PFO and IPS were obtained. Transesophageal echocardiography showed IPS in 28 and PFO in 15 patients. If bubbles appeared in the left heart within the first 4 cardiac cycles (the 4th cardiac cycle rule) as compared to alternate cutoffs, a PFO was most accurately diagnosed by both 2D and 3DTTE. Bubbles appearing at or after the 5th cardiac cycle most accurately determined an IPS. 3D versus 2DTTE had a trend for a higher sensitivity (61% vs. 36%, P = 0.06), but similar specificity (94% vs. 91%) for IPS. Accuracy of 3DTTE was 84% and 2DTTE was 75% (P = 0.08) for IPS. For PFO, 2DTTE sensitivity (87%) and specificity (98%) did not differ (P = NS) from that of 3DTTE sensitivity (73%) and specificity (100%). This study demonstrates for the first time that the 4th cardiac cycle rule differentiates PFO and IPS most optimally by 2D and 3DTTE. 3DTTE appears to have higher sensitivity for diagnosing IPS. These data suggest that 3DTTE is preferable when IPS is to be diagnosed. Both methods are similar for diagnosing PFO. © 2013, Wiley Periodicals, Inc.

GnRH signalling pathways and GnRH-induced homologous desensitization in a gonadotrope cell line (alphaT3-1).

PubMed

Poulin, B; Rich, N; Mas, J L; Kordon, C; Enjalbert, A; Drouva, S V

1998-07-25

Exposure of the gonadotrope cells to gonadotropin-releasing hormone (GnRH) reduces their responsiveness to a new GnRH stimulation (homologous desensitization). The time frame as well as the mechanisms underlying this phenomenon are yet unclear. We studied in a gonadotrope cell line (alphaT3-1) the effects of short as well as long term GnRH pretreatments on the GnRH-induced phospholipases-C (PLC), -A2 (PLA2) and -D (PLD) activities, by measuring the production of IP3, total inositol phosphates (IPs), arachidonic acid (AA) and phosphatidylethanol (PEt) respectively. We demonstrated that although rapid desensitization of GnRH-induced IP3 formation did not occur in these cells, persistent stimulation of cells with GnRH or its analogue resulted in a time-dependent attenuation of GnRH-elicited IPs formation. GnRH-induced IPs desensitization was potentiated after direct activation of PKC by the phorbol ester TPA, suggesting the involvement of distinct mechanisms in the uncoupling exerted by either GnRH or TPA on GnRH-stimulated PI hydrolysis. The levels of individual phosphoinositides remained unchanged under any desensitization condition applied. Interestingly, while the GnRH-induced PLA2 activity was rapidly desensitized (2.5 min) after GnRH pretreatments, the neuropeptide-evoked PLD activation was affected at later times, indicating an important time-dependent contribution of these enzymatic activities in the sequential events underlying the GnRH-induced homologous desensitization processes in the gonadotropes. Under GnRH desensitization conditions, TPA was still able to induce PLD activation and to further potentiate the GnRH-evoked PLD activity. AlphaT3-1 cells possess several PKC isoforms which, except PKCzeta, were differentially down-regulated by TPA (PKCalpha, betaII, delta, epsilon, eta) or GnRH (PKCbetaII, delta, epsilon, eta). In spite of the presence of PKC inhibitors or down-regulation of PKC isoforms by TPA, the desensitizing effect of the neuropeptide on GnRH-induced IPs, AA and PEt formation remained unchanged. In conclusion, in alphaT3-1 cells the GnRH-induced homologous desensitization affects the GnRH coupling with PLC, PLA2 and PLD by mechanism(s) which do not implicate TPA-sensitive PKC isoforms, but likely reflect time-dependent modification(s) on the activation processes of the enzymes.

Differential principal component analysis of ChIP-seq.

PubMed

Ji, Hongkai; Li, Xia; Wang, Qian-fei; Ning, Yang

2013-04-23

We propose differential principal component analysis (dPCA) for analyzing multiple ChIP-sequencing datasets to identify differential protein-DNA interactions between two biological conditions. dPCA integrates unsupervised pattern discovery, dimension reduction, and statistical inference into a single framework. It uses a small number of principal components to summarize concisely the major multiprotein synergistic differential patterns between the two conditions. For each pattern, it detects and prioritizes differential genomic loci by comparing the between-condition differences with the within-condition variation among replicate samples. dPCA provides a unique tool for efficiently analyzing large amounts of ChIP-sequencing data to study dynamic changes of gene regulation across different biological conditions. We demonstrate this approach through analyses of differential chromatin patterns at transcription factor binding sites and promoters as well as allele-specific protein-DNA interactions.

CPM Is a Useful Cell Surface Marker to Isolate Expandable Bi-Potential Liver Progenitor Cells Derived from Human iPS Cells.

PubMed

Kido, Taketomo; Koui, Yuta; Suzuki, Kaori; Kobayashi, Ayaka; Miura, Yasushi; Chern, Edward Y; Tanaka, Minoru; Miyajima, Atsushi

2015-10-13

To develop a culture system for large-scale production of mature hepatocytes, liver progenitor cells (LPCs) with a high proliferation potential would be advantageous. We have found that carboxypeptidase M (CPM) is highly expressed in embryonic LPCs, hepatoblasts, while its expression is decreased along with hepatic maturation. Consistently, CPM expression was transiently induced during hepatic specification from human-induced pluripotent stem cells (hiPSCs). CPM(+) cells isolated from differentiated hiPSCs at the immature hepatocyte stage proliferated extensively in vitro and expressed a set of genes that were typical of hepatoblasts. Moreover, the CPM(+) cells exhibited a mature hepatocyte phenotype after induction of hepatic maturation and also underwent cholangiocytic differentiation in a three-dimensional culture system. These results indicated that hiPSC-derived CPM(+) cells share the characteristics of LPCs, with the potential to proliferate and differentiate bi-directionally. Thus, CPM is a useful marker for isolating hiPSC-derived LPCs, which allows development of a large-scale culture system for producing hepatocytes and cholangiocytes. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

CPM Is a Useful Cell Surface Marker to Isolate Expandable Bi-Potential Liver Progenitor Cells Derived from Human iPS Cells

PubMed Central

Kido, Taketomo; Koui, Yuta; Suzuki, Kaori; Kobayashi, Ayaka; Miura, Yasushi; Chern, Edward Y.; Tanaka, Minoru; Miyajima, Atsushi

2015-01-01

Summary To develop a culture system for large-scale production of mature hepatocytes, liver progenitor cells (LPCs) with a high proliferation potential would be advantageous. We have found that carboxypeptidase M (CPM) is highly expressed in embryonic LPCs, hepatoblasts, while its expression is decreased along with hepatic maturation. Consistently, CPM expression was transiently induced during hepatic specification from human-induced pluripotent stem cells (hiPSCs). CPM+ cells isolated from differentiated hiPSCs at the immature hepatocyte stage proliferated extensively in vitro and expressed a set of genes that were typical of hepatoblasts. Moreover, the CPM+ cells exhibited a mature hepatocyte phenotype after induction of hepatic maturation and also underwent cholangiocytic differentiation in a three-dimensional culture system. These results indicated that hiPSC-derived CPM+ cells share the characteristics of LPCs, with the potential to proliferate and differentiate bi-directionally. Thus, CPM is a useful marker for isolating hiPSC-derived LPCs, which allows development of a large-scale culture system for producing hepatocytes and cholangiocytes. PMID:26365514

Histone Deacetylase Inhibition Promotes Osteoblast Maturation by Altering the Histone H4 Epigenome and Reduces Akt Phosphorylation*

PubMed Central

Dudakovic, Amel; Evans, Jared M.; Li, Ying; Middha, Sumit; McGee-Lawrence, Meghan E.; van Wijnen, Andre J.; Westendorf, Jennifer J.

2013-01-01

Bone has remarkable regenerative capacity, but this ability diminishes during aging. Histone deacetylase inhibitors (HDIs) promote terminal osteoblast differentiation and extracellular matrix production in culture. The epigenetic events altered by HDIs in osteoblasts may hold clues for the development of new anabolic treatments for osteoporosis and other conditions of low bone mass. To assess how HDIs affect the epigenome of committed osteoblasts, MC3T3 cells were treated with suberoylanilide hydroxamic acid (SAHA) and subjected to microarray gene expression profiling and high-throughput ChIP-Seq analysis. As expected, SAHA induced differentiation and matrix calcification of osteoblasts in vitro. ChIP-Seq analysis revealed that SAHA increased histone H4 acetylation genome-wide and in differentially regulated genes, except for the 500 bp upstream of transcriptional start sites. Pathway analysis indicated that SAHA increased the expression of insulin signaling modulators, including Slc9a3r1. SAHA decreased phosphorylation of insulin receptor β, Akt, and the Akt substrate FoxO1, resulting in FoxO1 stabilization. Thus, SAHA induces genome-wide H4 acetylation and modulates the insulin/Akt/FoxO1 signaling axis, whereas it promotes terminal osteoblast differentiation in vitro. PMID:23940046

The new generation of beta-cells: replication, stem cell differentiation, and the role of small molecules.

PubMed

Borowiak, Malgorzata

2010-01-01

Diabetic patients suffer from the loss of insulin-secreting β-cells, or from an improper working β-cell mass. Due to the increasing prevalence of diabetes across the world, there is a compelling need for a renewable source of cells that could replace pancreatic β-cells. In recent years, several promising approaches to the generation of new β-cells have been developed. These include directed differentiation of pluripotent cells such as embryonic stem (ES) cells or induced pluripotent stem (iPS) cells, or reprogramming of mature tissue cells. High yield methods to differentiate cell populations into β-cells, definitive endoderm, and pancreatic progenitors, have been established using growth factors and small molecules. However, the final step of directed differentiation to generate functional, mature β-cells in sufficient quantities has yet to be achieved in vitro. Beside the needs of transplantation medicine, a renewable source of β-cells would also be important in terms of a platform to study the pathogenesis of diabetes, and to seek alternative treatments. Finally, by generating new β-cells, we could learn more details about pancreatic development and β-cell specification. This review gives an overview of pancreas ontogenesis in the perspective of stem cell differentiation, and highlights the critical aspects of small molecules in the generation of a renewable β-cell source. Also, it discusses longer term challenges and opportunities in moving towards a therapeutic goal for diabetes.

Effect of ionic activity products on the structure and composition of mineral self assembled on three-dimensional poly(lactide-co-glycolide) scaffolds

PubMed Central

Shin, Kyungsup; Jayasuriya, Ambalangodage C.; Kohn, David H.

2009-01-01

A biomimetic approach involving the self-assembly of mineral within the pores of three-dimensional porous polymer scaffolds is a promising strategy to integrate advantages of inorganic and organic phases into a single material for hard tissue engineering. Such a material enhances the ability of progenitor cells to differentiate down an osteoblast lineage in vitro and in vivo, compared with polymer scaffolds. The mechanisms regulating mineral formation in this one-step process, however, are poorly understood, especially the effects of ionic activity products (IP) of the mineralizing solution and incubation time. The aims of this study were to define the structure and composition of mineral formed within the pores of biodegradable polymer scaffolds as a function of IP and time. Three-dimensional poly(lactide-co-glycolide) scaffolds were fabricated by solvent casting/particulate leaching and incubated for 4–16 days in six variants of simulated body fluid whose IPs were varied by adjusting ionic concentrations. Scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy demonstrated the formation of carbonated apatite with sub-micrometer sized crystals that grew into spherical globules extending out of the scaffold pore surfaces. As IP increased, more mineral grew on the scaffold pore surfaces, but the apatite became less crystalline and the Ca/P molar ratio decreased from 1.63 ± 0.005 to 1.51 ± 0.002. Since morphology, composition, and structure of mineral are factors that affect cell function, this study demonstrates that the IP of the mineralizing solution is an important modulator of material properties, potentially leading to enhanced control of cell function. PMID:17584901

Essential and nonredundant roles for Diaphanous formins in cortical microtubule capture and directed cell migration.

PubMed

Daou, Pascale; Hasan, Salma; Breitsprecher, Dennis; Baudelet, Emilie; Camoin, Luc; Audebert, Stéphane; Goode, Bruce L; Badache, Ali

2014-03-01

Formins constitute a large family of proteins that regulate the dynamics and organization of both the actin and microtubule cytoskeletons. Previously we showed that the formin mDia1 helps tether microtubules at the cell cortex, acting downstream of the ErbB2 receptor tyrosine kinase. Here we further study the contributions of mDia1 and its two most closely related formins, mDia2 and mDia3, to cortical microtubule capture and ErbB2-dependent breast carcinoma cell migration. We find that depletion of each of these three formins strongly disrupts chemotaxis without significantly affecting actin-based structures. Further, all three formins are required for formation of cortical microtubules in a nonredundant manner, and formin proteins defective in actin polymerization remain active for microtubule capture. Using affinity purification and mass spectrometry analysis, we identify differential binding partners of the formin-homology domain 2 (FH2) of mDia1, mDia2, and mDia3, which may explain their nonredundant roles in microtubule capture. The FH2 domain of mDia1 specifically interacts with Rab6-interacting protein 2 (Rab6IP2). Further, mDia1 is required for cortical localization of Rab6IP2, and concomitant depletion of Rab6IP2 and IQGAP1 severely disrupts cortical capture of microtubules, demonstrating the coinvolvement of mDia1, IQGAP1, and Rab6IP2 in microtubule tethering at the leading edge.

Anaphase B

PubMed Central

Scholey, Jonathan M.; Civelekoglu-Scholey, Gul; Brust-Mascher, Ingrid

2016-01-01

Anaphase B spindle elongation is characterized by the sliding apart of overlapping antiparallel interpolar (ip) microtubules (MTs) as the two opposite spindle poles separate, pulling along disjoined sister chromatids, thereby contributing to chromosome segregation and the propagation of all cellular life. The major biochemical “modules” that cooperate to mediate pole–pole separation include: (i) midzone pushing or (ii) braking by MT crosslinkers, such as kinesin-5 motors, which facilitate or restrict the outward sliding of antiparallel interpolar MTs (ipMTs); (iii) cortical pulling by disassembling astral MTs (aMTs) and/or dynein motors that pull aMTs outwards; (iv) ipMT plus end dynamics, notably net polymerization; and (v) ipMT minus end depolymerization manifest as poleward flux. The differential combination of these modules in different cell types produces diversity in the anaphase B mechanism. Combinations of antagonist modules can create a force balance that maintains the dynamic pre-anaphase B spindle at constant length. Tipping such a force balance at anaphase B onset can initiate and control the rate of spindle elongation. The activities of the basic motor filament components of the anaphase B machinery are controlled by a network of non-motor MT-associated proteins (MAPs), for example the key MT cross-linker, Ase1p/PRC1, and various cell-cycle kinases, phosphatases, and proteases. This review focuses on the molecular mechanisms of anaphase B spindle elongation in eukaryotic cells and briefly mentions bacterial DNA segregation systems that operate by spindle elongation. PMID:27941648

Essential and nonredundant roles for Diaphanous formins in cortical microtubule capture and directed cell migration

PubMed Central

Daou, Pascale; Hasan, Salma; Breitsprecher, Dennis; Baudelet, Emilie; Camoin, Luc; Audebert, Stéphane; Goode, Bruce L.; Badache, Ali

2014-01-01

Formins constitute a large family of proteins that regulate the dynamics and organization of both the actin and microtubule cytoskeletons. Previously we showed that the formin mDia1 helps tether microtubules at the cell cortex, acting downstream of the ErbB2 receptor tyrosine kinase. Here we further study the contributions of mDia1 and its two most closely related formins, mDia2 and mDia3, to cortical microtubule capture and ErbB2-dependent breast carcinoma cell migration. We find that depletion of each of these three formins strongly disrupts chemotaxis without significantly affecting actin-based structures. Further, all three formins are required for formation of cortical microtubules in a nonredundant manner, and formin proteins defective in actin polymerization remain active for microtubule capture. Using affinity purification and mass spectrometry analysis, we identify differential binding partners of the formin-homology domain 2 (FH2) of mDia1, mDia2, and mDia3, which may explain their nonredundant roles in microtubule capture. The FH2 domain of mDia1 specifically interacts with Rab6-interacting protein 2 (Rab6IP2). Further, mDia1 is required for cortical localization of Rab6IP2, and concomitant depletion of Rab6IP2 and IQGAP1 severely disrupts cortical capture of microtubules, demonstrating the coinvolvement of mDia1, IQGAP1, and Rab6IP2 in microtubule tethering at the leading edge. PMID:24403606

Limitations of Using IL-17A and IFN-γ-Induced Protein 10 to Detect Bovine Tuberculosis

PubMed Central

Xin, Ting; Gao, Xintao; Yang, Hongjun; Li, Pingjun; Liang, Qianqian; Hou, Shaohua; Sui, Xiukun; Guo, Xiaoyu; Yuan, Weifeng; Zhu, Hongfei; Ding, Jiabo; Jia, Hong

2018-01-01

Bovine tuberculosis (bTB) is primarily caused by infection with Mycobacterium bovis, which belongs to the Mycobacterium tuberculosis complex. The airborne route is considered the most common for transmission of M. bovis, and more than 15% of cattle with bTB shed the Mycobacterium, which can be detect by nested PCR to amplify mycobacterial mpb70 from a nasal swab from a cow. To screen for cytokines fostering early and accurate detection of bTB, peripheral blood mononuclear cells were isolated from naturally M. bovis-infected, experimentally M. bovis 68002-infected, and uninfected cattle, then these cells were stimulated by PPD-B, CFP-10-ESAT-6 (CE), or phosphate-buffered saline (PBS) for 6 h. The levels of interferon gamma (IFN-γ), IFN-γ-induced protein 10 (IP-10), IL-6, IL-12, IL-17A, and tumor necrosis factor alpha mRNA were measured using real-time PCR. To explore the cytokines associated with different periods of M. bovis infection, cattle were divided into three groups: PCR-positive, PCR-negative, and uninfected using the tuberculin skin test, CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test, IFN-γ release assay (IGRA), CFP-10/ESAT-6 (CE)-based IGRA, and nested PCR. The expression of IP-10, IL-17A, and IFN-γ proteins induced by PPD-B, CE, or PBS was detected by ELISA. The results showed that levels of PPD-B-stimulated IL-17A and IP-10 (mRNA and protein), and CE-induced IP-10 (mRNA and protein) were significantly higher in cattle naturally or experimentally infected with M. bovis than in those that were uninfected. The levels of PPD-B- or CE-induced IL-17A and IP-10 (protein) could be used to differentiate M. bovis-infected calves from uninfected ones for 6 to 30 weeks post-infection, whereas PPD-B- and CE-induced IP-10 and IL-17A mRNA expression could be used to differentiate M. bovis-infected calves from uninfected ones between 6 and 58 weeks post-infection. However, CE-induced IL-17A (protein) was not a reliable indicator of M. bovis infection in cattle that were confirmed positive for infection by nested PCR. Furthermore, the levels of PPD-B- or CE-induced IP-10 and IL-17A protein were lower than IFN-γ in M. bovis-infected cattle. Therefore, IL-17A and IP-10 protein are not suitable biomarkers for bTB. Antigen-induced IP-10 mRNA should be analyzed further for their potential to be used in the diagnosis of bTB. PMID:29560355

MLF1 interacting protein: a potential gene therapy target for human prostate cancer?

PubMed

Zhang, Lei; Ji, Guoqing; Shao, Yuzhang; Qiao, Shaoyi; Jing, Yuming; Qin, Rongliang; Sun, Huiming; Shao, Chen

2015-02-01

Here, we investigated the role of one gene that has been previously associated with human prostate carcinoma cells-myelodysplasia/myeloid leukemia factor 1 interacting protein (MLF1IP)-in order to better ascertain its role in human prostate carcinogenesis. The prostate cancer cell line PC-3 was lentivirally transfected to silence endogenous MLF1IP gene expression, which was confirmed by real-time quantitative PCR (RT-qPCR). Cellomics ArrayScan VTI imaging and MTT assays were conducted to assess cell proliferation. Cell cycle phase arrest and apoptosis were assayed by flow cytometry. Colony formation was assessed by fluorescence microscopy. MLF1IP gene expression was also analyzed by RT-qPCR in sixteen prostate cancer tissue samples and six healthy control prostate tissue samples from human patients. Cell proliferation was significantly inhibited in MLF1IP-silenced cells relative to control cells. G1 phase, S and G2/M phase cell counts were not significantly changed in MLF1IP-silenced cells relative to control cells. Apoptosis was significantly increased in MLF1IP-silenced cells, while MLF1IP-silenced cells displayed a significantly reduced number of cell colonies, compared to control cells. The 16 human prostate cancer tissue samples revealed no clear upregulation or downregulation in MLF1IP gene expression. MLF1IP significantly promotes prostate cancer cell proliferation and colony formation and significantly inhibits apoptosis without affecting cell cycle phase arrest. Further study is required to conclusively determine whether MLF1IP is upregulated in human prostate cancer tumors and to determine the precise cellular mechanism(s) for MLF1IP in prostate carcinogenesis.

Conditioned medium: a new alternative for cryopreservation of equine umbilical cord mesenchymal stem cells.

PubMed

Maia, Leandro; Dias, Marianne Camargos; de Moraes, Carolina Nogueira; de Paula Freitas-Dell'Aqua, Camila; da Mota, Ligia S L Silveira; Santiloni, Valquíria; da Cruz Landim-Alvarenga, Fernanda

2017-03-01

Cryopreservation is a feasible alternative to maintaining several cell lines, particularly for immediate therapeutic use, transportation of samples, and implementation of new in vitro studies. This work parts from the hypothesis that the medium of cryopreservation composed by 90% of conditioned medium (CM) supports cryopreservation of equine umbilical cord intervascular matrix mesenchymal stem cells (UCIM-MSCs), allowing the maintenance of the biological properties for the establishment of cell banks intended for therapeutic use and in vitro studies. Thus, we evaluated the viability, apoptosis/necrosis rates, immunophenotypic profile (IP), chromosomal stability, clonicity, and differentiation potential of UCIM-MSCs cryopreserved with four different mediums (with FBS: M1, M3, M4 and without FBS: M2). After 3 months of cryopreservation, samples were thawed and analyzed. The potential of differentiation in the mesodermal lineages, clonicity, and the chromosomal stability were maintained after cryopreservation of UCIM-MSCs with medium containing FBS. Changes (P < 0.05) at IP for some markers were observed at cells cryopreserved with medium M1-M3. Only the UCIM-MSCs cryopreserved with the CM (M4) had similar viability post-thaw (P = 0.23) when compared with fresh cells. We proved the hypothesis that the medium of cryopreservation containing CM supports the cryopreservation of UCIM-MSCs, at the experimental conditions, being the medium that better maintains the biological characteristics observed at fresh cells. Thus, future studies of UCIM-MSCs secretome should be conducted to better understand the beneficial and protective effects of the CM during the freezing process. © 2017 International Federation for Cell Biology.

MHC-matched induced pluripotent stem cells can attenuate cellular and humoral immune responses but are still susceptible to innate immunity in pigs.

PubMed

Mizukami, Yoshihisa; Abe, Tomoyuki; Shibata, Hiroaki; Makimura, Yukitoshi; Fujishiro, Shuh-hei; Yanase, Kimihide; Hishikawa, Shuji; Kobayashi, Eiji; Hanazono, Yutaka

2014-01-01

Recent studies have revealed negligible immunogenicity of induced pluripotent stem (iPS) cells in syngeneic mice and in autologous monkeys. Therefore, human iPS cells would not elicit immune responses in the autologous setting. However, given that human leukocyte antigen (HLA)-matched allogeneic iPS cells would likely be used for medical applications, a more faithful model system is needed to reflect HLA-matched allogeneic settings. Here we examined whether iPS cells induce immune responses in the swine leukocyte antigen (SLA)-matched setting. iPS cells were generated from the SLA-defined C1 strain of Clawn miniature swine, which were confirmed to develop teratomas in mice, and transplanted into the testes (n = 4) and ovary (n = 1) of C1 pigs. No teratomas were found in pigs on 47 to 125 days after transplantation. A Mixed lymphocyte reaction revealed that T-cell responses to the transplanted MHC-matched (C1) iPS cells were significantly lower compared to allogeneic cells. The humoral immune responses were also attenuated in the C1-to-C1 setting. More importantly, even MHC-matched iPS cells were susceptible to innate immunity, NK cells and serum complement. iPS cells lacked the expression of SLA class I and sialic acids. The in vitro cytotoxic assay showed that C1 iPS cells were targeted by NK cells and serum complement of C1. In vivo, the C1 iPS cells developed larger teratomas in NK-deficient NOG (T-B-NK-) mice (n = 10) than in NK-competent NOD/SCID (T-B-NK+) mice (n = 8) (p<0.01). In addition, C1 iPS cell failed to form teratomas after incubation with the porcine complement-active serum. Taken together, MHC-matched iPS cells can attenuate cellular and humoral immune responses, but still susceptible to innate immunity in pigs.

Dose-dependent role of novel agents emodin and BTB14431 in colonic cancer treatment in rats.

PubMed

Braumann, Chris; Koplin, Gerold; Geier, Caroline; Höhn, Philipp; Pohlenz, Jana; Dubiel, Wolfgang; Rogalla, Stephan

2017-12-01

BTB14431 is an in silico homolog to emodin. Both were found to possess anti-tumor effects in vitro. The aim of this work was to analyze the tumor suppressing effects of both molecules in an intraperitoneal (ip) and intravenous (iv) treated rat model (WAG-Rij). A tumor cell suspension (CC531) was applied at the cecum after laparotomy and at the back. The rats where treated twice a day over 1 week with BTB14431, emodin and isotone sodium chloride solution (control). Treatment was applied iv or ip in a variety of dosages. Peripheral blood samples were taken before tumor application and on day 7. Twenty-one days after the last day of therapy animals were euthanized and tumor growth was evaluated. Data showed an insignificant decrease of tumor growth after iv and ip treatment with low doses of BTB14431 and emodin. Differential blood analysis showed apoptosis. Increased doses of emodin clearly raised mortality rate. Apoptosis was verified but no tumor-suppressing effects could be observed for iv and ip treatment with both agents in contrast to in vitro studies in our model. Establishing a successful ip treatment model for emotion and BTB14331 requires further studies.

Inositol Hexakisphosphate Kinase-3 Regulates the Morphology and Synapse Formation of Cerebellar Purkinje Cells via Spectrin/Adducin

PubMed Central

Fu, Chenglai; Xu, Jing; Li, Ruo-Jing; Crawford, Joshua A.; Khan, A. Basit; Ma, Ting Martin; Cha, Jiyoung Y.; Snowman, Adele M.; Pletnikov, Mikhail V.

2015-01-01

The inositol hexakisphosphate kinases (IP6Ks) are the principal enzymes that generate inositol pyrophosphates. There are three IP6Ks (IP6K1, 2, and 3). Functions of IP6K1 and IP6K2 have been substantially delineated, but little is known of IP6K3's role in normal physiology, especially in the brain. To elucidate functions of IP6K3, we generated mice with targeted deletion of IP6K3. We demonstrate that IP6K3 is highly concentrated in the brain in cerebellar Purkinje cells. IP6K3 physiologically binds to the cytoskeletal proteins adducin and spectrin, whose mutual interactions are perturbed in IP6K3-null mutants. Consequently, IP6K3 knock-out cerebella manifest abnormalities in Purkinje cell structure and synapse number, and the mutant mice display deficits in motor learning and coordination. Thus, IP6K3 is a major determinant of cytoskeletal disposition and function of cerebellar Purkinje cells. SIGNIFICANCE STATEMENT We identified and cloned a family of three inositol hexakisphosphate kinases (IP6Ks) that generate the inositol pyrophosphates, most notably 5-diphosphoinositol pentakisphosphate (IP7). Of these, IP6K3 has been least characterized. In the present study we generated IP6K3 knock-out mice and show that IP6K3 is highly expressed in cerebellar Purkinje cells. IP6K3-deleted mice display defects of motor learning and coordination. IP6K3-null mice manifest aberrations of Purkinje cells with a diminished number of synapses. IP6K3 interacts with the cytoskeletal proteins spectrin and adducin whose altered disposition in IP6K3 knock-out mice may mediate phenotypic features of the mutant mice. These findings afford molecular/cytoskeletal mechanisms by which the inositol polyphosphate system impacts brain function. PMID:26245967

Reduced osteoblast activity in the mice lacking TR4 nuclear receptor leads to osteoporosis.

PubMed

Lin, Shin-Jen; Ho, Hsin-Chiu; Lee, Yi-Fen; Liu, Ning-Chun; Liu, Su; Li, Gonghui; Shyr, Chih-Rong; Chang, Chawnshang

2012-06-07

Early studies suggested that TR4 nuclear receptor might play important roles in the skeletal development, yet its detailed mechanism remains unclear. We generated TR4 knockout mice and compared skeletal development with their wild type littermates. Primary bone marrow cells were cultured and we assayed bone differentiation by alkaline phosphatase and alizarin red staining. Primary calvaria were cultured and osteoblastic marker genes were detected by quantitative PCR. Luciferase reporter assays, chromatin immunoprecipitation (ChIP) assays, and electrophoretic mobility shift assays (EMSA) were performed to demonstrate TR4 can directly regulate bone differentiation marker osteocalcin. We first found mice lacking TR4 might develop osteoporosis. We then found that osteoblast progenitor cells isolated from bone marrow of TR4 knockout mice displayed reduced osteoblast differentiation capacity and calcification. Osteoblast primary cultures from TR4 knockout mice calvaria also showed higher proliferation rates indicating lower osteoblast differentiation ability in mice after loss of TR4. Mechanism dissection found the expression of osteoblast markers genes, such as ALP, type I collagen alpha 1, osteocalcin, PTH, and PTHR was dramatically reduced in osteoblasts from TR4 knockout mice as compared to those from TR4 wild type mice. In vitro cell line studies with luciferase reporter assay, ChIP assay, and EMSA further demonstrated TR4 could bind directly to the promoter region of osteocalcin gene and induce its gene expression at the transcriptional level in a dose dependent manner. Together, these results demonstrate TR4 may function as a novel transcriptional factor to play pathophysiological roles in maintaining normal osteoblast activity during the bone development and remodeling, and disruption of TR4 function may result in multiple skeletal abnormalities.

Regulation of 1, 4, 5-triphosphate receptor channel gating dynamics by mutant presenilin in Alzheimer's disease cells

NASA Astrophysics Data System (ADS)

Wei, Fang; Li, Xiang; Cai, Meichun; Liu, Yanping; Jung, Peter; Shuai, Jianwei

2017-06-01

In neurons of patients with Alzheimer's disease, the intracellular Ca2+ concentration is increased by its release from the endoplasmic reticulum via the inositol 1, 4, 5-triphosphate receptor (IP3R). In this paper, we discuss the IP3R gating dynamics in familial Alzheimer's disease (FAD) cells induced with presenilin mutation PS1. By fitting the parameters of an IP3R channel model to experimental data of the open probability, the mean open time and the mean closed time of IP3R channels, in control cells and FAD mutant cells, we suggest that the interaction of presenilin mutation PS1 with IP3R channels leads the decrease in the unbinding rates of IP3 and the activating Ca2+ from IP3Rs. As a result, the increased affinities of IP3 and activating Ca2+ for IP3R channels induce the increase in the Ca2+ signal in FAD mutant cells. Specifically, the PS1 mutation decreases the IP3 dissociation rate of IP3R channels significantly in FAD mutant cells. Our results suggest possible novel targets for FAD therapeutic intervention.

High-content screening of small compounds on human embryonic stem cells.

PubMed

Barbaric, Ivana; Gokhale, Paul J; Andrews, Peter W

2010-08-01

Human ES (embryonic stem) cells and iPS (induced pluripotent stem) cells have been heralded as a source of differentiated cells that could be used in the treatment of degenerative diseases, such as Parkinson's disease or diabetes. Despite the great potential for their use in regenerative therapy, the challenge remains to understand the basic biology of these remarkable cells, in order to differentiate them into any functional cell type. Given the scale of the task, high-throughput screening of agents and culture conditions offers one way to accelerate these studies. The screening of small-compound libraries is particularly amenable to such high-throughput methods. Coupled with high-content screening technology that enables simultaneous assessment of multiple cellular features in an automated and quantitative way, this approach is proving powerful in identifying both small molecules as tools for manipulating stem cell fates and novel mechanisms of differentiation not previously associated with stem cell biology. Such screens performed on human ES cells also demonstrate the usefulness of human ES/iPS cells as cellular models for pharmacological testing of drug efficacy and toxicity, possibly a more imminent use of these cells than in regenerative medicine.

The fused in sarcoma protein forms cytoplasmic aggregates in motor neurons derived from integration-free induced pluripotent stem cells generated from a patient with familial amyotrophic lateral sclerosis carrying the FUS-P525L mutation.

PubMed

Liu, Xinxiu; Chen, Jiayu; Liu, Wenchao; Li, Xiaogang; Chen, Qi; Liu, Tao; Gao, Shaorong; Deng, Min

2015-07-01

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that primarily affects motor neurons (MNs) and has no effective treatment. Mutations in the fused in sarcoma (FUS) gene and abnormal aggregation of FUS protein have been reported in ALS. However, the mechanisms involved in ALS are poorly understood. Clinical drug trails have failed due to a lack of appropriate disease models, including a lack of access to MNs from ALS patients. Induced pluripotent stem (iPS) cells derived from patients with ALS provide an indispensable resource for in vitro mechanistic studies and for future patient-specific cell-based therapies. Previous reports demonstrated that viral-based ALS-iPS cells generated from fibroblasts harvested from Caucasian populations are ideal for basic research; however, ALS-iPS cells are precluded from cell-based therapeutic applications because of the risks associated with the integration of viral sequences into the genome and inconvenience associated with dermal biopsies. To establish a model for use in clinical applications, using episomal vectors, we generated an integration-free iPS cell line from peripheral blood mononuclear cells (PBMCs) harvested from a familial ALS (FALS) patient carrying the FUS-P525L mutation and a healthy control. Furthermore, we successfully differentiated ALS patient-specific iPS cells into MNs and subsequently detected cytoplasmic mislocalization and formation of FUS protein aggregates in MNs due to the FUS-P525L mutation. Our findings offer a cell-based disease model for use in further elucidating ALS pathogenesis and provide a tool for exploring gene repair coupled with cell replacement therapy.

From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium

PubMed Central

Reichman, Sacha; Terray, Angélique; Slembrouck, Amélie; Nanteau, Céline; Orieux, Gaël; Habeler, Walter; Nandrot, Emeline F.; Sahel, José-Alain; Monville, Christelle; Goureau, Olivier

2014-01-01

Progress in retinal-cell therapy derived from human pluripotent stem cells currently faces technical challenges that require the development of easy and standardized protocols. Here, we developed a simple retinal differentiation method, based on confluent human induced pluripotent stem cells (hiPSC), bypassing embryoid body formation and the use of exogenous molecules, coating, or Matrigel. In 2 wk, we generated both retinal pigmented epithelial cells and self-forming neural retina (NR)-like structures containing retinal progenitor cells (RPCs). We report sequential differentiation from RPCs to the seven neuroretinal cell types in maturated NR-like structures as floating cultures, thereby revealing the multipotency of RPCs generated from integration-free hiPSCs. Furthermore, Notch pathway inhibition boosted the generation of photoreceptor precursor cells, crucial in establishing cell therapy strategies. This innovative process proposed here provides a readily efficient and scalable approach to produce retinal cells for regenerative medicine and for drug-screening purposes, as well as an in vitro model of human retinal development and disease. PMID:24912154

Ischemic preconditioning modulates the expression of several genes, leading to the overproduction of IL-1Ra, iNOS, and Bcl-2 in a human model of liver ischemia-reperfusion.

PubMed

Barrier, Alain; Olaya, Natalia; Chiappini, Franck; Roser, François; Scatton, Olivier; Artus, Cédric; Franc, Brigitte; Dudoit, Sandrine; Flahault, Antoine; Debuire, Brigitte; Azoulay, Daniel; Lemoine, Antoinette

2005-10-01

Ischemia triggers an inflammatory response that precipitates cell death during reperfusion. Several studies have shown that tissues are protected by ischemic preconditioning (IP) consisting of 10 min of ischemia followed by 10 min of reperfusion just before ischemia. The molecular basis of this protective effect is poorly understood. We used cDNA arrays (20K) to compare global gene expression in liver biopsies from living human liver donors who underwent IP (n=7) or not (n=7) just before liver devascularization. Microarray data were analyzed using pairedt test with a type I error rate fixed at alpha = 2.5 10(6) (Bonferroni correction). We found that 60 genes were differentially expressed (36 over- and 24 underexpressed in preconditioning group). After IP, the most significantly overexpressed gene was IL-1Ra. This was confirmed by immunoblotting. Differentially expressed were genes involved in apoptosis (NOD2, ephrin-A1, and calpain) and in the carbohydrate metabolism. A significant increase in the amount of the anti-apoptotic protein Bcl-2 in preconditioned livers but no change in the cleavage of procaspase-3, -8, and -9 was observed. We also observed an increase in the amount in the inducible nitric oxide synthase. Therefore, the benefits of IP may be associated with the overproduction of IL-1Ra, Bcl-2, and NO countering the proinflammatory and proapoptotic effects generated during ischemia-reperfusion.

Transcriptional suppression of microRNA-27a contributes to laryngeal cancer differentiation via GSK-3β-involved Wnt/β-catenin pathway

PubMed Central

Chen, Sheng; Sun, Yuan-Yuan; Zhang, Zhao-Xiong; Li, Yun-Hui; Xu, Zhen-Ming; Fu, Wei-Neng

2017-01-01

miR-27a regulates cell differentiation in a variety of diseases. However, whether and how miR-27a participates in laryngeal cancer cell differentiation remains unknown. Therefore, we explored role and molecular mechanism of miR-27a in laryngeal cancer differentiation in the study. We found that miR-27a expression was inversely correlated with laryngeal cancer differentiation degree based on the clinical pathological diagnosis of each patient. miR-27 asignificantly rescued differentiation and inhibited β-catenin, LEF1, OCT4 and SOX2 in Wnt/β-catenin pathway in all-trans-retinoic acid (ATRA)-induced laryngeal cancer cells. Bindings of RARα to miR-27a and miR-27a to GSK-3β were confirmed by ChIP and Luciferase reporter assays, respectively. In conclusion, miR-27a is a negative regulator in laryngeal cancer differentiation. RARα-mediated miR-27a transcriptional inactivation releases the inhibition of miR-27a on GSK-3β leading to laryngeal cancer differentiation through GSK-3β-involved Wnt/β-catenin pathway, suggesting that miR-27a is a usefully therapeutic target at least in ATRA-induced laryngeal cancer differentiation. PMID:28122350

ERP44 inhibits human lung cancer cell migration mainly via IP3R2.

PubMed

Huang, Xue; Jin, Meng; Chen, Ying-Xiao; Wang, Jun; Zhai, Kui; Chang, Yan; Yuan, Qi; Yao, Kai-Tai; Ji, Guangju

2016-06-01

Cancer cell migration is involved in tumour metastasis. However, the relationship between calcium signalling and cancer migration is not well elucidated. In this study, we used the human lung adenocarcinoma A549 cell line to examine the role of endoplasmic reticulum protein 44 (ERP44), which has been reported to regulate calcium release inside of the endoplasmic reticulum (ER), in cell migration. We found that the inositol 1,4,5-trisphosphate receptors (IP3Rs/ITPRs) inhibitor 2-APB significantly inhibited A549 cell migration by inhibiting cell polarization and pseudopodium protrusion, which suggests that Ca2+ is necessary for A549 cell migration. Similarly, the overexpression of ERP44 reduced intracellular Ca2+ release via IP3Rs, altered cell morphology and significantly inhibited the migration of A549 cells. These phenomena were primarily dependent on IP3R2 because wound healing in A549 cells with IP3R2 rather than IP3R1 or IP3R3 siRNA was markedly inhibited. Moreover, the overexpression of ERP44 did not affect the migration of the human neuroblastoma cell line SH-SY5Y, which mainly expresses IP3R1. Based on the above observations, we conclude that ERP44 regulates A549 cell migration mainly via an IP3R2-dependent pathway.

ERP44 inhibits human lung cancer cell migration mainly via IP3R2

PubMed Central

Zhai, Kui; Chang, Yan; Yuan, Qi; Yao, Kai-Tai; Ji, Guangju

2016-01-01

Cancer cell migration is involved in tumour metastasis. However, the relationship between calcium signalling and cancer migration is not well elucidated. In this study, we used the human lung adenocarcinoma A549 cell line to examine the role of endoplasmic reticulum protein 44 (ERP44), which has been reported to regulate calcium release inside of the endoplasmic reticulum (ER), in cell migration. We found that the inositol 1,4,5-trisphosphate receptors (IP3Rs/ITPRs) inhibitor 2-APB significantly inhibited A549 cell migration by inhibiting cell polarization and pseudopodium protrusion, which suggests that Ca2+ is necessary for A549 cell migration. Similarly, the overexpression of ERP44 reduced intracellular Ca2+ release via IP3Rs, altered cell morphology and significantly inhibited the migration of A549 cells. These phenomena were primarily dependent on IP3R2 because wound healing in A549 cells with IP3R2 rather than IP3R1 or IP3R3 siRNA was markedly inhibited. Moreover, the overexpression of ERP44 did not affect the migration of the human neuroblastoma cell line SH-SY5Y, which mainly expresses IP3R1. Based on the above observations, we conclude that ERP44 regulates A549 cell migration mainly via an IP3R2-dependent pathway. PMID:27347718

Limitations and possibilities of low cell number ChIP-seq

PubMed Central

2012-01-01

Background Chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) offers high resolution, genome-wide analysis of DNA-protein interactions. However, current standard methods require abundant starting material in the range of 1–20 million cells per immunoprecipitation, and remain a bottleneck to the acquisition of biologically relevant epigenetic data. Using a ChIP-seq protocol optimised for low cell numbers (down to 100,000 cells / IP), we examined the performance of the ChIP-seq technique on a series of decreasing cell numbers. Results We present an enhanced native ChIP-seq method tailored to low cell numbers that represents a 200-fold reduction in input requirements over existing protocols. The protocol was tested over a range of starting cell numbers covering three orders of magnitude, enabling determination of the lower limit of the technique. At low input cell numbers, increased levels of unmapped and duplicate reads reduce the number of unique reads generated, and can drive up sequencing costs and affect sensitivity if ChIP is attempted from too few cells. Conclusions The optimised method presented here considerably reduces the input requirements for performing native ChIP-seq. It extends the applicability of the technique to isolated primary cells and rare cell populations (e.g. biobank samples, stem cells), and in many cases will alleviate the need for cell culture and any associated alteration of epigenetic marks. However, this study highlights a challenge inherent to ChIP-seq from low cell numbers: as cell input numbers fall, levels of unmapped sequence reads and PCR-generated duplicate reads rise. We discuss a number of solutions to overcome the effects of reducing cell number that may aid further improvements to ChIP performance. PMID:23171294

Establishment of oct4:gfp transgenic zebrafish line for monitoring cellular multipotency by GFP fluorescence.

PubMed

Kato, Hiroyuki; Abe, Kota; Yokota, Shinpei; Matsuno, Rinta; Mikekado, Tsuyoshi; Yokoi, Hayato; Suzuki, Tohru

2015-01-01

The establishment of induced pluripotent stem (iPS) cell technology in fish could facilitate the establishment of novel cryopreservation techniques for storing selected aquaculture strains as frozen cells. In order to apply iPS cell technology to fish, we established a transgenic zebrafish line, Tg(Tru.oct4:EGFP), using green fluorescent protein (GFP) expression under the control of the oct4 gene promoter as a marker to evaluate multipotency in iPS cell preparations. We used the oct4 promoter from fugu (Takifugu rubripes) due to the compact nature of the fugu genome and to facilitate future applications of this technology in marine fishes. During embryogenesis, maternal GFP fluorescence was observed at the cleavage stage and zygotic GFP expression was observed from the start of the shield stage until approximately 24 h after fertilization. gfp messenger RNA (mRNA) was expressed by whole embryonic cells at the shield stage, and then restricted to the caudal neural tube in the latter stages of embryogenesis. These observations showed that GFP fluorescence and the regulation of gfp mRNA expression by the exogenous fugu oct4 promoter are well suited for monitoring endogenous oct4 mRNA expression in embryos. Bisulfite sequencing revealed that the rate of CpG methylation in the transgenic oct4 promoter was high in adult cells (98%) and low in embryonic cells (37%). These findings suggest that, as with the endogenous oct4 promoter, demethylation and methylation both take place normally in the transgenic oct4 promoter during embryogenesis. The embryonic cells harvested at the shield stage formed embryonic body-like cellular aggregates and maintained GFP fluorescence for 6 d when cultured on Transwell-COL Permeable Supports or a feeder layer of adult fin cells. Loss of GFP fluorescence by cultured cells was correlated with cellular differentiation. We consider that the Tg(Tru.oct4:EGFP) zebrafish line established here is well suited for monitoring multipotency in multipotent zebrafish cell cultures and for iPS cell preparation.

The transcription factor Nerfin-1 prevents reversion of neurons into neural stem cells.

PubMed

Froldi, Francesca; Szuperak, Milan; Weng, Chen-Fang; Shi, Wei; Papenfuss, Anthony T; Cheng, Louise Y

2015-01-15

Cellular dedifferentiation is the regression of a cell from a specialized state to a more multipotent state and is implicated in cancer. However, the transcriptional network that prevents differentiated cells from reacquiring stem cell fate is so far unclear. Neuroblasts (NBs), the Drosophila neural stem cells, are a model for the regulation of stem cell self-renewal and differentiation. Here we show that the Drosophila zinc finger transcription factor Nervous fingers 1 (Nerfin-1) locks neurons into differentiation, preventing their reversion into NBs. Following Prospero-dependent neuronal specification in the ganglion mother cell (GMC), a Nerfin-1-specific transcriptional program maintains differentiation in the post-mitotic neurons. The loss of Nerfin-1 causes reversion to multipotency and results in tumors in several neural lineages. Both the onset and rate of neuronal dedifferentiation in nerfin-1 mutant lineages are dependent on Myc- and target of rapamycin (Tor)-mediated cellular growth. In addition, Nerfin-1 is required for NB differentiation at the end of neurogenesis. RNA sequencing (RNA-seq) and chromatin immunoprecipitation (ChIP) analysis show that Nerfin-1 administers its function by repression of self-renewing-specific and activation of differentiation-specific genes. Our findings support the model of bidirectional interconvertibility between neural stem cells and their post-mitotic progeny and highlight the importance of the Nerfin-1-regulated transcriptional program in neuronal maintenance. © 2015 Froldi et al.; Published by Cold Spring Harbor Laboratory Press.

Mechanism of proteasomal degradation of inositol trisphosphate receptors in CHO-K1 cells.

PubMed

Bhanumathy, Cunnigaiper D; Nakao, Steven K; Joseph, Suresh K

2006-02-10

myo-Inositol 1,4,5-trisphosphate receptor (IP3R) degradation occurs in response to carbachol (Cch) stimulation of CHO-K1 cells. The response was mediated by endogenous muscarinic receptors and was blocked by atropine or proteasomal inhibitors. We have used these cells to identify the sites of ubiquitination on IP3Rs and study the role of Ca2+ and substrate recognition properties of the degradation system using exogenously expressed IP3R constructs. Employing caspase-3 for IP3R cleavage, we show that Cch promotes polyubiquitination in the N-terminal domain and monoubiquitination in the C-terminal domain. The addition of extracellular Ca2+ to Ca2+-depleted Chinese hamster ovary (CHO) cells initiates IP3R degradation provided Cch is present. This effect is inhibited by thapsigargin. The data suggest that both a sustained elevation of IP3 and a minimal content of Ca2+ in the endoplasmic reticulum lumen is required to initiate IP3R degradation. Transient transfection of IP3R constructs into CHO cells indicated the selective degradation of only the SI+ splice variant of the type I IP3R. This was also the splice form present endogenously in these cells. A pore-defective, nonfunctional SI+ IP3R mutant (D2550A) was also degraded in Cch-stimulated cells. The Cch-mediated response in CHO cells provides a convenient model system to further analyze the Ca2+ dependence and structural requirements of the IP3R proteasomal degradation pathway.

Characterization of inositol phosphates in carrot (Daucus carota L. ) cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rincon, M.; Chen, Q.; Boss, W.F.

1989-01-01

We have shown previously that inositol-1,4,5-trisphosphate (IP{sub 3}) stimulates an efflux of {sup 45}Ca{sup 2+} from fusogenic carrot protoplasts. In light of these results, we suggested that IP{sub 3} might serve as a second messenger for the mobilization of intracellular Ca{sup 2+} in higher plant cells. To determine whether or not IP{sub 3} and other inositol phosphates were present in the carrot cells, the cells were labeled with myo-(2-{sup 3}H)inositol for 18 hours and extracted with ice-cold 10% trichloroacetic acid. The inositol metabolites were separated by anion exchange chromatography and by paper electrophoresis. We found that ({sup 3}H)inositol metabolites coelutedmore » with inositol bisphosphate (IP{sub 2}) and IP{sub 3} when separated by anion exchange chromatography. However, we could not detect IP{sub 2} or IP{sub 3} when the inositol metabolites were analyzed by paper electrophoresis even though the polyphosphoinositides, which are the source of IP{sub 2} and IP{sub 3}, were present in these cells. Thus, ({sup 3}H)inositol metabolites other than IP{sub 2} and IP{sub 3} had coeluted on the anion exchange columns. The data indicate that either IP{sub 3} is rapidly metabolized or that it is not present at a detectable level in the carrot cells.« less

Nuclear factor I-C reciprocally regulates adipocyte and osteoblast differentiation via control of canonical Wnt signaling.

PubMed

Zhou, Jie; Wang, Shan; Qi, Qi; Yang, Xiaoyue; Zhu, Endong; Yuan, Hairui; Li, Xuemei; Liu, Ying; Li, Xiaoxia; Wang, Baoli

2017-05-01

Nuclear factor I-C (NFIC) has recently been identified as an important player in osteogenesis and bone homeostasis in vivo However, the molecular mechanisms involved have yet to be defined. In the current study, Nfic expression was altered in primary marrow stromal cells and established progenitor lines after adipogenic and osteogenic treatment. Overexpression of Nfic in stromal cells ST2, mesenchymal cells C3H10T1/2, and primary marrow stromal cells inhibited adipogenic differentiation, whereas it promoted osteogenic differentiation. Conversely, silencing of endogenous Nfic in the cell lines enhanced adipogenic differentiation, whereas it blocked osteogenic differentiation. Mechanism investigations revealed that Nfic overexpression promoted nuclear translocation of β-catenin and increased nuclear protein levels of β-catenin and transcription factor 7-like 2 (TCF7L2). Promoter studies and the chromatin immunoprecipitation (ChIP) assay revealed that NFIC directly binds to the promoter of low-density lipoprotein receptor-related protein 5 (Lrp5) and thereafter transactivates the promoter. Finally, inactivation of canonical Wnt signaling in ST2 attenuated the inhibition of adipogenic differentiation and stimulation of osteogenic differentiation by NFIC. Our study suggests that NFIC balances adipogenic and osteogenic differentiation from progenitor cells through controlling canonical Wnt signaling and highlights the potential of NFIC as a target for new therapies to control metabolic disorders like osteoporosis and obesity.-Zhou, J., Wang, S., Qi, Q., Yang, X., Zhu, E., Yuan, H., Li, X., Liu, Y., Li, X., Wang, B. Nuclear factor I-C reciprocally regulates adipocyte and osteoblast differentiation via control of canonical Wnt signaling. © FASEB.

CCAAT/enhancer-binding protein β regulates the repression of type II collagen expression during the differentiation from proliferative to hypertrophic chondrocytes.

PubMed

Ushijima, Takahiro; Okazaki, Ken; Tsushima, Hidetoshi; Iwamoto, Yukihide

2014-01-31

CCAAT/enhancer-binding protein β (C/EBPβ) is a transcription factor that promotes hypertrophic differentiation by stimulating type X collagen and matrix metalloproteinase 13 during chondrocyte differentiation. However, the effect of C/EBPβ on proliferative chondrocytes is unclear. Here, we investigated whether C/EBPβ represses type II collagen (COL2A1) expression and is involved in the regulation of sex-determining region Y-type high mobility group box 9 (SOX9), a crucial factor for transactivation of Col2a1. Endogenous expression of C/EBPβ in the embryonic growth plate and differentiated ATDC5 cells were opposite to those of COL2A1 and SOX9. Overexpression of C/EBPβ by adenovirus vector in ATDC5 cells caused marked repression of Col2a1. The expression of Sox9 mRNA and nuclear protein was also repressed, resulting in decreased binding of SOX9 to the Col2a1 enhancer as shown by a ChIP assay. Knockdown of C/EBPβ by lentivirus expressing shRNA caused significant stimulation of these genes in ATDC5 cells. Reporter assays demonstrated that C/EBPβ repressed transcriptional activity of Col2a1. Deletion and mutation analysis showed that the C/EBPβ core responsive element was located between +2144 and +2152 bp within the Col2a1 enhancer. EMSA and ChIP assays also revealed that C/EBPβ directly bound to this region. Ex vivo organ cultures of mouse limbs transfected with C/EBPβ showed that the expression of COL2A1 and SOX9 was reduced upon ectopic C/EBPβ expression. Together, these results indicated that C/EBPβ represses the transcriptional activity of Col2a1 both directly and indirectly through modulation of Sox9 expression. This consequently promotes the phenotypic conversion from proliferative to hypertrophic chondrocytes during chondrocyte differentiation.

[Embryonic stem cells - a scientific by-product of the assisted reproduction technology?].

PubMed

Sterthaus, Oliver; Zhang, Hong; De Geyter, Christian

2009-12-01

The differentiation potential of embryonic stem (ES) cells seems to be higher when compared to adult stem cells, which mainly differentiate into certain tissue types only. ES cells have the potential to play an important role in regenerative medicine as demonstrated with murine ES cells. However, with human embryonic stem cells (hESC) several obstacles still have to be overcome, when these are to be used in clinical applications. The expansion of hESC, safety issues as well as the immune-tolerance after transplantation are all problems that still have to be solved. Since 2005 the derivation of hESC lines from super-numerous embryos has become permitted in Switzerland, albeit under strictly restrictive guidelines. In 2008 the Basler hESC laboratory was successful in derivating the first hESC line with a normal chromosome complement in Switzerland (CHES2). Now, new applications allow the personalized establishment of immune-tolerant stem cells, which lead to the replacement of therapeutic cloning by induced pluripotent stem cells (iPS).

Naïve-like conversion enhances the difference in innate in vitro differentiation capacity between rabbit ES cells and iPS cells

PubMed Central

HONSHO, Kimiko; HIROSE, Michiko; HATORI, Masanori; YASMIN, Lubna; IZU, Haruna; MATOBA, Shogo; TOGAYACHI, Sumie; MIYOSHI, Hiroyuki; SANKAI, Tadashi; OGURA, Atsuo; HONDA, Arata

2014-01-01

Quality evaluation of pluripotent stem cells using appropriate animal models needs to be improved for human regenerative medicine. Previously, we demonstrated that although the in vitro neural differentiating capacity of rabbit induced pluripotent stem cells (iPSCs) can be mitigated by improving their baseline level of pluripotency, i.e., by converting them into the so-called “naïve-like” state, the effect after such conversion of rabbit embryonic stem cells (ESCs) remains to be elucidated. Here we found that naïve-like conversion enhanced the differences in innate in vitro differentiation capacity between ESCs and iPSCs. Naïve-like rabbit ESCs exhibited several features indicating pluripotency, including the capacity for teratoma formation. They differentiated into mature oligodendrocytes much more effectively (3.3–7.2 times) than naïve-like iPSCs. This suggests an inherent variation in differentiation potential in vitro among PSC lines. When naïve-like ESCs were injected into preimplantation rabbit embryos, although they contributed efficiently to forming the inner cell mass of blastocysts, no chimeric pups were obtained. Thus, in vitro neural differentiation following naïve-like conversion is a promising option for determining the quality of PSCs without the need to demonstrate chimeric contribution. These results provide an opportunity to evaluate which pluripotent stem cells or treatments are best suited for therapeutic use. PMID:25345855

Generation of Isogenic Human iPS Cell Line Precisely Corrected by Genome Editing Using the CRISPR/Cas9 System.

PubMed

Grobarczyk, Benjamin; Franco, Bénédicte; Hanon, Kevin; Malgrange, Brigitte

2015-10-01

Genome engineering and human iPS cells are two powerful technologies, which can be combined to highlight phenotypic differences and identify pathological mechanisms of complex diseases by providing isogenic cellular material. However, very few data are available regarding precise gene correction in human iPS cells. Here, we describe an optimized stepwise protocol to deliver CRISPR/Cas9 plasmids in human iPS cells. We highlight technical issues especially those associated to human stem cell culture and to the correction of a point mutation to obtain isogenic iPS cell line, without inserting any resistance cassette. Based on a two-steps clonal isolation protocol (mechanical picking followed by enzymatic dissociation), we succeed to select and expand corrected human iPS cell line with a great efficiency (more than 2% of the sequenced colonies). This protocol can also be used to obtain knock-out cell line from healthy iPS cell line by the NHEJ pathway (with about 15% efficiency) and reproduce disease phenotype. In addition, we also provide protocols for functional validation tests after every critical step.

Efficient genomic correction methods in human iPS cells using CRISPR-Cas9 system.

PubMed

Li, Hongmei Lisa; Gee, Peter; Ishida, Kentaro; Hotta, Akitsu

2016-05-15

Precise gene correction using the CRISPR-Cas9 system in human iPS cells holds great promise for various applications, such as the study of gene functions, disease modeling, and gene therapy. In this review article, we summarize methods for effective editing of genomic sequences of iPS cells based on our experiences correcting dystrophin gene mutations with the CRISPR-Cas9 system. Designing specific sgRNAs as well as having efficient transfection methods and proper detection assays to assess genomic cleavage activities are critical for successful genome editing in iPS cells. In addition, because iPS cells are fragile by nature when dissociated into single cells, a step-by-step confirmation during the cell recovery process is recommended to obtain an adequate number of genome-edited iPS cell clones. We hope that the techniques described here will be useful for researchers from diverse backgrounds who would like to perform genome editing in iPS cells. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Molecular Mechanisms of Induced Pluripotency

PubMed Central

Muchkaeva, I.A.; Dashinimaev, E.B.; Terskikh, V.V.; Sukhanov, Y.V.; Vasiliev, A.V.

2012-01-01

In this review the distinct aspects of somatic cell reprogramming are discussed. The molecular mechanisms of generation of induced pluripotent stem (iPS) cells from somatic cells via the introduction of transcription factors into adult somatic cells are considered. Particular attention is focused on the generation of iPS cells without genome modifications via the introduction of the mRNA of transcription factors or the use of small molecules. Furthermore, the strategy of direct reprogramming of somatic cells omitting the generation of iPS cells is considered. The data concerning the differences between ES and iPS cells and the problem of epigenetic memory are also discussed. In conclusion, the possibility of using iPS cells in regenerative medicine is considered. PMID:22708059

Generation and Characterization of Induced Pluripotent Stem Cells from Aid-Deficient Mice

PubMed Central

Shimamoto, Ren; Amano, Naoki; Ichisaka, Tomoko; Watanabe, Akira; Yamanaka, Shinya; Okita, Keisuke

2014-01-01

It has been shown that DNA demethylation plays a pivotal role in the generation of induced pluripotent stem (iPS) cells. However, the underlying mechanism of this action is still unclear. Previous reports indicated that activation-induced cytidine deaminase (Aid, also known as Aicda) is involved in DNA demethylation in several developmental processes, as well as cell fusion-mediated reprogramming. Based on these reports, we hypothesized that Aid may be involved in the DNA demethylation that occurs during the generation of iPS cells. In this study, we examined the function of Aid in iPS cell generation using Aid knockout (Aid −/−) mice expressing a GFP reporter under the control of a pluripotent stem cell marker, Nanog. By introducing Oct3/4, Sox2, Klf4 and c-Myc, Nanog-GFP-positive iPS cells could be generated from the fibroblasts and primary B cells of Aid −/− mice. Their induction efficiency was similar to that of wild-type (Aid +/+) iPS cells. The Aid −/− iPS cells showed normal proliferation and gave rise to chimeras, indicating their capacity for self-renewal and pluripotency. A comprehensive DNA methylation analysis showed only a few differences between Aid +/+ and Aid −/− iPS cells. These data suggest that Aid does not have crucial functions in DNA demethylation during iPS cell generation. PMID:24718089

Inositol pyrophosphates promote tumor growth and metastasis by antagonizing liver kinase B1

PubMed Central

Rao, Feng; Xu, Jing; Fu, Chenglai; Cha, Jiyoung Y.; Gadalla, Moataz M.; Xu, Risheng; Barrow, James C.; Snyder, Solomon H.

2015-01-01

The inositol pyrophosphates, molecular messengers containing an energetic pyrophosphate bond, impact a wide range of biologic processes. They are generated primarily by a family of three inositol hexakisphosphate kinases (IP6Ks), the principal product of which is diphosphoinositol pentakisphosphate (IP7). We report that IP6K2, via IP7 synthesis, is a major mediator of cancer cell migration and tumor metastasis in cell culture and in intact mice. IP6K2 acts by enhancing cell-matrix adhesion and decreasing cell–cell adhesion. This action is mediated by IP7-elicited nuclear sequestration and inactivation of the tumor suppressor liver kinase B1 (LKB1). Accordingly, inhibitors of IP6K2 offer promise in cancer therapy. PMID:25617365

Brg1 coordinates multiple processes during retinogenesis and is a tumor suppressor in retinoblastoma

DOE PAGES

Aldiri, Issam; Ajioka, Itsuki; Xu, Beisi; ...

2015-12-01

Retinal development requires precise temporal and spatial coordination of cell cycle exit, cell fate specification, cell migration and differentiation. When this process is disrupted, retinoblastoma, a developmental tumor of the retina, can form. Epigenetic modulators are central to precisely coordinating developmental events, and many epigenetic processes have been implicated in cancer. Studying epigenetic mechanisms in development is challenging because they often regulate multiple cellular processes; therefore, elucidating the primary molecular mechanisms involved can be difficult. Here we explore the role of Brg1 (Smarca4) in retinal development and retinoblastoma in mice using molecular and cellular approaches. Brg1 was found to regulatemore » retinal size by controlling cell cycle length, cell cycle exit and cell survival during development. Brg1 was not required for cell fate specification but was required for photoreceptor differentiation and cell adhesion/polarity programs that contribute to proper retinal lamination during development. The combination of defective cell differentiation and lamination led to retinal degeneration in Brg1-deficient retinae. Despite the hypocellularity, premature cell cycle exit, increased cell death and extended cell cycle length, retinal progenitor cells persisted in Brg1-deficient retinae, making them more susceptible to retinoblastoma. In conclusion, ChIP-Seq analysis suggests that Brg1 might regulate gene expression through multiple mechanisms.« less

Brg1 coordinates multiple processes during retinogenesis and is a tumor suppressor in retinoblastoma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aldiri, Issam; Ajioka, Itsuki; Xu, Beisi

Retinal development requires precise temporal and spatial coordination of cell cycle exit, cell fate specification, cell migration and differentiation. When this process is disrupted, retinoblastoma, a developmental tumor of the retina, can form. Epigenetic modulators are central to precisely coordinating developmental events, and many epigenetic processes have been implicated in cancer. Studying epigenetic mechanisms in development is challenging because they often regulate multiple cellular processes; therefore, elucidating the primary molecular mechanisms involved can be difficult. Here we explore the role of Brg1 (Smarca4) in retinal development and retinoblastoma in mice using molecular and cellular approaches. Brg1 was found to regulatemore » retinal size by controlling cell cycle length, cell cycle exit and cell survival during development. Brg1 was not required for cell fate specification but was required for photoreceptor differentiation and cell adhesion/polarity programs that contribute to proper retinal lamination during development. The combination of defective cell differentiation and lamination led to retinal degeneration in Brg1-deficient retinae. Despite the hypocellularity, premature cell cycle exit, increased cell death and extended cell cycle length, retinal progenitor cells persisted in Brg1-deficient retinae, making them more susceptible to retinoblastoma. In conclusion, ChIP-Seq analysis suggests that Brg1 might regulate gene expression through multiple mechanisms.« less

Efficient and Reproducible Myogenic Differentiation from Human iPS Cells: Prospects for Modeling Miyoshi Myopathy In Vitro

PubMed Central

Tanaka, Akihito; Woltjen, Knut; Miyake, Katsuya; Hotta, Akitsu; Ikeya, Makoto; Yamamoto, Takuya; Nishino, Tokiko; Shoji, Emi; Sehara-Fujisawa, Atsuko; Manabe, Yasuko; Fujii, Nobuharu; Hanaoka, Kazunori; Era, Takumi; Yamashita, Satoshi; Isobe, Ken-ichi; Kimura, En; Sakurai, Hidetoshi

2013-01-01

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70–90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs. PMID:23626698

Stem/progenitor cells from inflamed human dental pulp retain tissue regeneration potential

PubMed Central

Alongi, Dominick J; Yamaza, Takayoshi; Song, Yingjie; Fouad, Ashraf F; Romberg, Elaine E; Shi, Songtao; Tuan, Rocky S; Huang, George T-J

2011-01-01

Background Potent stem/progenitor cells have been isolated from normal human dental pulps termed dental pulp stem cells (DPSCs). However, it is unknown whether these cells exist in inflamed pulps (IPs). Aims To determine whether DPSCs can be identified and isolated from IPs; and if they can be successfully cultured, whether they retain tissue regeneration potential in vivo. Materials & methods DPSCs from freshly collected normal pulps (NPs) and IPs were characterized in vitro and their tissue regeneration potential tested using an in vivo study model. Results The immunohistochemical analysis showed that IPs expressed higher levels of mesenchymal stem cell markers STRO-1, CD90, CD105 and CD146 compared with NPs (p < 0.05). Flow cytometry analysis showed that DPSCs from both NPs and IPs expressed moderate to high levels of CD146, stage-specific embryonic antigen-4, CD73 and CD166. Total population doubling of DPSCs-IPs (44.6 ± 2.9) was lower than that of DPSCs-NPs (58.9 ± 2.5) (p < 0.05), and DPSCs-IPs appeared to have a decreased osteo/dentinogenic potential compared with DPSCs-NPs based on the mineral deposition in cultures. Nonetheless, DPSCs-IPs formed pulp/dentin complexes similar to DPSCs-NPs when transplanted into immunocompromised mice. Conclusion DPSCs-IPs can be isolated and their mesenchymal stem cell marker profiles are similar to those from NPs. Although some stem cell properties of DPSCs-IPs were altered, cells from some samples remained potent in tissue regeneration in vivo. PMID:20465527

Human induced pluripotent stem cells labeled with fluorescent magnetic nanoparticles for targeted imaging and hyperthermia therapy for gastric cancer.

PubMed

Li, Chao; Ruan, Jing; Yang, Meng; Pan, Fei; Gao, Guo; Qu, Su; Shen, You-Lan; Dang, Yong-Jun; Wang, Kan; Jin, Wei-Lin; Cui, Da-Xiang

2015-09-01

Human induced pluripotent stem (iPS) cells exhibit great potential for generating functional human cells for medical therapies. In this paper, we report for use of human iPS cells labeled with fluorescent magnetic nanoparticles (FMNPs) for targeted imaging and synergistic therapy of gastric cancer cells in vivo. Human iPS cells were prepared and cultured for 72 h. The culture medium was collected, and then was co-incubated with MGC803 cells. Cell viability was analyzed by the MTT method. FMNP-labeled human iPS cells were prepared and injected into gastric cancer-bearing nude mice. The mouse model was observed using a small-animal imaging system. The nude mice were irradiated under an external alternating magnetic field and evaluated using an infrared thermal mapping instrument. Tumor sizes were measured weekly. iPS cells and the collected culture medium inhibited the growth of MGC803 cells. FMNP-labeled human iPS cells targeted and imaged gastric cancer cells in vivo, as well as inhibited cancer growth in vivo through the external magnetic field. FMNP-labeled human iPS cells exhibit considerable potential in applications such as targeted dual-mode imaging and synergistic therapy for early gastric cancer.

Inference of hierarchical regulatory network of estrogen-dependent breast cancer through ChIP-based data

PubMed Central

2010-01-01

Background Global profiling of in vivo protein-DNA interactions using ChIP-based technologies has evolved rapidly in recent years. Although many genome-wide studies have identified thousands of ERα binding sites and have revealed the associated transcription factor (TF) partners, such as AP1, FOXA1 and CEBP, little is known about ERα associated hierarchical transcriptional regulatory networks. Results In this study, we applied computational approaches to analyze three public available ChIP-based datasets: ChIP-seq, ChIP-PET and ChIP-chip, and to investigate the hierarchical regulatory network for ERα and ERα partner TFs regulation in estrogen-dependent breast cancer MCF7 cells. 16 common TFs and two common new TF partners (RORA and PITX2) were found among ChIP-seq, ChIP-chip and ChIP-PET datasets. The regulatory networks were constructed by scanning the ChIP-peak region with TF specific position weight matrix (PWM). A permutation test was performed to test the reliability of each connection of the network. We then used DREM software to perform gene ontology function analysis on the common genes. We found that FOS, PITX2, RORA and FOXA1 were involved in the up-regulated genes. We also conducted the ERα and Pol-II ChIP-seq experiments in tamoxifen resistance MCF7 cells (denoted as MCF7-T in this study) and compared the difference between MCF7 and MCF7-T cells. The result showed very little overlap between these two cells in terms of targeted genes (21.2% of common genes) and targeted TFs (25% of common TFs). The significant dissimilarity may indicate totally different transcriptional regulatory mechanisms between these two cancer cells. Conclusions Our study uncovers new estrogen-mediated regulatory networks by mining three ChIP-based data in MCF7 cells and ChIP-seq data in MCF7-T cells. We compared the different ChIP-based technologies as well as different breast cancer cells. Our computational analytical approach may guide biologists to further study the underlying mechanisms in breast cancer cells or other human diseases. PMID:21167036

HSP90 regulates cell survival via inositol hexakisphosphate kinase-2

PubMed Central

Chakraborty, Anutosh; Koldobskiy, Michael A.; Sixt, Katherine M.; Juluri, Krishna R.; Mustafa, Asif K.; Snowman, Adele M.; van Rossum, Damian B.; Patterson, Randen L.; Snyder, Solomon H.

2008-01-01

Heat-shock proteins (HSPs) are abundant, inducible proteins best known for their ability to maintain the conformation of proteins and to refold damaged proteins. Some HSPs, especially HSP90, can be antiapoptotic and the targets of anticancer drugs. Inositol hexakisphosphate kinase-2 (IP6K2), one of a family of enzymes generating the inositol pyrophosphate IP7 [diphosphoinositol pentakisphosphate (5-PP-IP5)], mediates apoptosis. Increased IP6K2 activity sensitizes cancer cells to stressors, whereas its depletion blocks cell death. We now show that HSP90 physiologically binds IP6K2 and inhibits its catalytic activity. Drugs and selective mutations that abolish HSP90–IP6K2 binding elicit activation of IP6K2, leading to cell death. Thus, the prosurvival actions of HSP90 reflect the inhibition of IP6K2, suggesting that selectively blocking this interaction could provide effective and safer modes of chemotherapy. PMID:18195352

Parallel Inhibition of Dopamine Amacrine Cells and Intrinsically Photosensitive Retinal Ganglion Cells in a Non-Image-Forming Visual Circuit of the Mouse Retina

PubMed Central

Vuong, Helen E.; Hardi, Claudia N.; Barnes, Steven

2015-01-01

An inner retinal microcircuit composed of dopamine (DA)-containing amacrine cells and melanopsin-containing, intrinsically photosensitive retinal ganglion cells (M1 ipRGCs) process information about the duration and intensity of light exposures, mediating light adaptation, circadian entrainment, pupillary reflexes, and other aspects of non-image-forming vision. The neural interaction is reciprocal: M1 ipRGCs excite DA amacrine cells, and these, in turn, feed inhibition back onto M1 ipRGCs. We found that the neuropeptide somatostatin [somatotropin release inhibiting factor (SRIF)] also inhibits the intrinsic light response of M1 ipRGCs and postulated that, to tune the bidirectional interaction of M1 ipRGCs and DA amacrine cells, SRIF amacrine cells would provide inhibitory modulation to both cell types. SRIF amacrine cells, DA amacrine cells, and M1 ipRGCs form numerous contacts. DA amacrine cells and M1 ipRGCs express the SRIF receptor subtypes sst2A and sst4 respectively. SRIF modulation of the microcircuit was investigated with targeted patch-clamp recordings of DA amacrine cells in TH–RFP mice and M1 ipRGCs in OPN4–EGFP mice. SRIF increases K+ currents, decreases Ca2+ currents, and inhibits spike activity in both cell types, actions reproduced by the selective sst2A agonist L-054,264 (N-[(1R)-2-[[[(1S*,3R*)-3-(aminomethyl)cyclohexyl]methyl]amino]-1-(1H-indol-3-ylmethyl)-2-oxoethyl]spiro[1H-indene-1,4′-piperidine]-1′-carboxamide) in DA amacrine cells and the selective sst4 agonist L-803,087 (N2-[4-(5,7-difluoro-2-phenyl-1H-indol-3-yl)-1-oxobutyl]-l-arginine methyl ester trifluoroacetate) in M1 ipRGCs. These parallel actions of SRIF may serve to counteract the disinhibition of M1 ipRGCs caused by SRIF inhibition of DA amacrine cells. This allows the actions of SRIF on DA amacrine cells to proceed with adjusting retinal DA levels without destabilizing light responses by M1 ipRGCs, which project to non-image-forming targets in the brain. SIGNIFICANCE STATEMENT Amacrine cells form multiple microcircuits in the inner retina to mediate visual processing, although their organization and function remain incompletely understood. The somatostatin [somatotropin release inhibiting factor (SRIF)]- and dopamine (DA)-releasing amacrine cells act globally, and, in this study, they are shown to interact and modulate the light response of intrinsically photosensitive retinal ganglion cells (ipRGCs). SRIF amacrine cells target both DA amacrine cells and M1 ipRGCs for inhibition. The parallel actions of SRIF may serve to compensate for the loss of DA-mediated inhibition of M1 ipRGCs. This inhibitory tuning is of particular importance because the DA system mediates a broad range of light adaptational actions in the retina and M1 ipRGCs project to brain areas that influence sleep, mood, cognition, circadian entrainment, and pupillary reflexes. PMID:26631476

Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors

PubMed Central

Hiramatsu, Kunihiko; Sasagawa, Satoru; Outani, Hidetatsu; Nakagawa, Kanako; Yoshikawa, Hideki; Tsumaki, Noriyuki

2011-01-01

Repair of cartilage injury with hyaline cartilage continues to be a challenging clinical problem. Because of the limited number of chondrocytes in vivo, coupled with in vitro de-differentiation of chondrocytes into fibrochondrocytes, which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells. PMID:21293062

Origins and implications of pluripotent stem cell variability and heterogeneity

PubMed Central

Cahan, Patrick; Daley, George Q.

2014-01-01

Pluripotent stem cells constitute a platform to model disease and developmental processes and can potentially be used in regenerative medicine. However, not all pluripotent cell lines are equal in their capacity to differentiate into desired cell types in vitro. Genetic and epigenetic variations contribute to functional variability between cell lines and heterogeneity within clones. These genetic and epigenetic variations could ‘lock’ the pluripotency network resulting in residual pluripotent cells or alter the signalling response of developmental pathways leading to lineage bias. The molecular contributors to functional variability and heterogeneity in both embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are only beginning to emerge, yet they are crucial to the future of the stem cell field. PMID:23673969

IpsA, a novel LacI-type regulator, is required for inositol-derived lipid formation in Corynebacteria and Mycobacteria.

PubMed

Baumgart, Meike; Luder, Kerstin; Grover, Shipra; Gätgens, Cornelia; Besra, Gurdyal S; Frunzke, Julia

2013-12-30

The development of new drugs against tuberculosis and diphtheria is focused on disrupting the biogenesis of the cell wall, the unique architecture of which confers resistance against current therapies. The enzymatic pathways involved in the synthesis of the cell wall by these pathogens are well understood, but the underlying regulatory mechanisms are largely unknown. Here, we characterize IpsA, a LacI-type transcriptional regulator conserved among Mycobacteria and Corynebacteria that plays a role in the regulation of cell wall biogenesis. IpsA triggers myo-inositol formation by activating ino1, which encodes inositol phosphate synthase. An ipsA deletion mutant of Corynebacterium glutamicum cultured on glucose displayed significantly impaired growth and presented an elongated cell morphology. Further studies revealed the absence of inositol-derived lipids in the cell wall and a complete loss of mycothiol biosynthesis. The phenotype of the C. glutamicum ipsA deletion mutant was complemented to different extend by homologs from Corynebacterium diphtheriae (dip1969) and Mycobacterium tuberculosis (rv3575), indicating the conserved function of IpsA in the pathogenic species. Additional targets of IpsA with putative functions in cell wall biogenesis were identified and IpsA was shown to bind to a conserved palindromic motif within the corresponding promoter regions. Myo-inositol was identified as an effector of IpsA, causing the dissociation of the IpsA-DNA complex in vitro. This characterization of IpsA function and of its regulon sheds light on the complex transcriptional control of cell wall biogenesis in the mycolata taxon and generates novel targets for drug development.

A Facile Method to Establish Human Induced Pluripotent Stem Cells From Adult Blood Cells Under Feeder-Free and Xeno-Free Culture Conditions: A Clinically Compliant Approach

PubMed Central

Chou, Bin-Kuan; Gu, Haihui; Gao, Yongxing; Dowey, Sarah N.; Wang, Ying; Shi, Jun; Li, Yanxin; Ye, Zhaohui; Cheng, Tao

2015-01-01

Reprogramming human adult blood mononuclear cells (MNCs) cells by transient plasmid expression is becoming increasingly popular as an attractive method for generating induced pluripotent stem (iPS) cells without the genomic alteration caused by genome-inserting vectors. However, its efficiency is relatively low with adult MNCs compared with cord blood MNCs and other fetal cells and is highly variable among different adult individuals. We report highly efficient iPS cell derivation under clinically compliant conditions via three major improvements. First, we revised a combination of three EBNA1/OriP episomal vectors expressing five transgenes, which increased reprogramming efficiency by ≥10–50-fold from our previous vectors. Second, human recombinant vitronectin proteins were used as cell culture substrates, alleviating the need for feeder cells or animal-sourced proteins. Finally, we eliminated the previously critical step of manually picking individual iPS cell clones by pooling newly emerged iPS cell colonies. Pooled cultures were then purified based on the presence of the TRA-1-60 pluripotency surface antigen, resulting in the ability to rapidly expand iPS cells for subsequent applications. These new improvements permit a consistent and reliable method to generate human iPS cells with minimal clonal variations from blood MNCs, including previously difficult samples such as those from patients with paroxysmal nocturnal hemoglobinuria. In addition, this method of efficiently generating iPS cells under feeder-free and xeno-free conditions allows for the establishment of clinically compliant iPS cell lines for future therapeutic applications. PMID:25742692

Tetramethylpyrazine promotes SH-SY5Y cell differentiation into neurons through epigenetic regulation of Topoisomerase IIβ.

PubMed

Yan, Y; Zhao, J; Cao, C; Jia, Z; Zhou, N; Han, S; Wang, Y; Xu, Y; Zhao, J; Yan, Y; Cui, H

2014-10-10

Tetramethylpyrazine (TMP) is an active compound extracted from the traditional Chinese medicinal herb Chuanxiong. Recently, it has been reported that TMP enhances neurogenesis, and promotes neural stem cell differentiation toward neurons. However, its molecular basis remains unknown. Topoisomerase IIβ (TopoIIβ) is a nuclear enzyme with an essential role in neuronal development. This study aimed to investigate whether TopoIIβ is involved in TMP-induced neuronal differentiation. We examined the effect of TMP on neuronal differentiation of SH-SY5Y cells. It was found that TMP inhibited cell proliferation and induced G0/G1 cell cycle arrest. TMP promoted SH-SY5Y cells to differentiate toward post-mitotic neurons characterized by long, out-branched neurites and up-regulated neuronal markers, microtubule-associated protein 2 (MAP2) and tau. Meanwhile, we demonstrated that TopoIIβ was highly expressed following TMP treatment. To unravel how TMP affects TopoIIβ expression, two chromatin active markers, acetylated histone H3 (Ac-H3) and acetylated histone H4 (Ac-H4) were examined in this study. Our data showed that the levels of Ac-H3 and Ac-H4 were positively correlated with TopoIIβ expression in the processes of neuronal differentiation. We further performed chromatin immunoprecipitation (ChIP) analysis and identified that TMP enhanced the recruitment of Ac-H3 and Ac-H4 to the TopoIIβ gene promoter region. Therefore, we concluded that TMP may stimulate neuronal differentiation of SH-SY5Y cells through epigenetic regulation of TopoIIβ. These results suggest a novel molecular mechanism underlying TMP-promoted neuronal differentiation. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Human iPS cell-derived astrocyte transplants preserve respiratory function after spinal cord injury

PubMed Central

Li, Ke; Javed, Elham; Scura, Daniel; Hala, Tamara J.; Seetharam, Suneil; Falnikar, Aditi; Richard, Jean-Philippe; Chorath, Ashley; Maragakis, Nicholas J.; Wright, Megan C.; Lepore, Angelo C.

2015-01-01

Transplantation-based replacement of lost and/or dysfunctional astrocytes is a promising therapy for spinal cord injury (SCI) that has not been extensively explored, despite the integral roles played by astrocytes in the central nervous system (CNS). Induced pluripotent stem (iPS) cells are a clinically-relevant source of pluripotent cells that both avoid ethical issues of embryonic stem cells and allow for homogeneous derivation of mature cell types in large quantities, potentially in an autologous fashion. Despite their promise, the iPS cell field is in its infancy with respect to evaluating in vivo graft integration and therapeutic efficacy in SCI models. Astrocytes express the major glutamate transporter, GLT1, which is responsible for the vast majority of glutamate uptake in spinal cord. Following SCI, compromised GLT1 expression/function can increase susceptibility to excitotoxicity. We therefore evaluated intraspinal transplantation of human iPS cell-derived astrocytes (hIPSAs) following cervical contusion SCI as a novel strategy for reconstituting GLT1 expression and for protecting diaphragmatic respiratory neural circuitry. Transplant-derived cells showed robust long-term survival post-injection and efficiently differentiated into astrocytes in injured spinal cord of both immunesuppressed mice and rats. However, the majority of transplant-derived astrocytes did not express high levels of GLT1, particularly at early times post-injection. To enhance their ability to modulate extracellular glutamate levels, we engineered hIPSAs with lentivirus to constitutively express GLT1. Overexpression significantly increased GLT1 protein and functional GLT1-mediated glutamate uptake levels in hIPSAs both in vitro and in vivo post-transplantation. Compared to human fibroblast control and unmodified hIPSA transplantation, GLT1-overexpressing hIPSAs reduced (1) lesion size within the injured cervical spinal cord, (2) morphological denervation by respiratory phrenic motor neurons at the diaphragm neuromuscular junction, and (3) functional diaphragm denervation as measured by recording of spontaneous EMGs and evoked compound muscle action potentials. Our findings demonstrate that hiPSA transplantation is a therapeutically-powerful approach for SCI. PMID:26216662

Human iPS cell-derived astrocyte transplants preserve respiratory function after spinal cord injury.

PubMed

Li, Ke; Javed, Elham; Scura, Daniel; Hala, Tamara J; Seetharam, Suneil; Falnikar, Aditi; Richard, Jean-Philippe; Chorath, Ashley; Maragakis, Nicholas J; Wright, Megan C; Lepore, Angelo C

2015-09-01

Transplantation-based replacement of lost and/or dysfunctional astrocytes is a promising therapy for spinal cord injury (SCI) that has not been extensively explored, despite the integral roles played by astrocytes in the central nervous system (CNS). Induced pluripotent stem (iPS) cells are a clinically-relevant source of pluripotent cells that both avoid ethical issues of embryonic stem cells and allow for homogeneous derivation of mature cell types in large quantities, potentially in an autologous fashion. Despite their promise, the iPS cell field is in its infancy with respect to evaluating in vivo graft integration and therapeutic efficacy in SCI models. Astrocytes express the major glutamate transporter, GLT1, which is responsible for the vast majority of glutamate uptake in spinal cord. Following SCI, compromised GLT1 expression/function can increase susceptibility to excitotoxicity. We therefore evaluated intraspinal transplantation of human iPS cell-derived astrocytes (hIPSAs) following cervical contusion SCI as a novel strategy for reconstituting GLT1 expression and for protecting diaphragmatic respiratory neural circuitry. Transplant-derived cells showed robust long-term survival post-injection and efficiently differentiated into astrocytes in injured spinal cord of both immunesuppressed mice and rats. However, the majority of transplant-derived astrocytes did not express high levels of GLT1, particularly at early times post-injection. To enhance their ability to modulate extracellular glutamate levels, we engineered hIPSAs with lentivirus to constitutively express GLT1. Overexpression significantly increased GLT1 protein and functional GLT1-mediated glutamate uptake levels in hIPSAs both in vitro and in vivo post-transplantation. Compared to human fibroblast control and unmodified hIPSA transplantation, GLT1-overexpressing hIPSAs reduced (1) lesion size within the injured cervical spinal cord, (2) morphological denervation by respiratory phrenic motor neurons at the diaphragm neuromuscular junction, and (3) functional diaphragm denervation as measured by recording of spontaneous EMGs and evoked compound muscle action potentials. Our findings demonstrate that hiPSA transplantation is a therapeutically-powerful approach for SCI. Copyright © 2015 Elsevier Inc. All rights reserved.

Calcium sources used by post-natal human myoblasts during initial differentiation.

PubMed

Arnaudeau, Serge; Holzer, Nicolas; König, Stéphane; Bader, Charles R; Bernheim, Laurent

2006-08-01

Increases in cytoplasmic Ca(2+) are crucial for inducing the initial steps of myoblast differentiation that ultimately lead to fusion; yet the mechanisms that produce this elevated Ca(2+) have not been fully resolved. For example, it is still unclear whether the increase comes exclusively from membrane Ca(2+) influx or also from Ca(2+) release from internal stores. To address this, we investigated early differentiation of myoblast clones each derived from single post-natal human satellite cells. Initial differentiation was assayed by immunostaining myonuclei for the transcription factor MEF2. When Ca(2+) influx was eliminated by using low external Ca(2+) media, we found that approximately half the clones could still differentiate. Of the clones that required influx of external Ca(2+), most clones used T-type Ca(2+) channels, but others used store-operated channels as influx-generating mechanisms. On the other hand, clones that differentiated in low external Ca(2+) relied on Ca(2+) release from internal stores through IP(3) receptors. Interestingly, by following clones over time, we observed that some switched their preferred Ca(2+) source: clones that initially used calcium release from internal stores to differentiate later required Ca(2+) influx and inversely. In conclusion, we show that human myoblasts can use three alternative mechanisms to increase cytoplasmic Ca(2+) at the onset of the differentiation process: influx through T-types Ca(2+) channels, influx through store operated channels and release from internal stores through IP(3) receptors. In addition, we suggest that, probably because Ca(2+) elevation is essential during initial differentiation, myoblasts may be able to select between these alternate Ca(2+) pathways.

iPS cells to model CDKL5-related disorders

PubMed Central

Amenduni, Mariangela; De Filippis, Roberta; Cheung, Aaron Y L; Disciglio, Vittoria; Epistolato, Maria Carmela; Ariani, Francesca; Mari, Francesca; Mencarelli, Maria Antonietta; Hayek, Youssef; Renieri, Alessandra; Ellis, James; Meloni, Ilaria

2011-01-01

Rett syndrome (RTT) is a progressive neurologic disorder representing one of the most common causes of mental retardation in females. To date mutations in three genes have been associated with this condition. Classic RTT is caused by mutations in the MECP2 gene, whereas variants can be due to mutations in either MECP2 or FOXG1 or CDKL5. Mutations in CDKL5 have been identified both in females with the early onset seizure variant of RTT and in males with X-linked epileptic encephalopathy. CDKL5 is a kinase protein highly expressed in neurons, but its exact function inside the cell is unknown. To address this issue we established a human cellular model for CDKL5-related disease using the recently developed technology of induced pluripotent stem cells (iPSCs). iPSCs can be expanded indefinitely and differentiated in vitro into many different cell types, including neurons. These features make them the ideal tool to study disease mechanisms directly on the primarily affected neuronal cells. We derived iPSCs from fibroblasts of one female with p.Q347X and one male with p.T288I mutation, affected by early onset seizure variant and X-linked epileptic encephalopathy, respectively. We demonstrated that female CDKL5-mutated iPSCs maintain X-chromosome inactivation and clones express either the mutant CDKL5 allele or the wild-type allele that serve as an ideal experimental control. Array CGH indicates normal isogenic molecular karyotypes without detection of de novo CNVs in the CDKL5-mutated iPSCs. Furthermore, the iPS cells can be differentiated into neurons and are thus suitable to model disease pathogenesis in vitro. PMID:21750574

iPS cells to model CDKL5-related disorders.

PubMed

Amenduni, Mariangela; De Filippis, Roberta; Cheung, Aaron Y L; Disciglio, Vittoria; Epistolato, Maria Carmela; Ariani, Francesca; Mari, Francesca; Mencarelli, Maria Antonietta; Hayek, Youssef; Renieri, Alessandra; Ellis, James; Meloni, Ilaria

2011-12-01

Rett syndrome (RTT) is a progressive neurologic disorder representing one of the most common causes of mental retardation in females. To date mutations in three genes have been associated with this condition. Classic RTT is caused by mutations in the MECP2 gene, whereas variants can be due to mutations in either MECP2 or FOXG1 or CDKL5. Mutations in CDKL5 have been identified both in females with the early onset seizure variant of RTT and in males with X-linked epileptic encephalopathy. CDKL5 is a kinase protein highly expressed in neurons, but its exact function inside the cell is unknown. To address this issue we established a human cellular model for CDKL5-related disease using the recently developed technology of induced pluripotent stem cells (iPSCs). iPSCs can be expanded indefinitely and differentiated in vitro into many different cell types, including neurons. These features make them the ideal tool to study disease mechanisms directly on the primarily affected neuronal cells. We derived iPSCs from fibroblasts of one female with p.Q347X and one male with p.T288I mutation, affected by early onset seizure variant and X-linked epileptic encephalopathy, respectively. We demonstrated that female CDKL5-mutated iPSCs maintain X-chromosome inactivation and clones express either the mutant CDKL5 allele or the wild-type allele that serve as an ideal experimental control. Array CGH indicates normal isogenic molecular karyotypes without detection of de novo CNVs in the CDKL5-mutated iPSCs. Furthermore, the iPS cells can be differentiated into neurons and are thus suitable to model disease pathogenesis in vitro.

Parallel factor ChIP provides essential internal control for quantitative differential ChIP-seq.

PubMed

Guertin, Michael J; Cullen, Amy E; Markowetz, Florian; Holding, Andrew N

2018-04-17

A key challenge in quantitative ChIP combined with high-throughput sequencing (ChIP-seq) is the normalization of data in the presence of genome-wide changes in occupancy. Analysis-based normalization methods were developed for transcriptomic data and these are dependent on the underlying assumption that total transcription does not change between conditions. For genome-wide changes in transcription factor (TF) binding, these assumptions do not hold true. The challenges in normalization are confounded by experimental variability during sample preparation, processing and recovery. We present a novel normalization strategy utilizing an internal standard of unchanged peaks for reference. Our method can be readily applied to monitor genome-wide changes by ChIP-seq that are otherwise lost or misrepresented through analytical normalization. We compare our approach to normalization by total read depth and two alternative methods that utilize external experimental controls to study TF binding. We successfully resolve the key challenges in quantitative ChIP-seq analysis and demonstrate its application by monitoring the loss of Estrogen Receptor-alpha (ER) binding upon fulvestrant treatment, ER binding in response to estrodiol, ER mediated change in H4K12 acetylation and profiling ER binding in patient-derived xenographs. This is supported by an adaptable pipeline to normalize and quantify differential TF binding genome-wide and generate metrics for differential binding at individual sites.

IpsA, a novel LacI-type regulator, is required for inositol-derived lipid formation in Corynebacteria and Mycobacteria

PubMed Central

2013-01-01

Background The development of new drugs against tuberculosis and diphtheria is focused on disrupting the biogenesis of the cell wall, the unique architecture of which confers resistance against current therapies. The enzymatic pathways involved in the synthesis of the cell wall by these pathogens are well understood, but the underlying regulatory mechanisms are largely unknown. Results Here, we characterize IpsA, a LacI-type transcriptional regulator conserved among Mycobacteria and Corynebacteria that plays a role in the regulation of cell wall biogenesis. IpsA triggers myo-inositol formation by activating ino1, which encodes inositol phosphate synthase. An ipsA deletion mutant of Corynebacterium glutamicum cultured on glucose displayed significantly impaired growth and presented an elongated cell morphology. Further studies revealed the absence of inositol-derived lipids in the cell wall and a complete loss of mycothiol biosynthesis. The phenotype of the C. glutamicum ipsA deletion mutant was complemented to different extend by homologs from Corynebacterium diphtheriae (dip1969) and Mycobacterium tuberculosis (rv3575), indicating the conserved function of IpsA in the pathogenic species. Additional targets of IpsA with putative functions in cell wall biogenesis were identified and IpsA was shown to bind to a conserved palindromic motif within the corresponding promoter regions. Myo-inositol was identified as an effector of IpsA, causing the dissociation of the IpsA-DNA complex in vitro. Conclusions This characterization of IpsA function and of its regulon sheds light on the complex transcriptional control of cell wall biogenesis in the mycolata taxon and generates novel targets for drug development. PMID:24377418

Calcium waves in a grid of clustered channels with synchronous IP3 binding and unbinding.

PubMed

Rückl, M; Rüdiger, S

2016-11-01

Calcium signals in cells occur at multiple spatial scales and variable temporal duration. However, a physical explanation for transitions between long-lasting global oscillations and localized short-term elevations (puffs) of cytoplasmic Ca 2+ is still lacking. Here we introduce a phenomenological, coarse-grained model for the calcium variable, which is represented by ordinary differential equations. Due to its small number of parameters, and its simplicity, this model allows us to numerically study the interplay of multi-scale calcium concentrations with stochastic ion channel gating dynamics even in larger systems. We apply this model to a single cluster of inositol trisphosphate (IP 3 ) receptor channels and find further evidence for the results presented in earlier work: a single cluster may be capable of producing different calcium release types, where long-lasting events are accompanied by unbinding of IP 3 from the receptor (Rückl et al., PLoS Comput. Biol. 11, e1003965 (2015)). Finally, we show the practicability of the model in a grid of 64 clusters which is computationally intractable with previous high-resolution models. Here long-lasting events can lead to synchronized oscillations and waves, while short events stay localized. The frequency of calcium releases as well as their coherence can thereby be regulated by the amplitude of IP 3 stimulation. Finally the model allows for a new explanation of oscillating [IP 3 ], which is not based on metabolic production and degradation of IP 3 .

Role of MyD88 in TLR agonist-induced functional alterations of human adipose tissue-derived mesenchymal stem cells.

PubMed

Yu, Sungsook; Cho, Hyun Hwa; Joo, Hye Joon; Bae, Yong Chan; Jung, Jin Sup

2008-10-01

Toll-like receptors (TLRs) sense microorganism components and are critical host mediators of inflammation during infection. Recently, TLRs have been reported to be involved in cell proliferation and differentiation. We previously reported that TLR agonists might affect proliferation and differentiation of human adipose tissue-derived mesenchymal stem cells (hASCs). In this study, we sought to determine whether TLR signaling is dependent on MyD88 in hASCs. The hASCs were downregulated using LV-GFP-miR-MyD88, a lentiviral construct inserted siRNA against human MyD88 that significantly inhibited cell proliferation. MyD88 downregulation reduced NF-kappaB activation and enhancement of osteogenic differentiation induced by peptidoglycan (PGN) more significantly than that induced by lipopolysaccharide (LPS). Although LPS- and PGN-induced cytokine secretions were decreased greatly by MyD88 downregulation, IFN-gamma-induced protein-10 (IP10) and IFNbeta expression were enhanced by LPS irrespective of the downregulation of MyD88. These results suggest that TLR signaling is mediated via MyD88-independent pathways as well as MyD88-dependent pathways in hASCs and that MyD88 contributes to the regulation of cell proliferation and differentiation in hASCs.

[Progress and application prospect of pig induced pluripotent stem cells].

PubMed

Yan, Yi-Bo; Zhang, Yan-Li; Qi, Wei-Wei; Wan, Yong-Jie; Fan, Yi-Xuan; Wang, Feng

2011-04-01

Pig has always been the focus of establishing a big ungulate animal ES cell lines because of its convenient source, genetic similarity with humans, and their importance in animal husbandry, but little development is achieved. Induced pluripotent stem cells technology creates a new method of reprogramming somatic cells to pluripotent state. As the pig iPS cells is established and perfected, pig ES cells will be established in the coming years. The pig iPS cells will give a hint on other livestock ES cells. On the other hand, pig iPS cells can be used to improve the efficiency of transgenic cloning pigs to conduct effective breeding and conservation of breeds. It is particularly important that the pig iPS cells can provide new model for human medical research, a new donor cells for human tissue and organ engineering, and have extensive and far-reaching impact on the biomedical field. Here, we briefly review the major progress of iPS cells, and emphasize current state of pig iPS cells and its application prospect in biomedicine and animal husbandry in order to provide a useful reference for researchers working in this area.

Inositol Hexakisphosphate Inhibits Osteoclastogenesis on RAW 264.7 Cells and Human Primary Osteoclasts

PubMed Central

Arriero, María del Mar; Ramis, Joana M.; Perelló, Joan; Monjo, Marta

2012-01-01

Background Inoxitol hexakisphosphate (IP6) has been found to have an important role in biomineralization and a direct effect inhibiting mineralization of osteoblasts in vitro without impairing extracellular matrix production and expression of alkaline phosphatase. IP6 has been proposed to exhibit similar effects to those of bisphosphonates on bone resorption, however, its direct effect on osteoclasts (OCL) is presently unknown. Methodology/Principal Findings The aim of the present study was to investigate the effect of IP6 on the RAW 264.7 monocyte/macrophage mouse cell line and on human primary osteoclasts. On one hand, we show that IP6 decreases the osteoclastogenesis in RAW 264.7 cells induced by RANKL, without affecting cell proliferation or cell viability. The number of TRAP positive cells and mRNA levels of osteoclast markers such as TRAP, calcitonin receptor, cathepsin K and MMP-9 was decreased by IP6 on RANKL-treated cells. On the contrary, when giving IP6 to mature osteoclasts after RANKL treatment, a significant increase of bone resorption activity and TRAP mRNA levels was found. On the other hand, we show that 1 µM of IP6 inhibits osteoclastogenesis of human peripheral blood mononuclear cells (PBMNC) and their resorption activity both, when given to undifferentiated and to mature osteoclasts. Conclusions/Significance Our results demonstrate that IP6 inhibits osteoclastogenesis on human PBMNC and on the RAW264.7 cell line. Thus, IP6 may represent a novel type of selective inhibitor of osteoclasts and prove useful for the treatment of osteoporosis. PMID:22905230

Myt1L Promotes Differentiation of Oligodendrocyte Precursor Cells and is Necessary for Remyelination After Lysolecithin-Induced Demyelination.

PubMed

Shi, Yanqing; Shao, Qi; Li, Zhenghao; Gonzalez, Ginez A; Lu, Fengfeng; Wang, Dan; Pu, Yingyan; Huang, Aijun; Zhao, Chao; He, Cheng; Cao, Li

2018-04-01

The differentiation and maturation of oligodendrocyte precursor cells (OPCs) is essential for myelination and remyelination in the CNS. The failure of OPCs to achieve terminal differentiation in demyelinating lesions often results in unsuccessful remyelination in a variety of human demyelinating diseases. However, the molecular mechanisms controlling OPC differentiation under pathological conditions remain largely unknown. Myt1L (myelin transcription factor 1-like), mainly expressed in neurons, has been associated with intellectual disability, schizophrenia, and depression. In the present study, we found that Myt1L was expressed in oligodendrocyte lineage cells during myelination and remyelination. The expression level of Myt1L in neuron/glia antigen 2-positive (NG2 + ) OPCs was significantly higher than that in mature CC1 + oligodendrocytes. In primary cultured OPCs, overexpression of Myt1L promoted, while knockdown inhibited OPC differentiation. Moreover, Myt1L was potently involved in promoting remyelination after lysolecithin-induced demyelination in vivo. ChIP assays showed that Myt1L bound to the promoter of Olig1 and transcriptionally regulated Olig1 expression. Taken together, our findings demonstrate that Myt1L is an essential regulator of OPC differentiation, thereby supporting Myt1L as a potential therapeutic target for demyelinating diseases.

Adrenaline inhibits osteogenesis via repressing miR-21 expression.

PubMed

Chen, Danying; Wang, Zuolin

2017-01-01

Sympathetic signaling is involved in bone homeostasis; however, the cellular and molecular mechanisms remain unknown. In this study, we found that the psychological stress mediator adrenaline inhibited osteogenic differentiation of human bone marrow-derived stem cells (hMSC) by reducing microRNA-21 (miR-21) expression. Briefly, adrenaline significantly inhibited the osteogenic differentiation of hMSCs, as observed with both Alizarin red staining and maker gene expression (RUNX2, OSX, OCN, and OPN). During this process, miR-21 was suppressed by adrenaline via inhibition of histone acetylation, as verified by H3K9Ac chromatin immunoprecipitation (ChIP) assay. MiR-21 was confirmed to promote hMSC osteogenic differentiation, and overexpression of miR-21 reversed the impeditive effect of adrenaline on hMSC osteogenic differentiation. Our results demonstrate that down-regulation of miR-21 is responsible for the adrenaline-mediated inhibition of hMSC osteogenic differentiation. These findings indicate a regulation of bone metabolism by psychological stress and also provide a molecular basis for psychological stress-associated bone diseases. © 2016 International Federation for Cell Biology.

Future perspective of induced pluripotent stem cells for diagnosis, drug screening and treatment of human diseases.

PubMed

Lian, Qizhou; Chow, Yenyen; Esteban, Miguel Angel; Pei, Duanqing; Tse, Hung-Fat

2010-07-01

Recent advances in stem cell biology have transformed the understanding of cell physiology and developmental biology such that it can now play a more prominent role in the clinical application of stem cell and regenerative medicine. Success in the generation of human induced pluripotent stem cells (iPS) as well as related emerging technology on the iPS platform provide great promise in the development of regenerative medicine. Human iPS cells show almost identical properties to human embryonic stem cells (ESC) in pluripotency, but avoid many of their limitations of use. In addition, investigations into reprogramming of somatic cells to pluripotent stem cells facilitate a deeper understanding of human stem cell biology. The iPS cell technology has offered a unique platform for studying the pathogenesis of human disease, pharmacological and toxicological testing, and cell-based therapy. Nevertheless, significant challenges remain to be overcome before the promise of human iPS cell technology can be realised.

Identification of Substituted Pyrimido[5,4-b]indoles as Selective Toll-Like Receptor 4 Ligands

PubMed Central

2013-01-01

A cell-based high-throughput screen to identify small molecular weight stimulators of the innate immune system revealed substituted pyrimido[5,4-b]indoles as potent NFκB activators. The most potent hit compound selectively stimulated Toll-like receptor 4 (TLR4) in human and mouse cells. Synthetic modifications of the pyrimido[5,4-b]indole scaffold at the carboxamide, N-3, and N-5 positions revealed differential TLR4 dependent production of NFκB and type I interferon associated cytokines, IL-6 and interferon γ-induced protein 10 (IP-10) respectively. Specifically, a subset of compounds bearing phenyl and substituted phenyl carboxamides induced lower IL-6 release while maintaining higher IP-10 production, skewing toward the type I interferon pathway. Substitution at N-5 with short alkyl substituents reduced the cytotoxicity of the leading hit compound. Computational studies supported that active compounds appeared to bind primarily to MD-2 in the TLR4/MD-2 complex. These small molecules, which stimulate innate immune cells with minimal toxicity, could potentially be used as adjuvants or immune modulators. PMID:23656327

AHCYL1 Is Mediated by Estrogen-Induced ERK1/2 MAPK Cell Signaling and MicroRNA Regulation to Effect Functional Aspects of the Avian Oviduct

PubMed Central

Ahn, Suzie E.; Lee, Sang In; Bazer, Fuller W.; Han, Jae Yong; Song, Gwonhwa

2012-01-01

S-adenosylhomocysteine hydrolase-like protein 1 (AHCYL1), also known as IP3 receptor-binding protein released with IP3 (IRBIT), regulates IP3-induced Ca2+ release into the cytoplasm of cells. AHCYL1 is a critical regulator of early developmental stages in zebrafish, but little is known about the function of AHCYL1 or hormonal regulation of expression of the AHCYL1 gene in avian species. Therefore, we investigated differential expression profiles of the AHCYL1 gene in various adult organs and in oviducts from estrogen-treated chickens. Chicken AHCYL1 encodes for a protein of 540 amino acids that is highly conserved and has considerable homology to mammalian AHCYL1 proteins (>94% identity). AHCYL1 mRNA was expressed abundantly in various organs of chickens. Further, the synthetic estrogen agonist induced AHCYL1 mRNA and protein predominantly in luminal and glandular epithelial cells of the chick oviduct. In addition, estrogen activated AHCYL1 through the ERK1/2 signal transduction cascade and that activated expression of AHCYL1 regulated genes affecting oviduct development in chicks as well as calcium release in epithelial cells of the oviduct. Also, microRNAs, miR-124a, miR-1669, miR-1710 and miR-1782 influenced AHCYL1 expression in vitro via its 3′-UTR which suggests that post-transcriptional events are involved in the regulation of AHCYL1 expression in the chick oviduct. In conclusion, these results indicate that AHCYL1 is a novel estrogen-stimulated gene expressed in epithelial cells of the chicken oviduct that likely affects growth, development and calcium metabolism of the mature oviduct of hens via an estrogen-mediated ERK1/2 MAPK cell signaling pathway. PMID:23145124

Lateral diffusion of inositol 1,4,5-trisphosphate receptor type 1 in Purkinje cells is regulated by calcium and actin filaments.

PubMed

Fukatsu, Kazumi; Bannai, Hiroko; Inoue, Takafumi; Mikoshiba, Katsuhiko

2010-09-01

Inositol 1,4,5-trisphosphate receptor type 1 (IP(3) R1) is an intracellular Ca(2+) release channel that plays crucial roles in the functions of Purkinje cells. The dynamics of IP(3) R1 on the endoplasmic reticulum membrane and the distribution of IP(3) R1 in neurons are thought to be important for the spatial regulation of Ca(2+) release. In this study, we analyzed the lateral diffusion of IP(3) R1 in Purkinje cells in cerebellar slice cultures using fluorescence recovery after photobleaching. In the dendrites of Purkinje cells, IP(3) R1 showed lateral diffusion with an effective diffusion constant of approximately 0.30 μm(2) /s, and the diffusion of IP(3) R1 was negatively regulated by actin filaments. We found that actin filaments were also involved in the regulation of IP(3) R1 diffusion in the spine of Purkinje cells. Glutamate or quisqualic acid stimulation, which activates glutamate receptors and leads to a Ca(2+) transient in Purkinje cells, decreased the diffusion of IP(3) R1 and increased the density of actin in spines. These findings indicate that the neuronal activity-dependent augmentation of actin contributes to the stabilization of IP(3) R1 in spines. © 2010 The Authors. Journal Compilation © 2010 International Society for Neurochemistry.

ANTIPROLIFERATIVE EFFECT OF INOSITOL HEXAPHOSPHATE ON HUMAN SKIN MELANOMA CELLS IN VITRO.

PubMed

Wawszczyk, Joanna; Kapral, Małgorzata; Lodowska, Jolanta; Jesse, Katarzyna; Hollek, Andrzej; Węglarz, Ludmiła

2015-01-01

Human malignant melanoma is a highly metastatic tumor with poor prognosis. The majority of metastatic melanomas are resistant to diverse chemotherapeutic agents. Consequently, the search for novel antimelanoma agents continues. In recent years, the interest in plants and their biologically active constituents as a source of novel potential drugs significantly increased. Inositol hexaphosphate (IP6) is a naturally occurring compound that has been shown to inhibit the growth of a wide variety of tumor cells in multiple experimental model systems. The aim of this study was to evaluate the antiproliferative and cytotoxic influence of IP6 on melanotic melanoma cells in vitro. The A2058 cells used as a model of human skin melanoma malignum were exposed to different concentrations of IP6 (0.1-5 mM) for a various period of time and their growth was determined by sulforhodamine B assay after 24, 48 and 72 h. The cytotoxicity of IP6 was measured at 24 and 72 h by XTT assay. IP6 has been found to cause dose-dependent growth suppression of A2058 melanoma cells. At low concentrations (0.1 and 0.5 mM) it did not exert any influence on the cell proliferation as compared to control cultures. Higher concentrations of IP6 (from 1 to 5 mM) had a statistically significant, suppressive effect on cell proliferation after 24 h incubation. When the experimental time period was increased up to 72 h, statistically significant inhibition of cell proliferation was monitored at all IP6 concentrations used. Data obtained from XTT assay indicated that IP6 had dose- and time-dependent cytotoxic effect on melanoma cells. The results demonstrate the antiproliferative and cytotoxic properties of IP6 in a wide range of concentrations on human A2058 melanoma cells. Hence, it can be suggested that IP6 could have a promising therapeutic significance in treating cancer.

cChIP-seq: a robust small-scale method for investigation of histone modifications.

PubMed

Valensisi, Cristina; Liao, Jo Ling; Andrus, Colin; Battle, Stephanie L; Hawkins, R David

2015-12-21

ChIP-seq is highly utilized for mapping histone modifications that are informative about gene regulation and genome annotations. For example, applying ChIP-seq to histone modifications such as H3K4me1 has facilitated generating epigenomic maps of putative enhancers. This powerful technology, however, is limited in its application by the large number of cells required. ChIP-seq involves extensive manipulation of sample material and multiple reactions with limited quality control at each step, therefore, scaling down the number of cells required has proven challenging. Recently, several methods have been proposed to overcome this limit but most of these methods require extensive optimization to tailor the protocol to the specific antibody used or number of cells being profiled. Here we describe a robust, yet facile method, which we named carrier ChIP-seq (cChIP-seq), for use on limited cell amounts. cChIP-seq employs a DNA-free histone carrier in order to maintain the working ChIP reaction scale, removing the need to tailor reactions to specific amounts of cells or histone modifications to be assayed. We have applied our method to three different histone modifications, H3K4me3, H3K4me1 and H3K27me3 in the K562 cell line, and H3K4me1 in H1 hESCs. We successfully obtained epigenomic maps for these histone modifications starting with as few as 10,000 cells. We compared cChIP-seq data to data generated as part of the ENCODE project. ENCODE data are the reference standard in the field and have been generated starting from tens of million of cells. Our results show that cChIP-seq successfully recapitulates bulk data. Furthermore, we showed that the differences observed between small-scale ChIP-seq data and ENCODE data are largely to be due to lab-to-lab variability rather than operating on a reduced scale. Data generated using cChIP-seq are equivalent to reference epigenomic maps from three orders of magnitude more cells. Our method offers a robust and straightforward approach to scale down ChIP-seq to as low as 10,000 cells. The underlying principle of our strategy makes it suitable for being applied to a vast range of chromatin modifications without requiring expensive optimization. Furthermore, our strategy of a DNA-free carrier can be adapted to most ChIP-seq protocols.

Uncertain translation, uncertain benefit and uncertain risk: ethical challenges facing first-in-human trials of induced pluripotent stem (ips) cells.

PubMed

Fung, Ronald K F; Kerridge, Ian H

2013-02-01

The discovery of induced pluripotent stem (iPS) cells in 2006 was heralded as a major breakthrough in stem cell research. Since then, progress in iPS cell technology has paved the way towards clinical application, particularly cell replacement therapy, which has refueled debate on the ethics of stem cell research. However, much of the discourse has focused on questions of moral status and potentiality, overlooking the ethical issues which are introduced by the clinical testing of iPS cell replacement therapy. First-in-human trials, in particular, raise a number of ethical concerns including informed consent, subject recruitment and harm minimisation as well as the inherent uncertainty and risks which are involved in testing medical procedures on humans for the first time. These issues, while a feature of any human research, become more complex in the case of iPS cell therapy, given the seriousness of the potential risks, the unreliability of available animal models, the vulnerability of the target patient group, and the high stakes of such an intensely public area of science. Our paper will present a detailed case study of iPS cell replacement therapy for Parkinson's disease to highlight these broader ethical and epistemological concerns. If we accept that iPS cell technology is fraught with challenges which go far beyond merely refuting the potentiality of the stem cell line, we conclude that iPS cell research should not replace, but proceed alongside embryonic and adult somatic stem cell research to promote cross-fertilisation of knowledge and better clinical outcomes. © 2011 Blackwell Publishing Ltd.

PI3K/Akt-dependent functions of TFII-I transcription factors in mouse embryonic stem cells.

PubMed

Chimge, Nyam-Osor; Makeyev, Aleksandr V; Waigel, Sabine J; Enkhmandakh, Badam; Bayarsaihan, Dashzeveg

2012-04-01

Activation of PI3K/Akt signaling is sufficient to maintain the pluripotency of mouse embryonic stem cells (mESC) and results in down-regulation of Gtf2i and Gtf2ird1 encoding TFII-I family transcription factors. To investigate how these genes might be involved in the process of embryonic stem cell differentiation, we performed expression microarray profiling of mESC upon inhibition of PI3K by LY294002. This analysis revealed significant alterations in expression of genes for specific subsets of chromatin-modifying enzymes. Surprisingly, genome-wide promoter ChIP-chip mapping indicated that the majority of differently expressed genes could be direct targets of TFII-I regulation. The data support the hypothesis that upregulation of TFII-I factors leads to activation of a specific group of developmental genes during mESC differentiation. © 2011 Wiley Periodicals, Inc.

MOBE-ChIP: Probing Cell Type-Specific Binding Through Large-Scale Chromatin Immunoprecipitation.

PubMed

Wang, Shenqi; Lau, On Sun

2018-01-01

In multicellular organisms, the initiation and maintenance of specific cell types often require the activity of cell type-specific transcriptional regulators. Understanding their roles in gene regulation is crucial but probing their DNA targets in vivo, especially in a genome-wide manner, remains a technical challenge with their limited expression. To improve the sensitivity of chromatin immunoprecipitation (ChIP) for detecting the cell type-specific signals, we have developed the Maximized Objects for Better Enrichment (MOBE)-ChIP, where ChIP is performed at a substantially larger experimental scale and under low background conditions. Here, we describe the procedure in the study of transcription factors in the model plant Arabidopsis. However, with some modifications, the technique should also be implemented in other systems. Besides cell type-specific studies, MOBE-ChIP can also be used as a general strategy to improve ChIP signals.

IP3R-mediated Ca2+ release regulates protein metabolism in Drosophila neuroendocrine cells: implications for development under nutrient stress.

PubMed

Megha; Hasan, Gaiti

2017-04-15

Successful completion of animal development is fundamentally reliant on nutritional cues. Surviving periods of nutritional insufficiency requires adaptations that are coordinated, in part, by neural circuits. As neuropeptides secreted by neuroendocrine (NE) cells modulate neural circuits, we investigated NE cell function during development under nutrient stress. Starved Drosophila larvae exhibited reduced pupariation if either insulin signaling or IP 3 /Ca 2+ signaling were downregulated in NE cells. Moreover, an IP 3 R (inositol 1,4,5-trisphosphate receptor) loss-of-function mutant displayed reduced protein synthesis, which was rescued by overexpression of either InR (insulin receptor) or IP 3 R in NE cells of the mutant, suggesting that the two signaling pathways might be functionally compensatory. Furthermore, cultured IP 3 R mutant NE cells, but not neurons, exhibited reduced protein translation. Thus cell-specific regulation of protein synthesis by IP 3 R in NE cells influences protein metabolism. We propose that this regulation helps developing animals survive in poor nutritional conditions. © 2017. Published by The Company of Biologists Ltd.

Stem cell transplantation therapy for multifaceted therapeutic benefits after stroke.

PubMed

Wei, Ling; Wei, Zheng Z; Jiang, Michael Qize; Mohamad, Osama; Yu, Shan Ping

2017-10-01

One of the exciting advances in modern medicine and life science is cell-based neurovascular regeneration of damaged brain tissues and repair of neuronal structures. The progress in stem cell biology and creation of adult induced pluripotent stem (iPS) cells has significantly improved basic and pre-clinical research in disease mechanisms and generated enthusiasm for potential applications in the treatment of central nervous system (CNS) diseases including stroke. Endogenous neural stem cells and cultured stem cells are capable of self-renewal and give rise to virtually all types of cells essential for the makeup of neuronal structures. Meanwhile, stem cells and neural progenitor cells are well-known for their potential for trophic support after transplantation into the ischemic brain. Thus, stem cell-based therapies provide an attractive future for protecting and repairing damaged brain tissues after injury and in various disease states. Moreover, basic research on naïve and differentiated stem cells including iPS cells has markedly improved our understanding of cellular and molecular mechanisms of neurological disorders, and provides a platform for the discovery of novel drug targets. The latest advances indicate that combinatorial approaches using cell based therapy with additional treatments such as protective reagents, preconditioning strategies and rehabilitation therapy can significantly improve therapeutic benefits. In this review, we will discuss the characteristics of cell therapy in different ischemic models and the application of stem cells and progenitor cells as regenerative medicine for the treatment of stroke. Copyright © 2017 Elsevier Ltd. All rights reserved.

Simple suspension culture system of human iPS cells maintaining their pluripotency for cardiac cell sheet engineering.

PubMed

Haraguchi, Yuji; Matsuura, Katsuhisa; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

2015-12-01

In this study, a simple three-dimensional (3D) suspension culture method for the expansion and cardiac differentiation of human induced pluripotent stem cells (hiPSCs) is reported. The culture methods were easily adapted from two-dimensional (2D) to 3D culture without any additional manipulations. When hiPSCs were directly applied to 3D culture from 2D in a single-cell suspension, only a few aggregated cells were observed. However, after 3 days, culture of the small hiPSC aggregates in a spinner flask at the optimal agitation rate created aggregates which were capable of cell passages from the single-cell suspension. Cell numbers increased to approximately 10-fold after 12 days of culture. The undifferentiated state of expanded hiPSCs was confirmed by flow cytometry, immunocytochemistry and quantitative RT-PCR, and the hiPSCs differentiated into three germ layers. When the hiPSCs were subsequently cultured in a flask using cardiac differentiation medium, expression of cardiac cell-specific genes and beating cardiomyocytes were observed. Furthermore, the culture of hiPSCs on Matrigel-coated dishes with serum-free medium containing activin A, BMP4 and FGF-2 enabled it to generate robust spontaneous beating cardiomyocytes and these cells expressed several cardiac cell-related genes, including HCN4, MLC-2a and MLC-2v. This suggests that the expanded hiPSCs might maintain the potential to differentiate into several types of cardiomyocytes, including pacemakers. Moreover, when cardiac cell sheets were fabricated using differentiated cardiomyocytes, they beat spontaneously and synchronously, indicating electrically communicative tissue. This simple culture system might enable the generation of sufficient amounts of beating cardiomyocytes for use in cardiac regenerative medicine and tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.

Safety and effects of two mistletoe preparations on production of Interleukin-6 and other immune parameters - a placebo controlled clinical trial in healthy subjects

PubMed Central

2011-01-01

Background In Germany, Iscucin® Populi (IP), a preparation from mistletoe growing on the poplar tree, is used in cancer therapy while Viscum Mali e planta tota (VM), a preparation from mistletoe growing on the apple tree, is used in patients with osteoarthritis. Since mistletoe preparations are suspected to induce production of potentially tumor promoting cytokines like interleukin (IL)-6, further studies on the immunological effects are of interest. Methods In this 3-armed randomized, double blind clinical trial healthy volunteers received increasing doses of either IP (strength F, 0.0125%, G, 0.25% and H, 5%, each for 4 weeks), or VM (1:1000 [D3], 1:100 [D2] and 2% each for 4 weeks) or placebo (isotonic solution) subcutaneously twice per week over a period of 12 weeks. Physical examination was performed weekly. Routine laboratory parameters and immunological parameters (C-reactive protein (CRP), differential blood count, lymphocyte subsets, immunoglobulins, IL-6 and tumor necrosis factor (TNF)-α) were analysed every 4 weeks. Results 71 subjects were included in the study (IP = 30, VM = 21, placebo = 20) of whom 69 concluded it according to protocol. Application of IP strengths G and H caused strong local reactions at the site of injection. In parallel, a distinct eosinophilia (p < 0.001 compared to placebo) occurred. Furthermore, application of all IP concentrations resulted in an increase of CD4 cell counts (p < 0.05) compared to placebo. Stimulation of IL-6 production, CRP or relevant deviations in other laboratory parameters were not observed. Because of local reactions, IP strengths G and H were considered less tolerable than placebo. VM 2% was slightly less tolerable than placebo, caused only mild local reactions and an only small increase in eosinophile counts. Conclusion Treatment with IP results in eosinophilia and an increase of CD4 cells but not in an increase of IL-6 or CRP. No safety concerns regarding the two mistletoe preparations have been raised by this study. EudraCT-Number 2007-002166-35. Trial registration ClinicalTrials.gov: NCT01378702 PMID:22114899

Tsg101 regulates PI(4,5)P2/Ca2+ signaling for HIV-1 Gag assembly

PubMed Central

Ehrlich, Lorna S.; Medina, Gisselle N.; Photiadis, Sara; Whittredge, Paul B.; Watanabe, Susan; Taraska, Justin W.; Carter, Carol A.

2014-01-01

Our previous studies identified the 1,4,5-inositol trisphosphate receptor (IP3R), a channel mediating release of Ca2+ from ER stores, as a cellular factor differentially associated with HIV-1 Gag that might facilitate ESCRT function in virus budding. Channel opening requires activation that is initiated by binding of 1,4,5-triphosphate (IP3), a product of phospholipase C (PLC)-mediated PI(4,5)P2 hydrolysis. The store emptying that follows stimulates store refilling which requires intact PI(4,5)P2. Raising cytosolic Ca2+ promotes viral particle production and our studies indicate that IP3R and the ER Ca2+ store are the physiological providers of Ca2+ for Gag assembly and release. Here, we show that Gag modulates ER store gating and refilling. Cells expressing Gag exhibited a higher cytosolic Ca2+ level originating from the ER store than control cells, suggesting that Gag induced release of store Ca2+. This property required the PTAP motif in Gag that recruits Tsg101, an ESCRT-1 component. Consistent with cytosolic Ca2+ elevation, Gag accumulation at the plasma membrane was found to require continuous IP3R activation. Like other IP3R channel modulators, Gag was detected in physical proximity to the ER and to endogenous IP3R, as indicated respectively by total internal reflection fluorescence (TIRF) and immunoelectron microscopy (IEM) or indirect immunofluorescence. Reciprocal co-immunoprecipitation suggested that Gag and IP3R proximity is favored when the PTAP motif in Gag is intact. Gag expression was also accompanied by increased PI(4,5)P2 accumulation at the plasma membrane, a condition favoring store refilling capacity. Supporting this notion, Gag particle production was impervious to treatment with 2-aminoethoxydiphenyl borate, an inhibitor of a refilling coupling interaction. In contrast, particle production by a Gag mutant lacking the PTAP motif was reduced. We conclude that a functional PTAP L domain, and by inference Tsg101 binding, confers Gag with an ability to modulate both ER store Ca2+ release and ER store refilling. PMID:24904548

Induction of pluripotency by defined factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okita, Keisuke, E-mail: okita@cira.kyoto-u.ac.jp; Yamanaka, Shinya; Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507

2010-10-01

Somatic cells can be reprogrammed into pluripotent stem cells by introducing a combination of several transcription factors. The induced pluripotent stem (iPS) cells from a patient's somatic cells could be useful source of cells for drug discovery and cell transplantation therapies. However, most human iPS cells are made by viral vectors, such as retrovirus and lentivirus, which integrate the reprogramming factors into host genomes and may increase the risk of tumor formation. Studies of the mechanisms underlying the reprogramming and establishment of non-integration methods contribute evidence to resolve the safety concerns associated with iPS cells. On the other hand, patient-specificmore » iPS cells have already been established and used for recapitulating disease pathology.« less

IP-10 protects while MIP-2 promotes experimental anesthetic hapten - induced hepatitis

PubMed Central

Njoku, Dolores B.; Li, Zhaoxia; Mellerson, Jenelle L; Sharma, Rajni; Talor, Monica V.; Barat, Nicole; Rose, Noel R.

2009-01-01

MIP-2 and IFN-γ inducible protein-10 (IP-10) and their respective receptors, CXCR2 and CXCR3, modulate tissue inflammation by recruiting neutrophils or T cells from the spleen or bone marrow. Yet, how these chemokines modulate diseases such as immune-mediated drug-induced liver injury (DILI) is essentially unknown. To investigate how chemokines modulate experimental DILI in our model we used susceptible BALB/c (WT) and IL-4−/− (KO) mice that develop significantly reduced hepatitis and splenic T cell priming to anesthetic haptens and self proteins following TFA-S100 immunizations. We detected CXCR2+ splenic granulocytes in all mice two weeks following immunizations; by 3 weeks, MIP-2 levels (p<0.001) and GR1+ cells were elevated in WT livers, suggesting MIP-2-recruited granulocytes. Elevated splenic CXCR3+ CD4+T cells were identified after 2 weeks in KO mice indicating elevated IP-10 levels which were confirmed during T cell priming. This result suggested that IP-10 reduced T cell priming to critical DILI antigens. Increased T cell proliferation following co-culture of TFA-S100-primed WT splenocytes with anti-IP-10 (p<0.05) confirmed that IP-10 reduced T cell priming to CYP2E1 and TFA. We propose that MIP-2 promotes and IP-10 protects against the development of hepatitis and T cell priming in this murine model. PMID:19131211

Inositol bisphosphate and inositol trisphosphate inhibit cell-to-cell passage of carboxyfluorescein in staminal hairs ofSetcreasea purpurea.

PubMed

Tucker, E B

1988-06-01

pH-buffered carboxyfluorescein (Buffered-CF) alone (control), or Buffered-CF solutions containing one of the following: (1)D-myo-inositol (I); (2)D-myo-inositol 2-monophosphate (IP1); (3)D-myo-inositol 1,4-bisphosphate (IP2); (4)D-myo-inositol 1,4,5-trisphosphate (IP3); (5)D-fructose 2,6-diphosphate (F-2,6P2) were microinjected into the terminal cells of staminal hairs ofSetcreasea purpurea Boom. Passage of the CF from this terminal cell along the chain of cells towards the filament was monitored for 5 min using fluorescence microscopy and quantified using computer-assisted fluorescence-intensity video analysis. Cell-to-cell transport of CF in hairs microinjected with Buffered-CF containing either I, IP1 or F-2,6P2 was similar to that in hairs microinjected with Buffered-CF only. On the other hand, cell-to-cell transport of CF in hairs microinjected with Buffered-CF containing either IP2 or IP3 was inhibited. These results indicate that polyphosphoinositols may be involved in the regulation of intercellular transport of low-molecular-weight, hydrophilic molecules in plants.

Functional myogenic engraftment from mouse iPS cells.

PubMed

Darabi, Radbod; Pan, Weihong; Bosnakovski, Darko; Baik, June; Kyba, Michael; Perlingeiro, Rita C R

2011-11-01

Direct reprogramming of adult fibroblasts to a pluripotent state has opened new possibilities for the generation of patient- and disease-specific stem cells. However the ability of induced pluripotent stem (iPS) cells to generate tissue that mediates functional repair has been demonstrated in very few animal models of disease to date. Here we present the proof of principle that iPS cells may be used effectively for the treatment of muscle disorders. We combine the generation of iPS cells with conditional expression of Pax7, a robust approach to derive myogenic progenitors. Transplantation of Pax7-induced iPS-derived myogenic progenitors into dystrophic mice results in extensive engraftment, which is accompanied by improved contractility of treated muscles. These findings demonstrate the myogenic regenerative potential of iPS cells and provide rationale for their future therapeutic application for muscular dystrophies.

Ethanol potentiates the genotoxicity of the food-derived mammary carcinogen PhIP in human estrogen receptor-positive mammary cells: mechanistic support for lifestyle factors (cooked red meat and ethanol) associated with mammary cancer.

PubMed

Malik, Durr-E-Shahwar; David, Rhiannon M; Gooderham, Nigel J

2018-04-01

Consumption of cooked/processed meat and ethanol are lifestyle risk factors in the aetiology of breast cancer. Cooking meat generates heterocyclic amines such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Epidemiology, mechanistic and animal studies indicate that PhIP is a mammary carcinogen that could be causally linked to breast cancer incidence; PhIP is DNA damaging, mutagenic and oestrogenic. PhIP toxicity involves cytochrome P450 (CYP1 family)-mediated metabolic activation to DNA-damaging species, and transcriptional responses through Aryl hydrocarbon receptor (AhR) and estrogen-receptor-α (ER-α). Ethanol consumption is a modifiable lifestyle factor strongly associated with breast cancer risk. Ethanol toxicity involves alcohol dehydrogenase metabolism to reactive acetaldehyde, and is also a substrate for CYP2E1, which when uncoupled generates reactive oxygen species (ROS) and DNA damage. Here, using human mammary cells that differ in estrogen-receptor status, we explore genotoxicity of PhIP and ethanol and mechanisms behind this toxicity. Treatment with PhIP (10 -7 -10 -4 M) significantly induced genotoxicity (micronuclei formation) preferentially in ER-α positive human mammary cell lines (MCF-7, ER-α+) compared to MDA-MB-231 (ER-α-) cells. PhIP-induced CYP1A2 in both cell lines but CYP1B1 was selectively induced in ER-α(+) cells. ER-α inhibition in MCF-7 cells attenuated PhIP-mediated micronuclei formation and CYP1B1 induction. PhIP-induced CYP2E1 and ROS via ER-α-STAT-3 pathway, but only in ER-α (+) MCF-7 cells. Importantly, simultaneous treatments of physiological concentrations ethanol (10 -3 -10 -1 M) with PhIP (10 -7 -10 -4 M) increased oxidative stress and genotoxicity in MCF-7 cells, compared to the individual chemicals. Collectively, these data offer a mechanistic basis for the increased risk of breast cancer associated with dietary cooked meat and ethanol lifestyle choices.

The role of DAB2IP in androgen receptor activation during prostate cancer progression.

PubMed

Wu, K; Liu, J; Tseng, S-F; Gore, C; Ning, Z; Sharifi, N; Fazli, L; Gleave, M; Kapur, P; Xiao, G; Sun, X; Oz, O K; Min, W; Alexandrakis, G; Yang, C-R; Hsieh, C-L; Wu, H-C; He, D; Xie, D; Hsieh, J-T

2014-04-10

Altered androgen-receptor (AR) expression and/or constitutively active AR are commonly associated with prostate cancer (PCa) progression. Targeting AR remains a focal point for designing new strategy of PCa therapy. Here, we have shown that DAB2IP, a novel tumor suppressor in PCa, can inhibit AR-mediated cell growth and gene activation in PCa cells via distinct mechanisms. DAB2IP inhibits the genomic pathway by preventing AR nuclear translocation or phosphorylation and suppresses the non-genomic pathway via its unique functional domain to inactivate c-Src. Also, DAB2IP is capable of suppressing AR activation in an androgen-independent manner. In addition, DAB2IP can inhibit several AR splice variants showing constitutive activity in PCa cells. In DAB2IP(-/-) mice, the prostate gland exhibits hyperplastic epithelia, in which AR becomes more active. Consistently, DAB2IP expression inversely correlates with AR activation status particularly in recurrent or metastatic PCa patients. Taken together, DAB2IP is a unique intrinsic AR modulator in normal cells, and likely can be further developed into a therapeutic agent for PCa.

Prospero-related homeobox 1 (Prox1) at the crossroads of diverse pathways during adult neural fate specification

PubMed Central

Stergiopoulos, Athanasios; Elkouris, Maximilianos; Politis, Panagiotis K.

2015-01-01

Over the last decades, adult neurogenesis in the central nervous system (CNS) has emerged as a fundamental process underlying physiology and disease. Recent evidence indicates that the homeobox transcription factor Prox1 is a critical intrinsic regulator of neurogenesis in the embryonic CNS and adult dentate gyrus (DG) of the hippocampus, acting in multiple ways and instructed by extrinsic cues and intrinsic factors. In the embryonic CNS, Prox1 is mechanistically involved in the regulation of proliferation vs. differentiation decisions of neural stem cells (NSCs), promoting cell cycle exit and neuronal differentiation, while inhibiting astrogliogenesis. During the complex differentiation events in adult hippocampal neurogenesis, Prox1 is required for maintenance of intermediate progenitors (IPs), differentiation and maturation of glutamatergic interneurons, as well as specification of DG cell identity over CA3 pyramidal fate. The mechanism by which Prox1 exerts multiple functions involves distinct signaling pathways currently not fully highlighted. In this mini-review, we thoroughly discuss the Prox1-dependent phenotypes and molecular pathways in adult neurogenesis in relation to different upstream signaling cues and cell fate determinants. In addition, we discuss the possibility that Prox1 may act as a cross-talk point between diverse signaling cascades to achieve specific outcomes during adult neurogenesis. PMID:25674048

Differential Regulation of Multiple Steps in Inositol 1,4,5-Trisphosphate Signaling by Protein Kinase C Shapes Hormone-stimulated Ca2+ Oscillations*

PubMed Central

Bartlett, Paula J.; Metzger, Walson; Gaspers, Lawrence D.; Thomas, Andrew P.

2015-01-01

How Ca2+ oscillations are generated and fine-tuned to yield versatile downstream responses remains to be elucidated. In hepatocytes, G protein-coupled receptor-linked Ca2+ oscillations report signal strength via frequency, whereas Ca2+ spike amplitude and wave velocity remain constant. IP3 uncaging also triggers oscillatory Ca2+ release, but, in contrast to hormones, Ca2+ spike amplitude, width, and wave velocity were dependent on [IP3] and were not perturbed by phospholipase C (PLC) inhibition. These data indicate that oscillations elicited by IP3 uncaging are driven by the biphasic regulation of the IP3 receptor by Ca2+, and, unlike hormone-dependent responses, do not require PLC. Removal of extracellular Ca2+ did not perturb Ca2+ oscillations elicited by IP3 uncaging, indicating that reloading of endoplasmic reticulum stores via plasma membrane Ca2+ influx does not entrain the signal. Activation and inhibition of PKC attenuated hormone-induced Ca2+ oscillations but had no effect on Ca2+ increases induced by uncaging IP3. Importantly, PKC activation and inhibition differentially affected Ca2+ spike frequencies and kinetics. PKC activation amplifies negative feedback loops at the level of G protein-coupled receptor PLC activity and/or IP3 metabolism to attenuate IP3 levels and suppress the generation of Ca2+ oscillations. Inhibition of PKC relieves negative feedback regulation of IP3 accumulation and, thereby, shifts Ca2+ oscillations toward sustained responses or dramatically prolonged spikes. PKC down-regulation attenuates phenylephrine-induced Ca2+ wave velocity, whereas responses to IP3 uncaging are enhanced. The ability to assess Ca2+ responses in the absence of PLC activity indicates that IP3 receptor modulation by PKC regulates Ca2+ release and wave velocity. PMID:26078455

Glial cells isolated from dorsal root ganglia express prostaglandin E(2) (EP(4)) and prostacyclin (IP) receptors.

PubMed

Ng, Kai Yu; Wong, Yung Hou; Wise, Helen

2011-07-01

Isolated cells from adult rat dorsal root ganglia (DRG) are frequently used as a model system to study responses of primary sensory neurons to nociceptor sensitizing agents such as prostaglandin E(2) and prostacyclin, which are presumed to act only on the neurons in typical mixed cell cultures. In the present study, we evaluated the expression of prostaglandin E(2) (EP(4)) and prostacyclin (IP) receptors in cultures of mixed DRG cells and in purified DRG glia. We show here that EP(4) and IP receptor agonists stimulated adenylyl cyclase activity in both mixed DRG cells and in purified DRG glia, and that these responses were specifically inhibited by EP(4) and IP receptor antagonists, respectively. The presence of EP(4) and IP receptors in DRG glia was further confirmed by the expression of EP(4) and IP receptor immunoreactivity and mRNA. With the increasing awareness of neuron-glial interactions within intact DRG and the use of isolated DRG cells in the study of mechanisms underlying nociception, it will be essential to consider the role played by EP(4) and IP receptor-expressing glial cells when evaluating prostanoid-induced sensitization of DRG neurons. Copyright © 2011 Elsevier B.V. All rights reserved.

Cell surface expression of channel catfish leukocyte immune-type receptors (IpLITRs) and recruitment of both Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 and SHP-2.

PubMed

Montgomery, Benjamin C S; Mewes, Jacqueline; Davidson, Chelsea; Burshtyn, Deborah N; Stafford, James L

2009-04-01

Channel catfish leukocyte immune-type receptors (IpLITRs) are immunoglobulin superfamily (IgSF) members believed to play a role in the control and coordination of cellular immune responses in teleost. Putative stimulatory and inhibitory IpLITRs are co-expressed by different types of catfish immune cells (e.g. NK cells, T cells, B cells, and macrophages) but their signaling potential has not been determined. Following cationic polymer-mediated transfections into human cell lines we examined the surface expression, tyrosine phosphorylation, and phosphatase recruitment potential of two types of putative inhibitory IpLITRs using 'chimeric' expression constructs and an epitope-tagged 'native' IpLITR. We also cloned and expressed the teleost Src homology 2 domain-containing protein tyrosine phosphatases (SHP)-1 and SHP-2 and examined their expression in adult tissues and developing zebrafish embryos. Co-immunoprecipitation experiments support the inhibitory signaling potential of distinct IpLITR-types that bound both SHP-1 and SHP-2 following the phosphorylation of tyrosine residues within their cytoplasmic tail (CYT) regions. Phosphatase recruitment by IpLITRs represents an important first step in understanding their influence on immune cell effector functions and suggests that certain inhibitory signaling pathways are conserved among vertebrates.

MLF1IP promotes normal erythroid proliferation and is involved in the pathogenesis of polycythemia vera.

PubMed

Feng, Gege; Zhang, Tianjiao; Liu, Jinqin; Ma, Xiaotang; Li, Bing; Yang, Lin; Zhang, Yue; Xu, Zefeng; Qin, Tiejun; Zhou, Jiaxi; Huang, Gang; Shi, Lihong; Xiao, Zhijian

2017-03-01

Myelodysplasia/myeloid leukemia factor 1-interacting protein (MLF1IP) appears to be an erythroid lineage-specific gene in mice; however, its role in normal erythropoiesis and erythropoietic disorders have not yet been elucidated. Here, we found that MLF1IP is abundantly expressed in human erythroid progenitor cells and that MLF1IP-deficiency reduces cell proliferation resulting from cell cycle arrest. Moreover, MLF1IP expression is exclusively elevated in CFU-E cells from polycythemia vera (PV) patients, and MLF1IP transgenic mice develop a PV-like disorder. Further analyses revealed that the erythroid progenitors and early-stage erythroblasts from these transgenic mice expand by up-regulating cyclin D2 and down-regulating p27 and p21. Thus, our data demonstrate that MLF1IP promotes erythroid proliferation and is involved in the pathogenesis of PV, suggesting that it might be a novel molecular target for erythropoietic disorders. © 2017 Federation of European Biochemical Societies.

Pro-inflammatory effects of the Th1 chemokine CXCL10 in acquired aplastic anaemia.

PubMed

Li, Junhong; Ge, Meili; Lu, Shihong; Shi, Jun; Li, Xingxin; Wang, Min; Huang, Jinbo; Shao, Yingqi; Huang, Zhendong; Zhang, Jing; Nie, Neng; Zheng, Yizhou

2017-06-01

CXCL10/IFN-γ-induced protein 10 (IP-10) and its corresponding receptor CXCR3 have long been considered to be involved in the pathophysiology of type 1 T (Th1) cell-orientated autoimmune diseases. However, the exact role of CXCL10 in the pathogenesis of aplastic anaemia (AA) has not been thoroughly studied. The aim of our study was to evaluate the plasma level of CXCL10 and its effects on CD4 + T cell differentiation in AA. In our study, we found that an elevated plasma level of CXCL10 was negatively correlated with platelet, absolute neutrophil and reticulocyte counts, while it was positively correlated with the proportion of lymphocytes in white blood cells in AA patients. To confirm the pro-inflammatory effects of CXCL10 in AA, we isolated CD4 + T cells and evaluated the function of CXCL10 in CD4 + T cell differentiation. In vitro stimulation experiments further revealed the pro-inflammatory role of CXCL10 in AA, partially by promoting the secretion of interferon (IFN)-γ and IL-17. In addition, CXCL10 significantly skewed CD4 + T cell differentiation to Th1 cells and T helper 17 (Th17) cells in AA patients, while it inhibited the differentiation of type 2 T (Th2) cells only in controls. The mRNA expression of transcription factors representative of T cell differentiation was detected by RT-PCR. Consistently, our results showed that after CXCL10 treatment, the expression of T-bet and RORγt was significantly enhanced, while the expression of GATA3 was inhibited. In conclusion, our results indicated that CXCL10, a pro-inflammatory chemokine, might be involved in the abnormal immune response in AA. Copyright © 2017 Elsevier Ltd. All rights reserved.

Combination of miRNA499 and miRNA133 Exerts a Synergic Effect on Cardiac Differentiation

PubMed Central

Pisano, Federica; Altomare, Claudia; Cervio, Elisabetta; Barile, Lucio; Rocchetti, Marcella; Ciuffreda, Maria Chiara; Malpasso, Giuseppe; Copes, Francesco; Mura, Manuela; Danieli, Patrizia; Viarengo, Gianluca; Zaza, Antonio; Gnecchi, Massimiliano

2015-01-01

Several studies have demonstrated that miRNA are involved in cardiac development, stem cell maintenance, and differentiation. In particular, it has been shown that miRNA133, miRNA1, and miRNA499 are involved in progenitor cell differentiation into cardiomyocytes. However, it is unknown whether different miRNA may act synergistically to improve cardiac differentiation. We used mouse P19 cells as a cardiogenic differentiation model. miRNA499, miRNA1, or miRNA133 were transiently over-expressed in P19 cells individually or in different combinations. The over-expression of miRNA499 alone increased the number of beating cells and the association of miRNA499 with miRNA133 exerted a synergistic effect, further increasing the number of beating cells. Real-time polymerase chain reaction showed that the combination of miRNA499 + 133 enhanced the expression of cardiac genes compared with controls. Western blot and immunocytochemistry for connexin43 and cardiac troponin T confirmed these findings. Importantly, caffeine responsiveness, a clear functional parameter of cardiac differentiation, was increased by miRNA499 in association with miRNA133 and was directly correlated with the activation of the cardiac troponin I isoform promoter. Cyclic contractions were reversibly abolished by extracellular calcium depletion, nifedipine, ryanodine, and IP3R blockade. Finally, we demonstrated that the use of miRNA499 + 133 induced cardiac differentiation even in the absence of dimethyl sulfoxide. Our results show that the areas spontaneously contracting possess electrophysiological and pharmacological characteristics compatible with true cardiac excitation-contraction coupling. The translational relevance of our findings was reinforced by the demonstration that the over-expression of miRNA499 and miRNA133 was also able to induce the differentiation of human mesenchymal stromal cells toward the cardiac lineage. Stem Cells 2015;33:1187–1199 PMID:25534971

Dynamic methylation and expression of Oct4 in early neural stem cells.

PubMed

Lee, Shih-Han; Jeyapalan, Jennie N; Appleby, Vanessa; Mohamed Noor, Dzul Azri; Sottile, Virginie; Scotting, Paul J

2010-09-01

Neural stem cells are a multipotent population of tissue-specific stem cells with a broad but limited differentiation potential. However, recent studies have shown that over-expression of the pluripotency gene, Oct4, alone is sufficient to initiate a process by which these can form 'induced pluripotent stem cells' (iPS cells) with the same broad potential as embryonic stem cells. This led us to examine the expression of Oct4 in endogenous neural stem cells, as data regarding its expression in neural stem cells in vivo are contradictory and incomplete. In this study we have therefore analysed the expression of Oct4 and other genes associated with pluripotency throughout development of the mouse CNS and in neural stem cells grown in vitro. We find that Oct4 is still expressed in the CNS by E8.5, but that this expression declines rapidly until it is undetectable by E15.5. This decline is coincident with the gradual methylation of the Oct4 promoter and proximal enhancer. Immunostaining suggests that the Oct4 protein is predominantly cytoplasmic in location. We also found that neural stem cells from all ages expressed the pluripotency associated genes, Sox2, c-Myc, Klf4 and Nanog. These data provide an explanation for the varying behaviour of cells from the early neuroepithelium at different stages of development. The expression of these genes also provides an indication of why Oct4 alone is sufficient to induce iPS formation in neural stem cells at later stages.

Perspectives on human clinical trials of therapies using iPS cells in Japan: reaching the forefront of stem-cell therapies.

PubMed

Song, Peipei; Inagaki, Yoshinori; Sugawara, Yasuhiko; Kokudo, Norihiro

2013-06-01

A research project involving sheets of retinal pigment epithelium constructed from iPS cells derived from patients with age-related maculopathy is one step closer to being approved for clinical trials by the Japanese Government. Now is the time to make therapies using iPS cells clinically available.

c-Myc inhibits myoblast differentiation and promotes myoblast proliferation and muscle fibre hypertrophy by regulating the expression of its target genes, miRNAs and lincRNAs.

PubMed

Luo, Wen; Chen, Jiahui; Li, Limin; Ren, Xueyi; Cheng, Tian; Lu, Shiyi; Lawal, Raman Akinyanju; Nie, Qinghua; Zhang, Xiquan; Hanotte, Olivier

2018-05-21

The transcription factor c-Myc is an important regulator of cellular proliferation, differentiation and embryogenesis. While c-Myc can inhibit myoblast differentiation, the underlying mechanisms remain poorly understood. Here, we found that c-Myc does not only inhibits myoblast differentiation but also promotes myoblast proliferation and muscle fibre hypertrophy. By performing chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we identified the genome-wide binding profile of c-Myc in skeletal muscle cells. c-Myc achieves its regulatory effects on myoblast proliferation and differentiation by targeting the cell cycle pathway. Additionally, c-Myc can regulate cell cycle genes by controlling miRNA expression of which dozens of miRNAs can also be regulated directly by c-Myc. Among these c-Myc-associated miRNAs (CAMs), the roles played by c-Myc-induced miRNAs in skeletal muscle cells are similar to those played by c-Myc, whereas c-Myc-repressed miRNAs play roles that are opposite to those played by c-Myc. The cell cycle, ERK-MAPK and Akt-mediated pathways are potential target pathways of the CAMs during myoblast differentiation. Interestingly, we identified four CAMs that can directly bind to the c-Myc 3' UTR and inhibit c-Myc expression, suggesting that a negative feedback loop exists between c-Myc and its target miRNAs during myoblast differentiation. c-Myc also potentially regulates many long intergenic noncoding RNAs (lincRNAs). Linc-2949 and linc-1369 are directly regulated by c-Myc, and both lincRNAs are involved in the regulation of myoblast proliferation and differentiation by competing for the binding of muscle differentiation-related miRNAs. Our findings do not only provide a genome-wide overview of the role the c-Myc plays in skeletal muscle cells but also uncover the mechanism of how c-Myc and its target genes regulate myoblast proliferation and differentiation, and muscle fibre hypertrophy.

SNF5 Is an Essential Executor of Epigenetic Regulation during Differentiation

PubMed Central

You, Jueng Soo; De Carvalho, Daniel D.; Dai, Chao; Liu, Minmin; Pandiyan, Kurinji; Zhou, Xianghong J.; Liang, Gangning; Jones, Peter A.

2013-01-01

Nucleosome occupancy controls the accessibility of the transcription machinery to DNA regulatory regions and serves an instructive role for gene expression. Chromatin remodelers, such as the BAF complexes, are responsible for establishing nucleosome occupancy patterns, which are key to epigenetic regulation along with DNA methylation and histone modifications. Some reports have assessed the roles of the BAF complex subunits and stemness in murine embryonic stem cells. However, the details of the relationships between remodelers and transcription factors in altering chromatin configuration, which ultimately affects gene expression during cell differentiation, remain unclear. Here for the first time we demonstrate that SNF5, a core subunit of the BAF complex, negatively regulates OCT4 levels in pluripotent cells and is essential for cell survival during differentiation. SNF5 is responsible for generating nucleosome-depleted regions (NDRs) at the regulatory sites of OCT4 repressed target genes such as PAX6 and NEUROG1, which are crucial for cell fate determination. Concurrently, SNF5 closes the NDRs at the regulatory regions of OCT4-activated target genes such as OCT4 itself and NANOG. Furthermore, using loss- and gain-of-function experiments followed by extensive genome-wide analyses including gene expression microarrays and ChIP-sequencing, we highlight that SNF5 plays dual roles during differentiation by antagonizing the expression of genes that were either activated or repressed by OCT4, respectively. Together, we demonstrate that SNF5 executes the switch between pluripotency and differentiation. PMID:23637628

SNF5 is an essential executor of epigenetic regulation during differentiation.

PubMed

You, Jueng Soo; De Carvalho, Daniel D; Dai, Chao; Liu, Minmin; Pandiyan, Kurinji; Zhou, Xianghong J; Liang, Gangning; Jones, Peter A

2013-04-01

Nucleosome occupancy controls the accessibility of the transcription machinery to DNA regulatory regions and serves an instructive role for gene expression. Chromatin remodelers, such as the BAF complexes, are responsible for establishing nucleosome occupancy patterns, which are key to epigenetic regulation along with DNA methylation and histone modifications. Some reports have assessed the roles of the BAF complex subunits and stemness in murine embryonic stem cells. However, the details of the relationships between remodelers and transcription factors in altering chromatin configuration, which ultimately affects gene expression during cell differentiation, remain unclear. Here for the first time we demonstrate that SNF5, a core subunit of the BAF complex, negatively regulates OCT4 levels in pluripotent cells and is essential for cell survival during differentiation. SNF5 is responsible for generating nucleosome-depleted regions (NDRs) at the regulatory sites of OCT4 repressed target genes such as PAX6 and NEUROG1, which are crucial for cell fate determination. Concurrently, SNF5 closes the NDRs at the regulatory regions of OCT4-activated target genes such as OCT4 itself and NANOG. Furthermore, using loss- and gain-of-function experiments followed by extensive genome-wide analyses including gene expression microarrays and ChIP-sequencing, we highlight that SNF5 plays dual roles during differentiation by antagonizing the expression of genes that were either activated or repressed by OCT4, respectively. Together, we demonstrate that SNF5 executes the switch between pluripotency and differentiation.

p38β, A novel regulatory target of Pokemon in hepatic cells.

PubMed

Chen, Zhe; Liu, Feng; Zhang, Nannan; Cao, Deliang; Liu, Min; Tan, Ying; Jiang, Yuyang

2013-06-27

Pokemon is an important proto-oncogene involved in various biological processes and cancer development, such as cell differentiation, tumorigenesis and metastasis. Pokemon is recognized as a transcription factor localized upstream of several oncogenes, regulating their expression. p38MAPKs act as key regulatory factors in cellular signaling pathways associated with inflammatory responses, cell proliferation, differentiation and survival. p38β, a member of p38MAPK family, is closely correlated with tumorigenesis, but the mechanism of activation remains unclear. In this study, we found overexpression of Pokemon promoted the growth, migration and invasion of HepG2 cells. However, a p38 inhibitor SB202190 efficiently attenuated the promoting effect of Pokemon in the HepG2 cells. Targeted expression or silencing of Pokemon changed cellular p38β protein level and phosphorylation of downstream ATF2 in the p38 signaling pathway. Both dual luciferase report assay and ChIP assay suggested that p38β is a novel regulatory target of the transcription factor Pokemon and positively regulated by Pokemon in hepatic cells.

p38β, A Novel Regulatory Target of Pokemon in Hepatic Cells

PubMed Central

Chen, Zhe; Liu, Feng; Zhang, Nannan; Cao, Deliang; Liu, Min; Tan, Ying; Jiang, Yuyang

2013-01-01

Pokemon is an important proto-oncogene involved in various biological processes and cancer development, such as cell differentiation, tumorigenesis and metastasis. Pokemon is recognized as a transcription factor localized upstream of several oncogenes, regulating their expression. p38MAPKs act as key regulatory factors in cellular signaling pathways associated with inflammatory responses, cell proliferation, differentiation and survival. p38β, a member of p38MAPK family, is closely correlated with tumorigenesis, but the mechanism of activation remains unclear. In this study, we found overexpression of Pokemon promoted the growth, migration and invasion of HepG2 cells. However, a p38 inhibitor SB202190 efficiently attenuated the promoting effect of Pokemon in the HepG2 cells. Targeted expression or silencing of Pokemon changed cellular p38β protein level and phosphorylation of downstream ATF2 in the p38 signaling pathway. Both dual luciferase report assay and ChIP assay suggested that p38β is a novel regulatory target of the transcription factor Pokemon and positively regulated by Pokemon in hepatic cells. PMID:23807508

Generation of human pluripotent stem cell-derived hepatocyte-like cells for drug toxicity screening.

PubMed

Takayama, Kazuo; Mizuguchi, Hiroyuki

2017-02-01

Because drug-induced liver injury is one of the main reasons for drug development failures, it is important to perform drug toxicity screening in the early phase of pharmaceutical development. Currently, primary human hepatocytes are most widely used for the prediction of drug-induced liver injury. However, the sources of primary human hepatocytes are limited, making it difficult to supply the abundant quantities required for large-scale drug toxicity screening. Therefore, there is an urgent need for a novel unlimited, efficient, inexpensive, and predictive model which can be applied for large-scale drug toxicity screening. Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are able to replicate indefinitely and differentiate into most of the body's cell types, including hepatocytes. It is expected that hepatocyte-like cells generated from human ES/iPS cells (human ES/iPS-HLCs) will be a useful tool for drug toxicity screening. To apply human ES/iPS-HLCs to various applications including drug toxicity screening, homogenous and functional HLCs must be differentiated from human ES/iPS cells. In this review, we will introduce the current status of hepatocyte differentiation technology from human ES/iPS cells and a novel method to predict drug-induced liver injury using human ES/iPS-HLCs. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Endothelial progenitor cells from human dental pulp-derived iPS cells as a therapeutic target for ischemic vascular diseases.

PubMed

Yoo, Chae Hwa; Na, Hee-Jun; Lee, Dong-Seol; Heo, Soon Chul; An, Yuri; Cha, Junghwa; Choi, Chulhee; Kim, Jae Ho; Park, Joo-Cheol; Cho, Yee Sook

2013-11-01

Human dental pulp cells (hDPCs) are a valuable source for the generation of patient-specific human induced pluripotent stem cells (hiPSCs). An advanced strategy for the safe and efficient reprogramming of hDPCs and subsequent lineage-specific differentiation is a critical step toward clinical application. In present research, we successfully generated hDPC-iPSCs using only two non-oncogenic factors: Oct4 and Sox2 (2F hDPC-hiPSCs) and evaluated the feasibility of hDPC-iPSCs as substrates for endothelial progenitor cells (EPCs), contributing to EPC-based therapies. Under conventional differentiation conditions, 2F hDPC-hiPSCs showed higher differentiation efficiency, compared to hiPSCs from other cell types, into multipotent CD34(+) EPCs (2F-hEPCs) capable to differentiate into functional endothelial and smooth muscle cells. The angiogenic and neovasculogenic activities of 2F-hEPCs were confirmed using a Matrigel plug assay in mice. In addition, the therapeutic effects of 2F-hEPC transplantation were confirmed in mouse models of hind-limb ischemia and myocardial infarction. Importantly, 2F-EPCs effectively integrated into newly formed vascular structures and enhanced neovascularization via likely both direct and indirect paracrine mechanisms. 2F hDPC-hiPSCs have a robust capability for the generation of angiogenic and vasculogenic EPCs, representing a strategy for patient-specific EPC therapies and disease modeling, particularly for ischemic vascular diseases. Copyright © 2013 Elsevier Ltd. All rights reserved.

RARα-PLZF oncogene inhibits C/EBPα function in myeloid cells

PubMed Central

Girard, Nathalie; Tremblay, Mathieu; Humbert, Magali; Grondin, Benoît; Haman, André; Labrecque, Jean; Chen, Bing; Chen, Zhu; Chen, Sai-Juan; Hoang, Trang

2013-01-01

In acute promyelocytic leukemia, granulocytic differentiation is arrested at the promyelocyte stage. The variant t(11;17) translocation produces two fusion proteins, promyelocytic leukemia zinc finger-retinoic acid receptor α (PLZF-RARα) and RARα-PLZF, both of which participate in leukemia development. Here we provide evidence that the activity of CCAAT/enhancer binding protein α (C/EBPα), a master regulator of granulocytic differentiation, is severely impaired in leukemic promyelocytes with the t(11;17) translocation compared with those associated with the t(15;17) translocation. We show that RARα-PLZF inhibits myeloid cell differentiation through interactions with C/EBPα tethered to DNA, using ChIP and DNA capture assays. Furthermore, RARα-PLZF recruits HDAC1 and causes histone H3 deacetylation at C/EBPα target loci, thereby decreasing the expression of C/EBPα target genes. In line with these results, HDAC inhibitors restore in part C/EBPα target gene expression. These findings provide molecular evidence for a mechanism through which RARα-PLZF acts as a modifier oncogene that subverts differentiation in the granulocytic lineage by associating with C/EBPα and inhibiting its activity. PMID:23898169

Distribution and developmental changes of ghrelin-immunopositive cells in the pancreas of African ostrich chicks (Struthio camelus).

PubMed

Wang, J X; Li, P; Zhang, X T; Ye, L X

2017-09-01

Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor (GHS-R), is produced by multiple cell types and affects feeding behavior, metabolic regulation, and energy balance. In the mammalian pancreas, the types of endocrine cells that are immunoreactive to ghrelin vary. However, little was known about its distribution and developmental changes in the pancreas of African ostrich chicks (Struthio camelus). In the present study, the distribution, morphological characteristics, and developmental changes of ghrelin-immunopositive (ghrelin-ip) cells in the pancreas of African ostrich chicks were investigated using immunohistochemistry. Ghrelin-ip cells were found in both the pancreatic islets and acinar cell regions. The greatest number of ghrelin-ip cells were found in the pancreatic islets, and were primarily observed at the periphery of the islets; some ghrelin-ip cells were also located in the central portion of the pancreatic islets. Interestingly, from postnatal d 1 to d 90, there was a steady decrease in the number of ghrelin-ip cells in the pancreatic islets and acinar cell regions. These results clearly demonstrated that ghrelin-ip cells exist and decreased with age in the African ostrich pancreas from postnatal d 1 to d90. Thus, these findings indicated that ghrelin may be involved in the development of the pancreas in the African ostrich. © 2017 Poultry Science Association Inc.

Concentration- and time-dependent genotoxicity profiles of isoprene monoepoxides and diepoxide, and the cross-linking potential of isoprene diepoxide in cells.

PubMed

Li, Yan; Pelah, Avishay; An, Jing; Yu, Ying-Xin; Zhang, Xin-Yu

2014-01-01

Isoprene, a possible carcinogen, is a petrochemical and a natural product being primarily produced by plants. It is biotransformed to 2-ethenyl-2-methyloxirane (IP-1,2-O) and 2-(1-methylethenyl)oxirane (IP-3,4-O), both of which can be further metabolized to 2-methyl-2,2'-bioxirane (MBO). MBO is mutagenic, but IP-1,2-O and IP-3,4-O are not. While IP-1,2-O has been reported being genotoxic, the genotoxicity of IP-3,4-O and MBO, and the cross-linking potential of MBO have not been examined. In the present study, we used the comet assay to investigate the concentration- and time-dependent genotoxicity profiles of the three metabolites and the cross-linking potential of MBO in human hepatocyte L02 cells. For the incubation time of 1 h, all metabolites showed positive concentration-dependent profiles with a potency rank order of IP-3,4-O > MBO > IP-1,2-O. In human hepatocellular carcinoma (HepG2) and human leukemia (HL60) cells, IP-3,4-O was still more potent in inducing DNA breaks than MBO at high concentrations (>200 μM), although at low concentrations (≤200 μM) IP-3,4-O exhibited slightly lower or similar potency to MBO. Interestingly, their time-dependent genotoxicity profiles (0.5-4 h) in L02 cells were different from each other: IP-1,2-O and MBO (200 μM) exhibited negative and positive profiles, respectively, with IP-3,4-O lying in between, namely, IP-3,4-O-caused DNA breaks did not change over the exposure time. Further experiments demonstrated that hydrolysis of IP-1,2-O contributed to the negative profile and MBO induced cross-links at high concentrations and long incubation times. Collectively, the results suggested that IP-3,4-O might play a significant role in the toxicity of isoprene.

Accounting for immunoprecipitation efficiencies in the statistical analysis of ChIP-seq data.

PubMed

Bao, Yanchun; Vinciotti, Veronica; Wit, Ernst; 't Hoen, Peter A C

2013-05-30

ImmunoPrecipitation (IP) efficiencies may vary largely between different antibodies and between repeated experiments with the same antibody. These differences have a large impact on the quality of ChIP-seq data: a more efficient experiment will necessarily lead to a higher signal to background ratio, and therefore to an apparent larger number of enriched regions, compared to a less efficient experiment. In this paper, we show how IP efficiencies can be explicitly accounted for in the joint statistical modelling of ChIP-seq data. We fit a latent mixture model to eight experiments on two proteins, from two laboratories where different antibodies are used for the two proteins. We use the model parameters to estimate the efficiencies of individual experiments, and find that these are clearly different for the different laboratories, and amongst technical replicates from the same lab. When we account for ChIP efficiency, we find more regions bound in the more efficient experiments than in the less efficient ones, at the same false discovery rate. A priori knowledge of the same number of binding sites across experiments can also be included in the model for a more robust detection of differentially bound regions among two different proteins. We propose a statistical model for the detection of enriched and differentially bound regions from multiple ChIP-seq data sets. The framework that we present accounts explicitly for IP efficiencies in ChIP-seq data, and allows to model jointly, rather than individually, replicates and experiments from different proteins, leading to more robust biological conclusions.

RdgB2 is required for dim-light input into intrinsically photosensitive retinal ganglion cells

PubMed Central

Walker, Marquis T.; Rupp, Alan; Elsaesser, Rebecca; Güler, Ali D.; Sheng, Wenlong; Weng, Shijun; Berson, David M.; Hattar, Samer; Montell, Craig

2015-01-01

A subset of retinal ganglion cells is intrinsically photosensitive (ipRGCs) and contributes directly to the pupillary light reflex and circadian photoentrainment under bright-light conditions. ipRGCs are also indirectly activated by light through cellular circuits initiated in rods and cones. A mammalian homologue (RdgB2) of a phosphoinositide transfer/exchange protein that functions in Drosophila phototransduction is expressed in the retinal ganglion cell layer. This raised the possibility that RdgB2 might function in the intrinsic light response in ipRGCs, which depends on a cascade reminiscent of Drosophila phototransduction. Here we found that under high light intensities, RdgB2−/− mutant mice showed normal pupillary light responses and circadian photoentrainment. Consistent with this behavioral phenotype, the intrinsic light responses of ipRGCs in RdgB2−/− were indistinguishable from wild-type. In contrast, under low-light conditions, RdgB2−/− mutants displayed defects in both circadian photoentrainment and the pupillary light response. The RdgB2 protein was not expressed in ipRGCs but was in GABAergic amacrine cells, which provided inhibitory feedback onto bipolar cells. We propose that RdgB2 is required in a cellular circuit that transduces light input from rods to bipolar cells that are coupled to GABAergic amacrine cells and ultimately to ipRGCs, thereby enabling ipRGCs to respond to dim light. PMID:26269578

Curcumin induces the differentiation of myeloid-derived suppressor cells and inhibits their interaction with cancer cells and related tumor growth

PubMed Central

Tu, Shui Ping; Jin, Huanyu; Shi, Jin Dong; Zhu, Li Ming; Suo, Ya; Lu, Gang; Liu, Anna; Wang, Timothy C.; Yang, Chung S.

2011-01-01

Myeloid-derived suppressor cells (MDSCs) accumulate in the spleen and tumors and contribute to tumor growth, angiogenesis and progression. In this study, we examined the effects of curcumin on the activation and differentiation of MDSCs, their interaction with human cancer cells and related tumor growth. Treatment with curcumin in the diet or by i.p. injection significantly inhibited tumorigenecity and tumor growth, decreased the percentages of MDSCs in the spleen, blood and tumor tissues, reduced IL-6 levels in the serum and tumor tissues in a human gastric cancer xenograft model and a mouse colon cancer allograft model. Curcumin treatment significantly inhibited cell proliferation and colony formation of cancer cells and decreased the secretion of murine interleukin (IL)-6 by MDSCs in a co-culture system. Curcumin treatment inhibited the expansion of MDSCs, the activation of Stat3 and NF-κB in MDSCs, and the secretion of IL-6 by MDSCs when MDSCs were cultured in the presence of IL-1β, or with cancer cell- or myofibroblast-conditioned medium. Furthermore, curcumin treatment polarized MDSCs toward a M1-like phenotype with an increased expression of CCR7 and decreased expression of dectin 1 in vivo and in vitro. Our results demonstrate that curcumin inhibits the accumulation of MDSCs and their interaction with cancer cells and induces the differentiation of MDSCs. The induction of MDSC differentiation and inhibition of the interaction of MDSCs with cancer cells are potential strategies for cancer prevention and therapy. PMID:22030090

A mathematical model of calcium dynamics in HSY cells

PubMed Central

Han, Jung Min; Tanimura, Akihiko; Kirk, Vivien; Sneyd, James

2017-01-01

Saliva is an essential part of activities such as speaking, masticating and swallowing. Enzymes in salivary fluid protect teeth and gums from infectious diseases, and also initiate the digestion process. Intracellular calcium (Ca2+) plays a critical role in saliva secretion and regulation. Experimental measurements of Ca2+ and inositol trisphosphate (IP3) concentrations in HSY cells, a human salivary duct cell line, show that when the cells are stimulated with adenosine triphosphate (ATP) or carbachol (CCh), they exhibit coupled oscillations with Ca2+ spike peaks preceding IP3 spike peaks. Based on these data, we construct a mathematical model of coupled Ca2+ and IP3 oscillations in HSY cells and perform model simulations of three different experimental settings to forecast Ca2+ responses. The model predicts that when Ca2+ influx from the extracellular space is removed, oscillations gradually slow down until they stop. The model simulation of applying a pulse of IP3 predicts that photolysis of caged IP3 causes a transient increase in the frequency of the Ca2+ oscillations. Lastly, when Ca2+-dependent activation of PLC is inhibited, we see an increase in the oscillation frequency and a decrease in the amplitude. These model predictions are confirmed by experimental data. We conclude that, although concentrations of Ca2+ and IP3 oscillate, Ca2+ oscillations in HSY cells are the result of modulation of the IP3 receptor by intracellular Ca2+, and that the period is modulated by the accompanying IP3 oscillations. PMID:28199326

Micro-chromatin Immunoprecipation (μChIP) Protocol for Real-time PCR Analysis of a Limited Amount of Cells.

PubMed

Gillotin, Sébastien; Guillemot, François

2016-06-20

Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) is an important strategy to study gene regulation. When availability of cells is limited, however, it can be useful to focus on specific genes to investigate in depth the role of transcription factors or histone marks. Unfortunately, performing ChIP experiments to study transcription factors' binding to DNA can be difficult when biological material is restricted. This protocol describes a robust method to perform μChIP for over-expressed or endogenous transcription factors using ~100,000 cells per ChIP experiment (Masserdotti et al ., 2015). We also describe optimization steps, which we think are critical for this protocol to work and which can be used to further reduce the number of cells.

Phospholipase C-β1 and β4 Contribute to Non-Genetic Cell-to-Cell Variability in Histamine-Induced Calcium Signals in HeLa Cells

PubMed Central

Ishida, Sachiko; Matsu-ura, Toru; Fukami, Kiyoko; Michikawa, Takayuki; Mikoshiba, Katsuhiko

2014-01-01

A uniform extracellular stimulus triggers cell-specific patterns of Ca2+ signals, even in genetically identical cell populations. However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP3) concentration changes using a fluorescent IP3 sensor in single HeLa cells showing different patterns of histamine-induced Ca2+ oscillations in terms of the time constant of Ca2+ spike amplitude decay and the Ca2+ oscillation frequency. HeLa cells stimulated with histamine exhibited a considerable variation in the temporal pattern of Ca2+ signals and we found that there were cell-specific IP3 dynamics depending on the patterns of Ca2+ signals. RT-PCR and western blot analyses showed that phospholipase C (PLC)-β1, -β3, -β4, -γ1, -δ3 and -ε were expressed at relatively high levels in HeLa cells. Small interfering RNA-mediated silencing of PLC isozymes revealed that PLC-β1 and PLC-β4 were specifically involved in the histamine-induced IP3 increases in HeLa cells. Modulation of IP3 dynamics by knockdown or overexpression of the isozymes PLC-β1 and PLC-β4 resulted in specific changes in the characteristics of Ca2+ oscillations, such as the time constant of the temporal changes in the Ca2+ spike amplitude and the Ca2+ oscillation frequency, within the range of the cell-to-cell variability found in wild-type cell populations. These findings indicate that the heterogeneity in the process of IP3 production, rather than IP3-induced Ca2+ release, can cause cell-to-cell variability in the patterns of Ca2+ signals and that PLC-β1 and PLC-β4 contribute to generate cell-specific Ca2+ signals evoked by G protein-coupled receptor stimulation. PMID:24475116

miRNA-556-3p promotes human bladder cancer proliferation, migration and invasion by negatively regulating DAB2IP expression.

PubMed

Feng, Chen; Sun, Ping; Hu, Jing; Feng, Hua; Li, Mingqiu; Liu, Guibo; Pan, Yanming; Feng, Ying; Xu, Yongliang; Feng, Kejian; Feng, Yukuan

2017-06-01

MicroRNAs (miRNAs) play critical roles in tumorigenesis and metastasis by negatively regulating gene expression through complementary binding to the 3'-untranslated region of target mRNAs. The role of miRNAs in expression of the tumor suppressor DAB2IP in bladder cancer (BC) remains unknown. The aim of the present study was to identify miRNAs targeting DAB2IP and determine their expression and function in BC. We predicted candidate miRNAs targeting DAB2IP using TargetScan software. Dual-luciferase reporter assays confirmed that miRNA-556-3p directly regulated DAB2IP expression. Quantitative RT-PCR and RNase protection assays showed that endogenous miRNA-556-3p expression was significantly upregulated in clinical samples of BC patients and BC cell lines and western blot analysis indicated that DAB2IP expression in BC tissues and BC cell lines was concurrently downregulated. Gain or loss of function studies showed that upregulation of miRNA-556-3p promoted proliferation, invasion, migration and colony formation of BC cells, whereas downregulation resulted in opposite effects. Importantly, restoration of DAB2IP expression rescued the effects induced by miRNA-556-3p. Overexpression of miRNA-556-3p in BC cells not only decreased DAB2IP expression, but also markedly increased Ras GTPase activity and ERK1/2 phosphorylation level. These findings suggest that DAB2IP is a direct target of miRNA-556-3p, and endogenous miRNA-556-3p expression shows inverse correlation with simultaneous DAB2IP expression in BC tissues and cells. miRNA-556-3p functions as a tumor promoter in tumorigenesis and metastasis of BC by targeting DAB2IP. Moreover, miRNA-556-3p-mediated DAB2IP suppression plays an oncogenic role by partial activation of the Ras-ERK pathway.

Developing rods transplanted into the degenerating retina of Crx-knockout mice exhibit neural activity similar to native photoreceptors

PubMed Central

Homma, Kohei; Okamoto, Satoshi; Mandai, Michiko; Gotoh, Norimoto; Rajasimha, Harsha K.; Chang, Yi-Sheng; Chen, Shan; Li, Wei; Cogliati, Tiziana; Swaroop, Anand; Takahashi, Masayo

2013-01-01

Replacement of dysfunctional or dying photoreceptors offers a promising approach for retinal neurodegenerative diseases, including age-related macular degeneration and retinitis pigmentosa. Several studies have demonstrated the integration and differentiation of developing rod photoreceptors when transplanted in wild type or degenerating retina; however, the physiology and function of the donor cells are not adequately defined. Here, we describe the physiological properties of developing rod photoreceptors that are tagged with GFP driven by the promoter of rod differentiation factor, Nrl. GFP-tagged developing rods show Ca2+ responses and rectifier outward currents that are smaller than those observed in fully developed photoreceptors, suggesting their immature developmental state. These immature rods also exhibit hyperpolarization-activated current (Ih) induced by the activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. When transplanted into the subretinal space of wild type or retinal degeneration mice, GFP-tagged developing rods can integrate into the photoreceptor outer nuclear layer in wild-type mouse retina, and exhibit Ca2+ responses and membrane current comparable to native rod photoreceptors. A proportion of grafted rods develop rhodopsin-positive outer segment-like structures within two weeks after transplantation into the retina of Crx-knockout mice, and produce rectifier outward current and Ih upon membrane depolarization and hyperpolarization. GFP-positive rods derived from induced pluripotent stem (iPS) cells also display similar membrane current Ih as native developing rod photoreceptors, express rod-specific phototransduction genes, and HCN-1 channels. We conclude that Nrl-promoter driven GFP-tagged donor photoreceptors exhibit physiological characteristics of rods and that iPS cell-derived rods in vitro may provide a renewable source for cell replacement therapy. PMID:23495178

Rebamipide attenuates autoimmune arthritis severity in SKG mice via regulation of B cell and antibody production

PubMed Central

Byun, J-K; Moon, S-J; Jhun, J-Y; Kim, E-K; Park, J-S; Youn, J; Min, J-K; Park, S-H; Kim, H-Y; Cho, M-L

2014-01-01

Oxidative stress is involved in the pathophysiology of rheumatoid arthritis (RA). We investigated the therapeutic potential of rebamipide, a gastroprotective agent with a property of reactive oxygen species scavenger, on the development of inflammatory polyarthritis and the pathophysiological mechanisms by which rebamipide might confer anti-arthritic effects in SKG mice, an animal model of RA. Intraperitoneal (i.p.) injection of rebamipide attenuated the severity of clinical and histological arthritis. Rebampide treatment reduced the number of T helper type 1 (Th1), Th2, Th17, inducible T cell co-stimulator (ICOS)+ follicular helper T (Tfh) transitional type (T2) and mature B cells in the spleen, but increased the number of regulatory T (Treg), CD19+ CD1dhigh CD5high, CD19+ CD25high forkhead box protein 3 (FoxP3)+ regulatory B (Breg) cells, memory B cells, and transitional type 1 (T1) B cells. In addition, flow cytometric analysis revealed significantly decreased populations of FAS+GL-7+ germinal centre B cells and B220− CD138+ plasma cells in the spleens of rebamipide-treated SKG mice compared to controls. Rebamipide decreased germinal centre B cells and reciprocally induced Breg cells in a dose-dependent manner in vitro. Rebamipide-induced Breg cells had more suppressive capacity in relation to T cell proliferation and also inhibited Th17 differentiation from murine CD4+ T cells. Together, these data show that i.p. administration of rebamipide suppresses arthritis severity by inducing Breg and Treg cells and suppressing Tfh and Th17 cells in a murine model of RA. PMID:24749771

Epigenetic Marks Define the Lineage and Differentiation Potential of Two Distinct Neural Crest-Derived Intermediate Odontogenic Progenitor Populations

PubMed Central

Gopinathan, Gokul; Kolokythas, Antonia

2013-01-01

Epigenetic mechanisms, such as histone modifications, play an active role in the differentiation and lineage commitment of mesenchymal stem cells. In the present study, epigenetic states and differentiation profiles of two odontogenic neural crest-derived intermediate progenitor populations were compared: dental pulp (DP) and dental follicle (DF). ChIP on chip assays revealed substantial H3K27me3-mediated repression of odontoblast lineage genes DSPP and dentin matrix protein 1 (DMP1) in DF cells, but not in DP cells. Mineralization inductive conditions caused steep increases of mineralization and patterning gene expression levels in DP cells when compared to DF cells. In contrast, mineralization induction resulted in a highly dynamic histone modification response in DF cells, while there was only a subdued effect in DP cells. Both DF and DP progenitors featured H3K4me3-active marks on the promoters of early mineralization genes RUNX2, MSX2, and DLX5, while OSX, IBSP, and BGLAP promoters were enriched for H3K9me3 or H3K27me3. Compared to DF cells, DP cells expressed higher levels of three pluripotency-associated genes, OCT4, NANOG, and SOX2. Finally, gene ontology comparison of bivalent marks unique for DP and DF cells highlighted cell–cell attachment genes in DP cells and neurogenesis genes in DF cells. In conclusion, the present study indicates that the DF intermediate odontogenic neural crest lineage is distinguished from its DP counterpart by epigenetic repression of DSPP and DMP1 genes and through dynamic histone enrichment responses to mineralization induction. Findings presented here highlight the crucial role of epigenetic regulatory mechanisms in the terminal differentiation of odontogenic neural crest lineages. PMID:23379639

Stably Fluorescent Cell Line of Human Ovarian Epithelial Cancer Cells SK-OV-3ip-red.

PubMed

Konovalova, E V; Shulga, A A; Chumakov, S P; Khodarovich, Yu M; Woo, Eui-Jeon; Deev, S M

2017-11-01

Stable red fluorescing line of human ovarian epithelial cancer cells SK-OV-3ip-red was generated expressing gene coding for protein TurboFP635 (Katushka) fluorescing in the far-red spectrum region with excitation and emission peaks at 588 and 635 nm, respectively. Fluorescence of SK-OV-3ip-red line remained high during long-term cell culturing and after cryogenic freezing. The obtained cell line SK-OV-3ip-red can serve a basis for a model of a scattered tumor with numerous/extended metastases and used both for testing anticancer drugs inhibiting metastasis growth and for non-invasive monitoring of the growth dynamics with high precision.

Interferon-gamma improves impaired dentinogenic and immunosuppressive functions of irreversible pulpitis-derived human dental pulp stem cells

PubMed Central

Sonoda, Soichiro; Yamaza, Haruyoshi; Ma, Lan; Tanaka, Yosuke; Tomoda, Erika; Aijima, Reona; Nonaka, Kazuaki; Kukita, Toshio; Shi, Songtao; Nishimura, Fusanori; Yamaza, Takayoshi

2016-01-01

Clinically, irreversible pulpitis is treated by the complete removal of pulp tissue followed by replacement with artificial materials. There is considered to be a high potential for autologous transplantation of human dental pulp stem cells (DPSCs) in endodontic treatment. The usefulness of DPSCs isolated from healthy teeth is limited. However, DPSCs isolated from diseased teeth with irreversible pulpitis (IP-DPSCs) are considered to be suitable for dentin/pulp regeneration. In this study, we examined the stem cell potency of IP-DPSCs. In comparison with healthy DPSCs, IP-DPSCs expressed lower colony-forming capacity, population-doubling rate, cell proliferation, multipotency, in vivo dentin regeneration, and immunosuppressive activity, suggesting that intact IP-DPSCs may be inadequate for dentin/pulp regeneration. Therefore, we attempted to improve the impaired in vivo dentin regeneration and in vitro immunosuppressive functions of IP-DPSCs to enable dentin/pulp regeneration. Interferon gamma (IFN-γ) treatment enhanced in vivo dentin regeneration and in vitro T cell suppression of IP-DPSCs, whereas treatment with tumor necrosis factor alpha did not. Therefore, these findings suggest that IFN-γ may be a feasible modulator to improve the functions of impaired IP-DPSCs, suggesting that autologous transplantation of IFN-γ-accelerated IP-DPSCs might be a promising new therapeutic strategy for dentin/pulp tissue engineering in future endodontic treatment. PMID:26775677

Use of First-phase Insulin Secretion in Early Diagnosis of Thyroid Diabetes and Type 2 Diabetes Mellitus

PubMed Central

Meng, Li-Heng; Huang, Yao; Zhou, Jia; Liang, Xing-Huan; Xian, Jing; Li, Li; Qin, Ying-Fen

2017-01-01

Background: A relationship between hyperthyroidism and insulin secretion in type 2 diabetes mellitus (T2DM) has been reported. Therefore, this study explored the use of first-phase insulin secretion in the differential diagnosis of thyroid diabetes (TDM) and T2DM. Methods: In total, 101 patients with hyperthyroidism were divided into hyperthyroidism with normal glucose tolerance (TNGT), hyperthyroidism with impaired glucose regulation (TIGR), and diabetes (TDM) groups. Furthermore, 96 patients without hyperthyroidism were recruited as control groups (normal glucose tolerance [NGT], impaired glucose regulation [IGR], and T2DM). The following parameters were evaluated: homeostasis model assessment (HOMA)-IR, HOMA-β, modified β-cell function index (MBCI), peak insulin/fasting insulin (IP/I0), AUCins-OGTT, and AUCins-OGTT/AUCglu-OGTT from the oral glucose tolerance test (OGTT) insulin release test were utilized to assess the second-phase insulin secretion, while the IP/I0, AIR0′~10′, and AUCins-IVGTT from the intravenous glucose tolerance test (IVGTT) insulin release test were used to assess the first-phase insulin secretion. Results: In the OGTT, the HOMA-β values of the TNGT and TDM groups were higher than those of the NGT and T2DM groups (all P < 0.05). In the hyperthyroidism groups, the MBCI of the TDM group was lower than that of the TNGT and TIGR groups (all P < 0.05). Among the control groups, the MBCI values of the IGR and T2DM groups were lower than that of the normal glucose tolerance (NGT) group (all P < 0.05). In the IVGTT, insulin secretion peaked for all groups at 2–4 min, except for the T2DM group, which showed a low plateau and no secretion peak. The IP values of the TNGT, TIGR, and TDM groups were higher than those of the NGT, IGR, and T2DM groups (all P < 0.05). The Ip/I0, AIR0′~10′, and AUCins-IVGTT values of the TDM group were higher than those of the T2DM group but were lower than those of the TNGT, TIGR, NGR, and IGR groups (all P < 0.05). Compared with the other five groups, the Ip/I0, AIR0′~10′, and AUCins-IVGTT values of the T2DM group were significantly decreased (all P < 0.05). The Ip/I0 and AUCins-IVGTT values of the TNGT group were higher than those of the NGT group (all P < 0.05). Conclusions: β-cell function in TDM patients is superior to that in T2DM patients. First-phase insulin secretion could be used as an early diagnostic marker to differentiate TDM and T2DM. PMID:28345543

Inhibition by salmeterol and cilomilast of fluticasone-enhanced IP-10 release in airway epithelial cells.

PubMed

Reddy, P J; Aksoy, Mark O; Yang, Yi; Li, Xiu Xia; Ji, Rong; Kelsen, Steven G

2008-02-01

The CXC chemokines, IP-10/CXCL10 and IL-8/CXCL8, play a role in obstructive lung disease by attracting Th1/Tc1 lymphocytes and neutrophils, respectively. Inhaled corticosteroids (ICS) and long acting beta 2-agonists (LABA) are widely used. However, their effect(s) on the release of IP-10 and IL-8 by airway epithelial cells are poorly understood. This study examined the effects of fluticasone, salmeterol, and agents which raise intracellular cAMP (cilomilast and db-cAMP) on the expression of IP-10 and IL-8 protein and mRNA. Studies were performed in cultured human airway epithelial cells during cytokine-stimulated IP-10 and IL-8 release. Cytokine treatment (TNF-alpha, IL-1beta and IFN-gamma) increased IP-10 and IL-8 protein and mRNA levels. Fluticasone (0.1 nM to 1 microM) increased IP-10 but reduced IL-8 protein release without changing IP-10 mRNA levels assessed by real time RT-PCR. The combination of salmeterol (1 micro M) and cilomilast (1-10 mu M) reduced IP-10 but had no effect on IL-8 protein. Salmeterol alone (1 micro M) and db-cAMP alone (1 mM) antagonised the effects of fluticasone on IP-10 but not IL-8 protein. In human airway epithelial cells, inhibition by salmeterol of fluticasone-enhanced IP-10 release may be an important therapeutic effect of the LABA/ICS combination not present when the two drugs are used separately.

3D culturing and differentiation of SH-SY5Y neuroblastoma cells on bacterial nanocellulose scaffolds.

PubMed

Innala, Marcus; Riebe, Ilse; Kuzmenko, Volodymyr; Sundberg, Johan; Gatenholm, Paul; Hanse, Eric; Johannesson, Sara

2014-10-01

A new in vitro model, mimicking the complexity of nerve tissue, was developed based on a bacterial nanocellulose (BNC) scaffold that supports 3D culturing of neuronal cells. BNC is extracellularly excreted by Gluconacetobacter xylinus (G. xylinus) in the shape of long non-aggregated nanofibrils. The cellulose network created by G. xylinus has good mechanical properties, 99% water content, and the ability to be shaped into 3D structures by culturing in different molds. Surface modification with trimethyl ammonium beta-hydroxypropyl (TMAHP) to induce a positive surface charge, followed by collagen I coating, has been used to improve cell adhesion, growth, and differentiation on the scaffold. In the present study, we used SH-SY5Y neuroblastoma cells as a neuronal model. These cells attached and proliferated well on the BNC scaffold, as demonstrated by scanning electron microscopy (SEM) and the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay. Following neuronal differentiation, we demonstrated functional action potentials (APs) by electrophysiological recordings, indicating the presence of mature neurons on the scaffolds. In conclusion, we have demonstrated for the first time that neurons can attach, proliferate, and differentiate on BNC. This 3D model based on BNC scaffolds could possibly be used for developing in vitro disease models, when combined with human induced pluripotent stem (iPS) cells (derived from diseased patients) for detailed investigations of neurodegenerative disease mechanisms and in the search for new therapeutics.

Laser-Based Propagation of Human iPS and ES Cells Generates Reproducible Cultures with Enhanced Differentiation Potential

PubMed Central

Hohenstein Elliott, Kristi A.; Peterson, Cory; Soundararajan, Anuradha; Kan, Natalia; Nelson, Brandon; Spiering, Sean; Mercola, Mark; Bright, Gary R.

2012-01-01

Proper maintenance of stem cells is essential for successful utilization of ESCs/iPSCs as tools in developmental and drug discovery studies and in regenerative medicine. Standardization is critical for all future applications of stem cells and necessary to fully understand their potential. This study reports a novel approach for the efficient, consistent expansion of human ESCs and iPSCs using laser sectioning, instead of mechanical devices or enzymes, to divide cultures into defined size clumps for propagation. Laser-mediated propagation maintained the pluripotency, quality, and genetic stability of ESCs/iPSCs and led to enhanced differentiation potential. This approach removes the variability associated with ESC/iPSC propagation, significantly reduces the expertise, labor, and time associated with manual passaging techniques and provides the basis for scalable delivery of standardized ESC/iPSC lines. Adoption of standardized protocols would allow researchers to understand the role of genetics, environment, and/or procedural effects on stem cells and would ensure reproducible production of stem cell cultures for use in clinical/therapeutic applications. PMID:22701128

Resveratrol-induced autophagy is dependent on IP3Rs and on cytosolic Ca2.

PubMed

Luyten, Tomas; Welkenhuyzen, Kirsten; Roest, Gemma; Kania, Elzbieta; Wang, Liwei; Bittremieux, Mart; Yule, David I; Parys, Jan B; Bultynck, Geert

2017-06-01

Previous work revealed that intracellular Ca 2+ signals and the inositol 1,4,5-trisphosphate (IP 3 ) receptors (IP 3 R) are essential to increase autophagic flux in response to mTOR inhibition, induced by either nutrient starvation or rapamycin treatment. Here, we investigated whether autophagy induced by resveratrol, a polyphenolic phytochemical reported to trigger autophagy in a non-canonical way, also requires IP 3 Rs and Ca 2+ signaling. Resveratrol augmented autophagic flux in a time-dependent manner in HeLa cells. Importantly, autophagy induced by resveratrol (80μM, 2h) was completely abolished in the presence of 10μM BAPTA-AM, an intracellular Ca 2+ -chelating agent. To elucidate the IP 3 R's role in this process, we employed the recently established HEK 3KO cells lacking all three IP 3 R isoforms. In contrast to the HEK293 wt cells and to HEK 3KO cells re-expressing IP 3 R1, autophagic responses in HEK 3KO cells exposed to resveratrol were severely impaired. These altered autophagic responses could not be attributed to alterations in the mTOR/p70S6K pathway, since resveratrol-induced inhibition of S6 phosphorylation was not abrogated by chelating cytosolic Ca 2+ or by knocking out IP 3 Rs. Finally, we investigated whether resveratrol by itself induced Ca 2+ release. In permeabilized HeLa cells, resveratrol neither affected the sarco- and endoplasmic reticulum Ca 2+ ATPase (SERCA) activity nor the IP 3 -induced Ca 2+ release nor the basal Ca 2+ leak from the ER. Also, prolonged (4 h) treatment with 100μM resveratrol did not affect subsequent IP 3 -induced Ca 2+ release. However, in intact HeLa cells, although resveratrol did not elicit cytosolic Ca 2+ signals by itself, it acutely decreased the ER Ca 2+ -store content irrespective of the presence or absence of IP 3 Rs, leading to a dampened agonist-induced Ca 2+ signaling. In conclusion, these results reveal that IP 3 Rs and cytosolic Ca 2+ signaling are fundamentally important for driving autophagic flux, not only in response to mTOR inhibition but also in response to non-canonical autophagy inducers like resveratrol. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech. Copyright © 2017 Elsevier B.V. All rights reserved.

Human Decidua-Derived Mesenchymal Cells Are a Promising Source for the Generation and Cell Banking of Human Induced Pluripotent Stem Cells

PubMed Central

Shofuda, Tomoko; Kanematsu, Daisuke; Fukusumi, Hayato; Yamamoto, Atsuyo; Bamba, Yohei; Yoshitatsu, Sumiko; Suemizu, Hiroshi; Nakamura, Masato; Sugimoto, Yoshikazu; Furue, Miho Kusuda; Kohara, Arihiro; Akamatsu, Wado; Okada, Yohei; Okano, Hideyuki; Yamasaki, Mami; Kanemura, Yonehiro

2013-01-01

Placental tissue is a biomaterial with remarkable potential for use in regenerative medicine. It has a three-layer structure derived from the fetus (amnion and chorion) and the mother (decidua), and it contains huge numbers of cells. Moreover, placental tissue can be collected without any physical danger to the donor and can be matched with a variety of HLA types. The decidua-derived mesenchymal cells (DMCs) are highly proliferative fibroblast-like cells that express a similar pattern of CD antigens as bone marrow-derived mesenchymal cells (BM-MSCs). Here we demonstrated that induced pluripotent stem (iPS) cells could be efficiently generated from DMCs by retroviral transfer of reprogramming factor genes. DMC-hiPS cells showed equivalent characteristics to human embryonic stem cells (hESCs) in colony morphology, global gene expression profile (including human pluripotent stem cell markers), DNA methylation status of the OCT3/4 and NANOG promoters, and ability to differentiate into components of the three germ layers in vitro and in vivo. The RNA expression of XIST and the methylation status of its promoter region suggested that DMC-iPSCs, when maintained undifferentiated and pluripotent, had three distinct states: (1) complete X-chromosome reactivation, (2) one inactive X-chromosome, or (3) an epigenetic aberration. Because DMCs are derived from the maternal portion of the placenta, they can be collected with the full consent of the adult donor and have considerable ethical advantages for cell banking and the subsequent generation of human iPS cells for regenerative applications. PMID:26858858

The therapeutic potential of human adipose-derived mesenchymal stem cells producing CXCL10 in a mouse melanoma lung metastasis model.

PubMed

Mirzaei, Hamed; Salehi, Hossein; Oskuee, Reza Kazemi; Mohammadpour, Ali; Mirzaei, Hamid Reza; Sharifi, Mohammad Reza; Salarinia, Reza; Darani, Hossein Yousofi; Mokhtari, Mojgan; Masoudifar, Aria; Sahebkar, Amirhossein; Salehi, Rasoul; Jaafari, Mahmoud Reza

2018-04-10

Interferon γ-induced protein 10 kDa (IP-10) is a potent chemoattractant and has been suggested to enhance antitumor activity and mediate tumor regression through multiple mechanisms of action. Multiple lines of evidence have indicated that genetically-modified adult stem cells represent a potential source for cell-based cancer therapy. In the current study, we assessed therapeutic potential of human adipose derived mesenchymal stem cells (hADSC) genetically-modified to express IP-10 for the treatment of lung metastasis in an immunocompetent mouse model of metastatic melanoma. A Piggybac vector encoding IP-10 was employed to transfect hADSC ex vivo. Expression and bioactivity of the transgenic protein from hADSCs expressing IP-10 were confirmed prior to in vivo studies. Our results indicated that hADSCs expressing IP-10 could inhibit the growth of B16F10 melanoma cells and significantly prolonged survival. Immunohistochemistry analysis, TUNEL assay and western blot analysis indicated that hADSCs expressing IP-10 inhibited tumor cell growth, hindered tumor infiltration of Tregs, restricted angiogenesis and significantly prolonged survival. In conclusion, our results demonstrated that targeting metastatic tumor sites by hADSC expressing IP-10 could reduce melanoma tumor growth and lung metastasis. Copyright © 2018 Elsevier B.V. All rights reserved.

Abnormalities in human pluripotent cells due to reprogramming mechanisms

PubMed Central

Ma, Hong; Morey, Robert; O’Neil, Ryan C.; He, Yupeng; Daughtry, Brittany; Schultz, Matthew D.; Hariharan, Manoj; Nery, Joseph R.; Castanon, Rosa; Sabatini, Karen; Thiagarajan, Rathi D.; Tachibana, Masahito; Kang, Eunju; Tippner-Hedges, Rebecca; Ahmed, Riffat; Gutierrez, Nuria Marti; Van Dyken, Crystal; Polat, Alim; Sugawara, Atsushi; Sparman, Michelle; Gokhale, Sumita; Amato, Paula; Wolf, Don P.; Ecker, Joseph R.; Laurent, Louise C.; Mitalipov, Shoukhrat

2016-01-01

Human pluripotent stem cells hold potential for regenerative medicine, but available cell types have significant limitations. Although embryonic stem cells (ES cells) from in vitro fertilized embryos (IVF ES cells) represent the ‘gold standard’, they are allogeneic to patients. Autologous induced pluripotent stem cells (iPS cells) are prone to epigenetic and transcriptional aberrations. To determine whether such abnormalities are intrinsic to somatic cell reprogramming or secondary to the reprogramming method, genetically matched sets of human IVF ES cells, iPS cells and nuclear transfer ES cells (NT ES cells) derived by somatic cell nuclear transfer (SCNT) were subjected to genome-wide analyses. Both NT ES cells and iPS cells derived from the same somatic cells contained comparable numbers of de novo copy number variations. In contrast, DNA methylation and transcriptome profiles of NT ES cells corresponded closely to those of IVF ES cells, whereas iPS cells differed and retained residual DNA methylation patterns typical of parental somatic cells. Thus, human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal for cell replacement therapies. PMID:25008523

Inhibition of P-Glycoprotein Mediated Efflux in Caco-2 Cells by Phytic Acid.

PubMed

Li, Lujia; Fu, Qingxue; Xia, Mengxin; Xin, Lei; Shen, Hongyi; Li, Guowen; Ji, Guang; Meng, Qianchao; Xie, Yan

2018-01-31

Phytic acid (IP6) is a natural phosphorylated inositol, which is abundantly present in most cereal grains and seeds. This study investigated the effects of IP6 regulation on P-glycoprotein (P-gp) and its potential mechanisms using in situ and in vitro models. The effective permeability of the typical P-gp substrate rhodamine 123 (R123) in colon was significantly increased from (1.69 ± 0.22) × 10 -5 cm/s in the control group to (3.39 ± 0.417) × 10 -5 cm/s (p < 0.01) in the 3.5 mM IP6 group. Additionally, IP6 can concentration-dependently decrease the R123 efflux ratio in both Caco-2 and MDCK II-MDR1 cell monolayers and increase intracellular R123 accumulation in Caco-2 cells. Furthermore, IP6 noncompetitively inhibited P-gp by impacting R123 efflux kinetics. The noncompetitive inhibition of P-gp by IP6 was likely due to decreases in P-gp ATPase activity and P-gp molecular conformational changes induced by IP6. In summary, IP6 is a promising P-gp inhibitor candidate.

Functional dissociations between four basic arithmetic operations in the human posterior parietal cortex: A cytoarchitectonic mapping study

PubMed Central

Rosenberg-Lee, Miriam; Chang, Ting Ting; Young, Christina B; Wu, Sarah; Menon, Vinod

2011-01-01

Although lesion studies over the past several decades have focused on functional dissociations in posterior parietal cortex (PPC) during arithmetic, no consistent view has emerged of its differential involvement in addition, subtraction, multiplication, and division. To circumvent problems with poor anatomical localization, we examined functional overlap and dissociations in cytoarchitectonically-defined subdivisions of the intraparietal sulcus (IPS), superior parietal lobule (SPL) and angular gyrus (AG), across these four operations. Compared to a number identification control task, all operations except addition, showed a consistent profile of left posterior IPS activation and deactivation in the right posterior AG. Multiplication and subtraction differed significantly in right, but not left, IPS and AG activity, challenging the view that the left AG differentially subserves retrieval during multiplication. Although addition and multiplication both rely on retrieval, multiplication evoked significantly greater activation in right posterior IPS, as well as the prefrontal cortex, lingual and fusiform gyri, demonstrating that addition and multiplication engage different brain processes. Comparison of PPC responses to the two pairs of inverse operations: division vs. multiplication and subtraction vs. addition revealed greater activation of left lateral SPL during division, suggesting that processing inverse relations is operation specific. Our findings demonstrate that individual IPS, SPL and AG subdivisions are differentially modulated by the four arithmetic operations and they point to significant functional heterogeneity and individual differences in activation and deactivation within the PPC. Critically, these effects are related to retrieval, calculation and inversion, the three key cognitive processes that are differentially engaged by arithmetic operations. Our findings point to distributed representation of these processes in the human PPC and also help explain why lesion and previous imaging studies have yielded inconsistent findings. PMID:21616086

Functional dissociations between four basic arithmetic operations in the human posterior parietal cortex: a cytoarchitectonic mapping study.

PubMed

Rosenberg-Lee, Miriam; Chang, Ting Ting; Young, Christina B; Wu, Sarah; Menon, Vinod

2011-07-01

Although lesion studies over the past several decades have focused on functional dissociations in posterior parietal cortex (PPC) during arithmetic, no consistent view has emerged of its differential involvement in addition, subtraction, multiplication, and division. To circumvent problems with poor anatomical localization, we examined functional overlap and dissociations in cytoarchitectonically defined subdivisions of the intraparietal sulcus (IPS), superior parietal lobule (SPL) and angular gyrus (AG), across these four operations. Compared to a number identification control task, all operations except addition, showed a consistent profile of left posterior IPS activation and deactivation in the right posterior AG. Multiplication and subtraction differed significantly in right, but not left, IPS and AG activity, challenging the view that the left AG differentially subserves retrieval during multiplication. Although addition and multiplication both rely on retrieval, multiplication evoked significantly greater activation in right posterior IPS, as well as the prefrontal cortex, lingual and fusiform gyri, demonstrating that addition and multiplication engage different brain processes. Comparison of PPC responses to the two pairs of inverse operations: division versus multiplication and subtraction versus addition revealed greater activation of left lateral SPL during division, suggesting that processing inverse relations is operation specific. Our findings demonstrate that individual IPS, SPL and AG subdivisions are differentially modulated by the four arithmetic operations and they point to significant functional heterogeneity and individual differences in activation and deactivation within the PPC. Critically, these effects are related to retrieval, calculation and inversion, the three key cognitive processes that are differentially engaged by arithmetic operations. Our findings point to distribute representation of these processes in the human PPC and also help explain why lesion and previous imaging studies have yielded inconsistent findings. Copyright © 2011 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Orellana, S.A.; Trilivas, I.; Brown, J.H.

Carbachol and guanine nucleotides stimulate formation of the (/sup 3/H)inositol phosphates IP, IP2, and IP3 in saponin-permeabilized monolayers labelled with (/sup 3/H) inositol. Carbachol alone has little effect on formation of the (/sup 3/H) inositol phosphates (IPs), but GTP..gamma..S causes synergistic accumulation of (/sup 3/H)IPs to levels similar to those seen in intact cells. GTP, GppNHp, and GTP..gamma..S all support formation of the (/sup 3/H)IPs, with or without hormone, but GTP..gamma..S is the most effective. In the presence of GTP..gamma..S, the effect of carbachol is dose-dependent. Half-maximal and maximal accumulation of the (/sup 3/H)IPs occur at approx. 5 ..mu..M andmore » approx. 100 ..mu..M carbachol, respectively; values close to those seen in intact cells. GTP..gamma..S alone stimulates formation of the (/sup 3/H)IPs after a brief lag time. The combination of GTP..gamma..S and carbachol both increases the rate of, and decreases the lag in, formation of the (/sup 3/H)IPs. LiCl increases (/sup 3/H)IP and IP2, but not IP3, accumulation; while 2,3-diphosphoglycerate substantially increases that of (/sup 3/H)IP3. GTP..gamma..S and carbachol cause formation of (/sup 3/H)IPs in the absence of Ca/sup + +/, but formation induced by GTP..gamma..S with or without carbachol is Ca/sup + +/-sensitive over a range of physiological concentrations. Although carbachol, Ca/sup + +/, and GTP..gamma..S all have effects on formation of (/sup 3/H)IPs, GTP..gamma..S appears to be a primary and obligatory regulator of phosphoinositide hydrolysis in the permeabilized 1321N1 astrocytoma cell.« less

Stimulation of Inositol 1,4,5-Trisphosphate (IP3) Receptor Subtypes by Adenophostin A and Its Analogues

PubMed Central

Saleem, Huma; Tovey, Stephen C.; Riley, Andrew M.; Potter, Barry V. L.; Taylor, Colin W.

2013-01-01

Inositol 1,4,5-trisphosphate receptors (IP3R) are intracellular Ca2+ channels. Most animal cells express mixtures of the three IP3R subtypes encoded by vertebrate genomes. Adenophostin A (AdA) is the most potent naturally occurring agonist of IP3R and it shares with IP3 the essential features of all IP3R agonists, namely structures equivalent to the 4,5-bisphosphate and 6-hydroxyl of IP3. The two essential phosphate groups contribute to closure of the clam-like IP3-binding core (IBC), and thereby IP3R activation, by binding to each of its sides (the α- and β-domains). Regulation of the three subtypes of IP3R by AdA and its analogues has not been examined in cells expressing defined homogenous populations of IP3R. We measured Ca2+ release evoked by synthetic adenophostin A (AdA) and its analogues in permeabilized DT40 cells devoid of native IP3R and stably expressing single subtypes of mammalian IP3R. The determinants of high-affinity binding of AdA and its analogues were indistinguishable for each IP3R subtype. The results are consistent with a cation-π interaction between the adenine of AdA and a conserved arginine within the IBC α-domain contributing to closure of the IBC. The two complementary contacts between AdA and the α-domain (cation-π interaction and 3″-phosphate) allow activation of IP3R by an analogue of AdA (3″-dephospho-AdA) that lacks a phosphate group equivalent to the essential 5-phosphate of IP3. These data provide the first structure-activity analyses of key AdA analogues using homogenous populations of all mammalian IP3R subtypes. They demonstrate that differences in the Ca2+ signals evoked by AdA analogues are unlikely to be due to selective regulation of IP3R subtypes. PMID:23469136

Human IP10-scFv and DC-induced CTL synergistically inhibit the growth of glioma in a xenograft model.

PubMed

Wang, Xuan; Zhang, Fang-Cheng; Zhao, Hong-Yang; Lu, Xiao-Ling; Sun, Yun; Xiong, Zhi-Yong; Jiang, Xiao-Bing

2014-08-01

The epidermal growth factor receptor (EGFR) mutant of EGFRvIII is highly expressed in glioma cells, and the EGFRvIII-specific dendritic cell (DC)-induced tumor antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) may hold promise in cancer immunotherapy. Interferon (IFN)-γ-inducible protein (IP)-10 (IP-10) is a potent inhibitor of angiogenesis and can recruit CXCR3(+) T cells, including CD8(+) T cells, which are important for the control of tumor growth. In this study, we assessed if the combination of IP10-EGFRvIIIscFv with DC-induced CTLs would improve the therapeutic antitumor efficacy. IP10-scFv was generated by linking the human IP-10 gene with the DNA fragment for anti-EGFRvIIIscFv with a (Gly4Ser)3 flexible linker, purified by affinity chromatography, and characterized for its anti-EGFRvIII immunoreactivity and chemotactic activity. DCs were isolated from human peripheral blood monocyte cells and pulsed with EGFRvIII-peptide, then co-cultured with autologous CD8(+) T cells. BALB/c-nu mice were inoculated with human glioma U87-EGFRvIII cells in the brain and treated intracranially with IP10-scFv and/or intravenously with DC-induced CTLs for evaluating the therapeutic effect. Treatment with both IP10-scFv and EGFRvIII peptide-pulsed, DC-induced CTL synergistically inhibited the growth of glioma and prolonged the survival of tumor-bearing mice, which was accompanied by the inhibition of tumor angiogenesis and enhancement of cytotoxicity, thereby increasing the numbers of brain-infiltrating lymphocytes (BILs) and prolonging the residence time of CTLs in the tumor.

Regulation of myeloid leukemia factor-1 interacting protein (MLF1IP) expression in glioblastoma.

PubMed

Hanissian, Silva H; Teng, Bin; Akbar, Umar; Janjetovic, Zorica; Zhou, Qihong; Duntsch, Christopher; Robertson, Jon H

2005-06-14

The myelodysplasia/myeloid leukemia factor 1-interacting protein MLF1IP is a novel gene which encodes for a putative transcriptional repressor. It is localized to human chromosome 4q35.1 and is expressed in both the nuclei and cytoplasm of cells. Northern and Western blot analyses have revealed MLF1IP to be present at very low amounts in normal brain tissues, whereas a number of human and rat glioblastoma (GBM) cell lines demonstrated a high level expression of the MLF1IP protein. Immunohistochemical analysis of rat F98 and C6 GBM tumor models showed that MLF1IP was highly expressed in the tumor core where it was co-localized with MLF1 and nestin. Moreover, MLF1IP expression was elevated in the contralateral brain where no tumor cells were detected. These observations, together with previous data demonstrating a role for MLF1IP in erythroleukemias, suggest a possible function for this protein in glioma pathogenesis and potentially in other types of malignancies.

Differential tissue-specific function of the Adora2b in cardio-protection

PubMed Central

Seo, Seong-wook; Koeppen, Michael; Bonney, Stephanie; Gobel, Merit; Thayer, Molly; Harter, Patrick N.; Ravid, Katya; Eltzschig, Holger K.; Mittelbronn, Michel; Walker, Lori; Eckle, Tobias

2015-01-01

The adenosine A2b-receptor (Adora2b) has been implicated in cardio-protection from myocardial ischemia. As such the Adora2b was found to be critical in ischemic preconditioning (IP) or ischemia reperfusion (IR) injury of the heart. While the Adora2b is present on various cells types, the tissue specific role of the Adora2b in cardio-protection is still unknown. To study the tissue specific role of Adora2b signaling on inflammatory cells, endothelia or myocytes during myocardial ischemia in vivo, we intercrossed floxed Adora2b mice with Lyz2-Cre+, VE-Cadherin-Cre+ or Myosin-Cre+ transgenic mice, respectively. Mice were exposed to 60 minutes of myocardial ischemia with or without IP (4×5min) followed by 120 minutes of reperfusion. Cardio-protection by IP was abolished in Adora2bf/f-VE-Cadherin-Cre+ or Adora2bf/f-Myosin-Cre+, indicating that Adora2bs signaling on endothelia or myocytes mediates IP. In contrast, primarily Adora2b signaling on inflammatory cells was necessary to provide cardio-protection in IR injury, indicated by significantly larger infarcts and higher troponin levels in Adora2bf/f-Lyz2-Cre+ mice only. Cytokine profiling of IR injury in Adora2bf/f-Lyz2-Cre+ mice pointed towards PMNs. Analysis of PMNs from Adora2bf/f-Lyz2-Cre+ confirmed PMNs as one source of identified tissue cytokines. Finally, adoptive transfer of Ador2b−/− PMNs revealed a critical role of the Adorab2 on PMNs in cardio-protection from IR-injury. Adora2b signaling mediates different types of cardio-protection in a tissue specific manner. These findings have implications for the use of Adora2b agonists in the treatment or prevention of myocardial injury by ischemia. PMID:26136425

GATA1 and PU.1 Bind to Ribosomal Protein Genes in Erythroid Cells: Implications for Ribosomopathies

PubMed Central

Amanatiadou, Elsa P.; Papadopoulos, Giorgio L.; Strouboulis, John; Vizirianakis, Ioannis S.

2015-01-01

The clear connection between ribosome biogenesis dysfunction and specific hematopoiesis-related disorders prompted us to examine the role of critical lineage-specific transcription factors in the transcriptional regulation of ribosomal protein (RP) genes during terminal erythroid differentiation. By applying EMSA and ChIP methodologies in mouse erythroleukemia cells we show that GATA1 and PU.1 bind in vitro and in vivo the proximal promoter region of the RPS19 gene which is frequently mutated in Diamond-Blackfan Anemia. Moreover, ChIPseq data analysis also demonstrates that several RP genes are enriched as potential GATA1 and PU.1 gene targets in mouse and human erythroid cells, with GATA1 binding showing an association with higher ribosomal protein gene expression levels during terminal erythroid differentiation in human and mouse. Our results suggest that RP gene expression and hence balanced ribosome biosynthesis may be specifically and selectively regulated by lineage specific transcription factors during hematopoiesis, a finding which may be clinically relevant to ribosomopathies. PMID:26447946

Epigenomic Landscape of Human Fetal Brain, Heart, and Liver.

PubMed

Yan, Liying; Guo, Hongshan; Hu, Boqiang; Li, Rong; Yong, Jun; Zhao, Yangyu; Zhi, Xu; Fan, Xiaoying; Guo, Fan; Wang, Xiaoye; Wang, Wei; Wei, Yuan; Wang, Yan; Wen, Lu; Qiao, Jie; Tang, Fuchou

2016-02-26

The epigenetic regulation of spatiotemporal gene expression is crucial for human development. Here, we present whole-genome chromatin immunoprecipitation followed by high throughput DNA sequencing (ChIP-seq) analyses of a wide variety of histone markers in the brain, heart, and liver of early human embryos shortly after their formation. We identified 40,181 active enhancers, with a large portion showing tissue-specific and developmental stage-specific patterns, pointing to their roles in controlling the ordered spatiotemporal expression of the developmental genes in early human embryos. Moreover, using sequential ChIP-seq, we showed that all three organs have hundreds to thousands of bivalent domains that are marked by both H3K4me3 and H3K27me3, probably to keep the progenitor cells in these organs ready for immediate differentiation into diverse cell types during subsequent developmental processes. Our work illustrates the potentially critical roles of tissue-specific and developmental stage-specific epigenomes in regulating the spatiotemporal expression of developmental genes during early human embryonic development. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Recruitment of Fanconi Anemia and Breast Cancer Proteins to DNA Damage Sites is differentially Governed by Replication

PubMed Central

Shen, Xi; Do, Huong; Li, Yongjian; Chung, Woo-Hyun; Tomasz, Maria; de Winter, Johan P.; Xia, Bing; Elledge, Stephen J.; Wang, Weidong; Li, Lei

2009-01-01

Summary Fanconi anemia (FA) is characterized by cellular hypersensivity to DNA crosslinking agents, but how the Fanconi pathway protects cells from DNA crosslinks and whether FA proteins act directly on crosslinks remains unclear. We developed a chromatin-IP-based strategy termed eChIP and detected association of multiple FA proteins with DNA crosslinks in vivo. Inter-dependence analyses revealed that crosslink-specific enrichment of various FA proteins is controlled by distinct mechanisms. BRCA-related FA proteins (BRCA2, FANCJ/BACH1, and FANCN/PALB2), but not FA core and I/D2 complexes, require replication for their crosslink association. FANCD2, but not FANCJ and FANCN, requires the FA core complex for its recruitment. FA core complex requires nucleotide excision repair proteins XPA and XPC for its association. Consistent with the distinct recruitment mechanism, recombination-independent crosslink repair was inversely affected in cells deficient of FANC-core versus BRCA-related FA proteins. Thus, FA proteins participate in distinct DNA damage response mechanisms governed by DNA replication status. PMID:19748364

An HDAC2-TET1 switch at distinct chromatin regions significantly promotes the maturation of pre-iPS to iPS cells

PubMed Central

Wei, Tingyi; Chen, Wen; Wang, Xiukun; Zhang, Man; Chen, Jiayu; Zhu, Songcheng; Chen, Long; Yang, Dandan; Wang, Guiying; Jia, Wenwen; Yu, Yangyang; Duan, Tao; Wu, Minjuan; Liu, Houqi; Gao, Shaorong; Kang, Jiuhong

2015-01-01

The maturation of induced pluripotent stem cells (iPS) is one of the limiting steps of somatic cell reprogramming, but the underlying mechanism is largely unknown. Here, we reported that knockdown of histone deacetylase 2 (HDAC2) specifically promoted the maturation of iPS cells. Further studies showed that HDAC2 knockdown significantly increased histone acetylation, facilitated TET1 binding and DNA demethylation at the promoters of iPS cell maturation-related genes during the transition of pre-iPS cells to a fully reprogrammed state. We also found that HDAC2 competed with TET1 in the binding of the RbAp46 protein at the promoters of maturation genes and knockdown of TET1 markedly prevented the activation of these genes. Collectively, our data not only demonstrated a novel intrinsic mechanism that the HDAC2-TET1 switch critically regulates iPS cell maturation, but also revealed an underlying mechanism of the interplay between histone acetylation and DNA demethylation in gene regulation. PMID:25934799

Cytomegalovirus (CMV) DNA Quantitation in Bronchoalveolar Lavage Fluid From Hematopoietic Stem Cell Transplant Recipients With CMV Pneumonia

PubMed Central

Stevens-Ayers, Terry; Travi, Giovanna; Huang, Meei-Li; Cheng, Guang-Shing; Xie, Hu; Leisenring, Wendy; Erard, Veronique; Seo, Sachiko; Kimball, Louise; Corey, Lawrence; Pergam, Steven A; Jerome, Keith R.

2017-01-01

Abstract Background. Quantitative cytomegalovirus (CMV) DNA–specific polymerase chain reaction (PCR) analysis is widely used as a surveillance method for hematopoietic stem cell transplant (HCT) recipients. However, no CMV DNA threshold exists in bronchoalveolar lavage (BAL) to differentiate pneumonia from pulmonary shedding. Methods. We tested archived BAL fluid samples from 132 HCT recipients with CMV pneumonia and 139 controls (100 patients with non-CMV pneumonia, 18 with idiopathic pneumonia syndrome [IPS], and 21 who were asymptomatic) by quantitative CMV and β-globin DNA–specific PCR. Results. Patients with CMV pneumonia had higher median viral loads (3.9 log10 IU/mL; interquartile range [IQR], 2.6–6.0 log10 IU/mL) than controls (0 log10 IU/mL [IQR, 0–1.6 log10 IU/mL] for patients with non-CMV pneumonia, 0 log10 IU/mL [IQR, 0–1.6 log10 IU/mL] for patients with IPS, and 1.63 log10 IU/mL [IQR, 0–2.5 log10 IU/mL] for patients who were asymptomatic; P < .001 for all comparisons to patients with CMV pneumonia). Receiver operating characteristic curve analyses and predictive models identified a cutoff CMV DNA level of 500 IU/mL to differentiate between CMV pneumonia and pulmonary shedding, using current CMV pneumonia prevalence figures. However, different levels may be appropriate in settings of very high or low CMV pneumonia prevalence. The presence of pulmonary copathogens, radiographic presentation, or pulmonary hemorrhage did not alter predictive values. Conclusion. CMV DNA load in BAL can be used to differentiate CMV pneumonia from pulmonary shedding. PMID:28181657

Cytomegalovirus (CMV) DNA Quantitation in Bronchoalveolar Lavage Fluid From Hematopoietic Stem Cell Transplant Recipients With CMV Pneumonia.

PubMed

Boeckh, Michael; Stevens-Ayers, Terry; Travi, Giovanna; Huang, Meei-Li; Cheng, Guang-Shing; Xie, Hu; Leisenring, Wendy; Erard, Veronique; Seo, Sachiko; Kimball, Louise; Corey, Lawrence; Pergam, Steven A; Jerome, Keith R

2017-05-15

Quantitative cytomegalovirus (CMV) DNA-specific polymerase chain reaction (PCR) analysis is widely used as a surveillance method for hematopoietic stem cell transplant (HCT) recipients. However, no CMV DNA threshold exists in bronchoalveolar lavage (BAL) to differentiate pneumonia from pulmonary shedding. We tested archived BAL fluid samples from 132 HCT recipients with CMV pneumonia and 139 controls (100 patients with non-CMV pneumonia, 18 with idiopathic pneumonia syndrome [IPS], and 21 who were asymptomatic) by quantitative CMV and β-globin DNA-specific PCR. Patients with CMV pneumonia had higher median viral loads (3.9 log10 IU/mL; interquartile range [IQR], 2.6-6.0 log10 IU/mL) than controls (0 log10 IU/mL [IQR, 0-1.6 log10 IU/mL] for patients with non-CMV pneumonia, 0 log10 IU/mL [IQR, 0-1.6 log10 IU/mL] for patients with IPS, and 1.63 log10 IU/mL [IQR, 0-2.5 log10 IU/mL] for patients who were asymptomatic; P < .001 for all comparisons to patients with CMV pneumonia). Receiver operating characteristic curve analyses and predictive models identified a cutoff CMV DNA level of 500 IU/mL to differentiate between CMV pneumonia and pulmonary shedding, using current CMV pneumonia prevalence figures. However, different levels may be appropriate in settings of very high or low CMV pneumonia prevalence. The presence of pulmonary copathogens, radiographic presentation, or pulmonary hemorrhage did not alter predictive values. CMV DNA load in BAL can be used to differentiate CMV pneumonia from pulmonary shedding. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

KChIP1 modulation of Kv4.3-mediated A-type K(+) currents and repetitive firing in hippocampal interneurons.

PubMed

Bourdeau, M L; Laplante, I; Laurent, C E; Lacaille, J-C

2011-03-10

Neuronal A-type K(+) channels regulate action potential waveform, back-propagation and firing frequency. In hippocampal CA1 interneurons located at the stratum lacunosum-moleculare/radiatum junction (LM/RAD), Kv4.3 mediates A-type K(+) currents and a Kv4 β-subunit of the Kv channel interacting protein (KChIP) family, KChIP1, appears specifically expressed in these cells. However, the functional role of this accessory subunit in A-type K(+) currents and interneuron excitability remains largely unknown. Thus, first we studied KChIP1 and Kv4.3 channel interactions in human embryonic kidney 293 (HEK293) cells and determined that KChIP1 coexpression modulated the biophysical properties of Kv4.3 A-type currents (faster recovery from inactivation, leftward shift of activation curve, faster rise time and slower decay) and this modulation was selectively prevented by KChIP1 short interfering RNA (siRNA) knockdown. Next, we evaluated the effects of KChIP1 down-regulation by siRNA on A-type K(+) currents in LM/RAD interneurons in slice cultures. Recovery from inactivation of A-type K(+) currents was slower after KChIP1 down-regulation but other properties were unchanged. In addition, down-regulation of KChIP1 levels did not affect action potential waveform and firing, but increased firing frequency during suprathreshold depolarizations, indicating that KChIP1 regulates interneuron excitability. The effects of KChIP1 down-regulation were cell-specific since CA1 pyramidal cells that do not express KChIP1 were unaffected. Overall, our findings suggest that KChIP1 interacts with Kv4.3 in LM/RAD interneurons, enabling faster recovery from inactivation of A-type currents and thus promoting stronger inhibitory control of firing during sustained activity. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

NANOG priming before full reprogramming may generate germ cell tumours.

PubMed

Grad, I; Hibaoui, Y; Jaconi, M; Chicha, L; Bergström-Tengzelius, R; Sailani, M R; Pelte, M F; Dahoun, S; Mitsiadis, T A; Töhönen, V; Bouillaguet, S; Antonarakis, S E; Kere, J; Zucchelli, M; Hovatta, O; Feki, A

2011-11-09

Reprogramming somatic cells into a pluripotent state brings patient-tailored, ethical controversy-free cellular therapy closer to reality. However, stem cells and cancer cells share many common characteristics; therefore, it is crucial to be able to discriminate between them. We generated two induced pluripotent stem cell (iPSC) lines, with NANOG pre-transduction followed by OCT3/4, SOX2, and LIN28 overexpression. One of the cell lines, CHiPS W, showed normal pluripotent stem cell characteristics, while the other, CHiPS A, though expressing pluripotency markers, failed to differentiate and gave rise to germ cell-like tumours in vivo. Comparative genomic hybridisation analysis of the generated iPS lines revealed that they were genetically more stable than human embryonic stem cell counterparts. This analysis proved to be predictive for the differentiation potential of analysed cells. Moreover, the CHiPS A line expressed a lower ratio of p53/p21 when compared to CHiPS W. NANOG pre-induction followed by OCT3/4, SOX2, MYC, and KLF4 induction resulted in the same tumour-inducing phenotype. These results underline the importance of a re-examination of the role of NANOG during reprogramming. Moreover, this reprogramming method may provide insights into primordial cell tumour formation and cancer stem cell transformation.

Conserved Kv4 N-terminal domain critical for effects of Kv channel-interacting protein 2.2 on channel expression and gating.

PubMed

Bähring, R; Dannenberg, J; Peters, H C; Leicher, T; Pongs, O; Isbrandt, D

2001-06-29

Association of Kv channel-interacting proteins (KChIPs) with Kv4 channels leads to modulation of these A-type potassium channels (An, W. F., Bowlby, M. R., Betty, M., Cao, J., Ling, H. P., Mendoza, G., Hinson, J. W., Mattsson, K. I., Strassle, B. W., Trimmer, J. S., and Rhodes, K. J. (2000) Nature 403, 553-556). We cloned a KChIP2 splice variant (KChIP2.2) from human ventricle. In comparison with KChIP2.1, coexpression of KChIP2.2 with human Kv4 channels in mammalian cells slowed the onset of Kv4 current inactivation (2-3-fold), accelerated the recovery from inactivation (5-7-fold), and shifted Kv4 steady-state inactivation curves by 8-29 mV to more positive potentials. The features of Kv4.2/KChIP2.2 currents closely resemble those of cardiac rapidly inactivating transient outward currents. KChIP2.2 stimulated the Kv4 current density in Chinese hamster ovary cells by approximately 55-fold. This correlated with a redistribution of immunoreactivity from perinuclear areas to the plasma membrane. Increased Kv4 cell-surface expression and current density were also obtained in the absence of KChIP2.2 when the highly conserved proximal Kv4 N terminus was deleted. The same domain is required for association of KChIP2.2 with Kv4 alpha-subunits. We propose that an efficient transport of Kv4 channels to the cell surface depends on KChIP binding to the Kv4 N-terminal domain. Our data suggest that the binding is necessary, but not sufficient, for the functional activity of KChIPs.

Infiltrating T cells promote renal cell carcinoma (RCC) progression via altering the estrogen receptor β-DAB2IP signals.

PubMed

Yeh, Chiuan-Ren; Ou, Zheng-Yu; Xiao, Guang-Qian; Guancial, Elizabeth; Yeh, Shuyuan

2015-12-29

Previous studies indicated the T cells, one of the most common types of immune cells existing in the microenvironment of renal cell carcinoma (RCC), may influence the progression of RCC. The potential linkage of T cells and the estrogen receptor beta (ERβ), a key player to impact RCC progression, however, remains unclear. Our results demonstrate that RCC cells can recruit more T cells than non-malignant kidney cells. Using an in vitro matrigel invasion system, we found infiltrating T cells could promote RCC cells invasion via increasing ERβ expression and transcriptional activity. Mechanism dissection suggested that co-culturing T cells with RCC cells released more T cell attraction factors, including IFN-γ, CCL3 and CCL5, suggesting a positive regulatory feed-back mechanism. Meanwhile, infiltrating T cells may also promote RCC cell invasion via increased ERβ and decreased DAB2IP expressions, and knocking down DAB2IP can then reverse the T cells-promoted RCC cell invasion. Together, our results suggest that infiltrating T cells may promote RCC cell invasion via increasing the RCC cell ERβ expression to inhibit the tumor suppressor DAB2IP signals. Further mechanism dissection showed that co-culturing T cells with RCC cells could produce more IGF-1 and FGF-7, which may enhance the ERβ transcriptional activity. The newly identified relationship between infiltrating T cells/ERβ/DAB2IP signals may provide a novel therapeutic target in the development of agents against RCC.

Intermediate progenitors are increased by lengthening of the cell cycle through calcium signaling and p53 expression in human neural progenitors

PubMed Central

García-García, Elisa; Pino-Barrio, María José; López-Medina, Laura; Martínez-Serrano, Alberto

2012-01-01

During development, neurons can be generated directly from a multipotent progenitor or indirectly through an intermediate progenitor (IP). This last mode of division amplifies the progeny of neurons. The mechanisms governing the generation and behavior of IPs are not well understood. In this work, we demonstrate that the lengthening of the cell cycle enhances the generation of neurons in a human neural progenitor cell system in vitro and also the generation and expansion of IPs. These IPs are insulinoma-associated 1 (Insm1)+/BTG family member 2 (Btg2)−, which suggests an increase in a self-amplifying IP population. Later the cultures express neurogenin 2 (Ngn2) and become neurogenic. The signaling responsible for this cell cycle modulation is investigated. It is found that the release of calcium from the endoplasmic reticulum to the cytosol in response to B cell lymphoma-extra large overexpression or ATP addition lengths the cell cycle and increases the number of IPs and, in turn, the final neuron outcome. Moreover, data suggest that the p53–p21 pathway is responsible for the changes in cell cycle. In agreement with this, increased p53 levels are necessary for a calcium-induced increase in neurons. Our findings contribute to understand how calcium signaling can modulate cell cycle length during neurogenesis. PMID:22323293

Tetramethylpyrazine induces SH-SY5Y cell differentiation toward the neuronal phenotype through activation of the PI3K/Akt/Sp1/TopoIIβ pathway.

PubMed

Yan, Yong-Xin; Zhao, Jun-Xia; Han, Shuo; Zhou, Na-Jing; Jia, Zhi-Qiang; Yao, Sheng-Jie; Cao, Cui-Li; Wang, Yan-Ling; Xu, Yan-Nan; Zhao, Juan; Yan, Yun-Li; Cui, Hui-Xian

2015-12-01

Tetramethylpyrazine (TMP) is an active compound extracted from the traditional Chinese medicinal herb Chuanxiong. Previously, we have shown that TMP induces human SH-SY5Y neuroblastoma cell differentiation toward the neuronal phenotype by targeting topoisomeraseIIβ (TopoIIβ), a protein implicated in neural development. In the present study, we aimed to elucidate whether the transcriptional factors specificity protein 1 (Sp1) and nuclear factor Y (NF-Y), in addition to the upstream signaling pathways ERK1/2 and PI3K/Akt, are involved in modulating TopoIIβ expression in the neuronal differentiation process. We demonstrated that SH-SY5Y cells treated with TMP (80μM) terminally differentiated into neurons, characterized by increased neuronal markers, tubulin βIII and microtubule associated protein 2 (MAP2), and increased neurite outgrowth, with no negative effect on cell survival. TMP also increased the expression of TopoIIβ, which was accompanied by increased expression of Sp1 in the differentiated neuron-like cells, whereas NF-Y protein levels remained unchanged following the differentiation progression. We also found that the phosphorylation level of Akt, but not ERK1/2, was significantly increased as a result of TMP stimulation. Furthermore, as established by chromatin immunoprecipitation (ChIP) assay, activation of the PI3K/Akt pathway increased Sp1 binding to the promoter of the TopoIIβ gene. Blockage of PI3K/Akt was shown to lead to subsequent inhibition of TopoIIβ expression and neuronal differentiation. Collectively, the results indicate that the PI3K/Akt/Sp1/TopoIIβ signaling pathway is necessary for TMP-induced neuronal differentiation. Our findings offer mechanistic insights into understanding the upstream regulation of TopoIIβ in neuronal differentiation, and suggest potential applications of TMP both in neuroscience research and clinical practice to treat relevant diseases of the nervous system. Copyright © 2015 Elsevier GmbH. All rights reserved.

Differential modulation of the discriminative stimulus effects of methamphetamine and cocaine by alprazolam and oxazepam in male and female rats.

PubMed

Spence, A L; Guerin, G F; Goeders, N E

2016-03-01

Drug users often combine benzodiazepines with psychostimulants, such as methamphetamine. However, very little research has been conducted on this type of polydrug use, particularly in female subjects. The present study was therefore designed to examine the effects of two benzodiazepines, alprazolam and oxazepam, on the discriminative stimulus effects of methamphetamine and cocaine in both male and female rats. Rats were trained to discriminate methamphetamine (1.0 mg/kg, ip) or cocaine (10 mg/kg, ip) from saline using a two-lever operant, food-reinforced, drug discrimination design. Pretreatment with oxazepam (5, 10 and 20 mg/kg, ip) significantly attenuated methamphetamine discrimination in both male and female rats. In contrast, however, the high dose of alprazolam (4 mg/kg, ip) actually augmented the subjective effects of lower doses of methamphetamine (0.125 and 0.25 mg/kg, ip). Oxazepam produced similar effects on the subjective effects of cocaine as with methamphetamine, significantly reducing cocaine discrimination in both male and female rats. However, neither the high nor low dose of alprazolam (2 and 4 mg/kg, ip) produced any apparent effect on cocaine discrimination. Finally, while similar results were observed in both male and female rats across these experiments, methamphetamine and cocaine discrimination were more sensitive to oxazepam in female subjects. The results of these experiments suggest that alprazolam and oxazepam can differentially affect the subjective effects of methamphetamine and cocaine. These results also demonstrate that alprazolam can differentially affect the discriminative stimulus effects of methamphetamine and cocaine. Copyright © 2015 Elsevier Ltd. All rights reserved.

β Subunits Functionally Differentiate Human Kv4.3 Potassium Channel Splice Variants

PubMed Central

Abbott, Geoffrey W.

2017-01-01

The human ventricular cardiomyocyte transient outward K+ current (Ito) mediates the initial phase of myocyte repolarization and its disruption is implicated in Brugada Syndrome and heart failure (HF). Human cardiac Ito is generated primarily by two Kv4.3 splice variants (Kv4.3L and Kv4.3S, diverging only by a C-terminal, S6-proximal, 19-residue stretch unique to Kv4.3L), which are differentially remodeled in HF, but considered functionally alike at baseline. Kv4.3 is regulated in human heart by β subunits including KChIP2b and KCNEs, but their effects were previously assumed to be Kv4.3 isoform-independent. Here, this assumption was tested experimentally using two-electrode voltage-clamp analysis of human subunits co-expressed in Xenopus laevis oocytes. Unexpectedly, Kv4.3L-KChIP2b channels exhibited up to 8-fold lower current augmentation, 40% slower inactivation, and 5 mV-shifted steady-state inactivation compared to Kv4.3S-KChIP2b. A synthetic peptide mimicking the 19-residue stretch diminished these differences, reinforcing the importance of this segment in mediating Kv4.3 regulation by KChIP2b. KCNE subunits induced further functional divergence, including a 7-fold increase in Kv4.3S-KCNE4-KChIP2b current compared to Kv4.3L-KCNE4-KChIP2b. The discovery of β-subunit-dependent functional divergence in human Kv4.3 splice variants suggests a C-terminal signaling hub is crucial to governing β-subunit effects upon Kv4.3, and demonstrates the potential significance of differential Kv4.3 gene-splicing and β subunit expression in myocyte physiology and pathobiology. PMID:28228734

β Subunits Functionally Differentiate Human Kv4.3 Potassium Channel Splice Variants.

PubMed

Abbott, Geoffrey W

2017-01-01

The human ventricular cardiomyocyte transient outward K + current ( I to ) mediates the initial phase of myocyte repolarization and its disruption is implicated in Brugada Syndrome and heart failure (HF). Human cardiac I to is generated primarily by two Kv4.3 splice variants (Kv4.3L and Kv4.3S, diverging only by a C-terminal, S6-proximal, 19-residue stretch unique to Kv4.3L), which are differentially remodeled in HF, but considered functionally alike at baseline. Kv4.3 is regulated in human heart by β subunits including KChIP2b and KCNEs, but their effects were previously assumed to be Kv4.3 isoform-independent. Here, this assumption was tested experimentally using two-electrode voltage-clamp analysis of human subunits co-expressed in Xenopus laevis oocytes. Unexpectedly, Kv4.3L-KChIP2b channels exhibited up to 8-fold lower current augmentation, 40% slower inactivation, and 5 mV-shifted steady-state inactivation compared to Kv4.3S-KChIP2b. A synthetic peptide mimicking the 19-residue stretch diminished these differences, reinforcing the importance of this segment in mediating Kv4.3 regulation by KChIP2b. KCNE subunits induced further functional divergence, including a 7-fold increase in Kv4.3S-KCNE4-KChIP2b current compared to Kv4.3L-KCNE4-KChIP2b. The discovery of β-subunit-dependent functional divergence in human Kv4.3 splice variants suggests a C-terminal signaling hub is crucial to governing β-subunit effects upon Kv4.3, and demonstrates the potential significance of differential Kv4.3 gene-splicing and β subunit expression in myocyte physiology and pathobiology.

Running TCP/IP over ATM Networks.

ERIC Educational Resources Information Center

Witt, Michael

1995-01-01

Discusses Internet protocol (IP) and subnets and describes how IP may operate over asynchronous transfer mode (ATM). Topics include TCP (transmission control protocol), ATM cells and adaptation layers, a basic architectural model for IP over ATM, address resolution, mapping IP to a subnet technology, and connection management strategy. (LRW)

Dynamic methylation and expression of Oct4 in early neural stem cells

PubMed Central

Lee, Shih-Han; Jeyapalan, Jennie N; Appleby, Vanessa; Mohamed Noor, Dzul Azri; Sottile, Virginie; Scotting, Paul J

2010-01-01

Neural stem cells are a multipotent population of tissue-specific stem cells with a broad but limited differentiation potential. However, recent studies have shown that over-expression of the pluripotency gene, Oct4, alone is sufficient to initiate a process by which these can form ‘induced pluripotent stem cells’ (iPS cells) with the same broad potential as embryonic stem cells. This led us to examine the expression of Oct4 in endogenous neural stem cells, as data regarding its expression in neural stem cells in vivo are contradictory and incomplete. In this study we have therefore analysed the expression of Oct4 and other genes associated with pluripotency throughout development of the mouse CNS and in neural stem cells grown in vitro. We find that Oct4 is still expressed in the CNS by E8.5, but that this expression declines rapidly until it is undetectable by E15.5. This decline is coincident with the gradual methylation of the Oct4 promoter and proximal enhancer. Immunostaining suggests that the Oct4 protein is predominantly cytoplasmic in location. We also found that neural stem cells from all ages expressed the pluripotency associated genes, Sox2, c-Myc, Klf4 and Nanog. These data provide an explanation for the varying behaviour of cells from the early neuroepithelium at different stages of development. The expression of these genes also provides an indication of why Oct4 alone is sufficient to induce iPS formation in neural stem cells at later stages. PMID:20646110

Specific receptor for inositol-1,4,5-trisphosphate in permeabilized rabbit neutrophils

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bradford, P.G.; Spat, A.; Rubin, R.P.

1986-03-05

Neutrophil chemotaxis and degranulation are resultant, in part, from the mobilization of intracellular calcium by inositol-1,4,5-trisphosphate ((1,4,5)IP/sub 3/), one of the products of chemoattractant-stimulated phospholipase C activity. High specific activity (ca. 40 Ci/mmol) (/sup 32/P)(1,4,5)IP/sub 3/ was prepared from (..gamma..-/sup 32/P)ATP-labeled human erythrocyte ghosts and was used in binding assays with saponin-permeabilized rabbit peritoneal neutrophils. At 4/sup 0/C and in the presence of inhibitors of the IP/sub 3/ 5-phosphomonoesterase, (/sup 32/P)(1,4,5)IP/sub 3/ rapidly associated with a specific binding component which saturated within 60s. Nonspecific binding, taken as the residual binding in the presence of 10 ..mu..M (1,4,5)IP/sub 3/, was 15%more » of the total. No specific binding was detected using intact cells. The specific binding to permeable cells was reversible (t/sup 1/2/ approx. 60s) and could be inhibited in a dose-dependent manner by (1,4,5)IP/sub 3/ (EC/sub 50/ = 30 nM) and by other calcium mobilizing inositol phosphates ((2,4,5)IP/sub 3/) but not by inactive analogs ((1,4)IP/sub 2/, (4,5)IP/sub 2/, (1)IP). The dose-responses of (1,4,5)IP/sub 3/ and (2,4,5)IP/sub 3/ in inhibiting (/sup 32/P)(1,4,5)IP/sub 3/ specific binding correlated well with their abilities to release Ca/sup 2 +/ from nonmitochondrial vesicular stores in the same preparation of cells, suggesting that the authors have identified the physiological receptor for (1,4,5)IP/sub 3/.« less

Extended Self-Renewal and Accelerated Reprogramming in the Absence of Kdm5b

PubMed Central

Hu, Gangqing; Yu, Zu-Xi; Liu, Chengyu

2013-01-01

Embryonic stem (ES) cell pluripotency is thought to be regulated in part by H3K4 methylation. However, it is unclear how H3K4 demethylation contributes to ES cell function and participates in induced pluripotent stem (iPS) cell reprogramming. Here, we show that KDM5B, which demethylates H3K4, is important for ES cell differentiation and presents a barrier to the reprogramming process. Depletion of Kdm5b leads to an extension in the self-renewal of ES cells in the absence of LIF. Transcriptome analysis revealed the persistent expression of pluripotency genes and underexpression of developmental genes during differentiation in the absence of Kdm5b, suggesting that KDM5B plays a key role in cellular fate changes. We also observed accelerated reprogramming of differentiated cells in the absence of Kdm5b, demonstrating that KDM5B is a barrier to the reprogramming process. Expression analysis revealed that mesenchymal master regulators associated with the epithelial-to-mesenchymal transition (EMT) are downregulated during reprogramming in the absence of Kdm5b. Moreover, global analysis of H3K4me3/2 revealed that enhancers of fibroblast genes are rapidly deactivated in the absence of Kdm5b, and genes associated with EMT lose H3K4me3/2 during the early reprogramming process. These findings provide functional insight into the role for KDM5B in regulating ES cell differentiation and as a barrier to the reprogramming process. PMID:24100015

Human Induced Hepatic Lineage-Oriented Stem Cells: Autonomous Specification of Human iPS Cells toward Hepatocyte-Like Cells without Any Exogenous Differentiation Factors

PubMed Central

Yanagi, Satoshi; Kato, Chika; Takashima, Ryokichi; Kobayashi, Eiji; Hagiwara, Keitaro; Ochiya, Takahiro

2015-01-01

Preparing targeted cells for medical applications from human induced pluripotent stem cells (hiPSCs) using growth factors, compounds, or gene transfer has been challenging. Here, we report that human induced hepatic lineage-oriented stem cells (hiHSCs) were generated and expanded as a new type of hiPSC under non-typical coculture with feeder cells in a chemically defined hiPSC medium at a very high density. Self-renewing hiHSCs expressed markers of both human embryonic stem cells (hESCs) and hepatocytes. Those cells were highly expandable, markedly enhancing gene expression of serum hepatic proteins and cytochrome P450 enzymes with the omission of FGF-2 from an undefined hiPSC medium. The hepatic specification of hiHSCs was not attributable to the genetic and epigenetic backgrounds of the starting cells, as they were established from distinct donors and different types of cells. Approximately 90% of hiHSCs autonomously differentiated to hepatocyte-like cells, even in a defined minimum medium without any of the exogenous growth factors necessary for hepatic specification. After 12 days of this culture, the differentiated cells significantly enhanced gene expression of serum hepatic proteins (ALB, SERPINA1, TTR, TF, FABP1, FGG, AGT, RBP4, and AHSG), conjugating enzymes (UGT2B4, UGT2B7, UGT2B10, GSTA2, and GSTA5), transporters (SULT2A1, SLC13A5, and SLCO2B1), and urea cycle-related enzymes (ARG1 and CPS1). In addition, the hepatocyte-like cells performed key functions of urea synthesis, albumin secretion, glycogen storage, indocyanine green uptake, and low-density lipoprotein uptake. The autonomous hepatic specification of hiHSCs was due to their culture conditions (coculture with feeder cells in a defined hiPSC medium at a very high density) in self-renewal rather than in differentiation. These results suggest the feasibility of preparing large quantities of hepatocytes as a convenient and inexpensive hiPSC differentiation. Our study also suggests the necessity of optimizing culture conditions to generate other specific lineage-oriented hiPSCs, allowing for a very simple differentiation. PMID:25875613

Inositol pyrophosphates mediate the DNA-PK/ATM-p53 cell death pathway by regulating CK2 phosphorylation of Tti1/Tel2

PubMed Central

Rao, Feng; Cha, Jiyoung; Xu, Jing; Xu, Risheng; Vandiver, M. Scott; Tyagi, Richa; Tokhunts, Robert; Koldobskiy, Michael A.; Fu, Chenglai; Barrow, Roxanne; Wu, Mingxuan; Fiedler, Dorothea; Barrow, James C.; Snyder, Solomon H.

2014-01-01

The apoptotic actions of p53 require its phosphorylation by a family of phosphoinositide-3-kinase-related-kinases (PIKKs), which include DNA-PKcs and ATM. These kinases are stabilized by the TTT (Tel2, Tti1, Tti2) co-chaperone family, whose actions are mediated by CK2 phosphorylation. The inositol pyrophosphates, such as 5-diphosphoinositol pentakisphosphate (IP7), are generated by a family of inositol hexakisphosphate kinases (IP6Ks) of which IP6K2 has been implicated in p53-associated cell death. In the present study we report a novel apoptotic signaling cascade linking CK2, TTT, the PIKKs, and p53. We demonstrate that IP7, formed by IP6K2, binds CK2 to enhance its phosphorylation of the TTT complex thereby stabilizing DNA-PKcs and ATM. This process stimulates p53 phosphorylation at serine-15 to activate the cell death program in human cancer cells and in murine B cells. PMID:24657168

Progenitor cells for regenerative medicine and consequences of ART and cloning-associated epimutations.

PubMed

Laprise, Shari L

2010-06-01

The "holy grail" of regenerative medicine is the identification of an undifferentiated progenitor cell that is pluripotent, patient specific, and ethically unambiguous. Such a progenitor cell must also be able to differentiate into functional, transplantable tissue, while avoiding the risks of immune rejection. With reports detailing aberrant genomic imprinting associated with assisted reproductive technologies (ART) and reproductive cloning, the idea that human embryonic stem cells (hESCs) derived from surplus in vitro fertilized embryos or nuclear transfer ESCs (ntESCs) harvested from cloned embryos may harbor dangerous epigenetic errors has gained attention. Various progenitor cell sources have been proposed for human therapy, from hESCs to ntESCs, and from adult stem cells to induced pluripotent stem cells (iPS and piPS cells). This review highlights the advantages and disadvantages of each of these technologies, with particular emphasis on epigenetic stability.

Vectors to Increase Production Efficiency of Inducible Pluripotent Stem Cell (iPSC) | NCI Technology Transfer Center | TTC

Cancer.gov

This invention describes the discovery that specific p53 isoform increase the number of inducible pluripotent stem cells (iPS). It is known that the activity of p53 regulates the self-renewal and pluripotency of normal and cancer stem cells, and also affects re-programming efficiency of iPS cells. This p53 isoform-based technology provides a more natural process of increasing iPS cell production than previous methods of decreasing p53. NCI seeks licensees for this technology.

An RCT Evaluating the Effects of Skills Training and Medication Type on Work Outcomes Among Patients With Schizophrenia.

PubMed

Glynn, Shirley M; Marder, Stephen R; Noordsy, Douglas L; O'Keefe, Christopher; Becker, Deborah R; Drake, Robert E; Sugar, Catherine A

2017-03-01

Although supported employment increases job acquisition for people with serious mental illness, data on participants' job tenure have been variable. This study evaluated the effects of a standardized work skills training program (the Workplace Fundamentals Module [WPFM]) on job tenure and other work outcomes among individuals receiving individual placement and support (IPS). The effects of two atypical antipsychotic medications on side effects were also tested. The primary hypothesis tested was that participants in IPS plus WPFM would have increased job tenure compared with those enrolled in IPS only, and the secondary hypothesis was that different antipsychotic medications would yield unique side effects. A 2×2 randomized controlled trial compared work outcomes, including job tenure, of participants receiving IPS with or without WPFM for up to two years after obtaining a job. Participants were also randomly assigned to olanzapine or risperidone. Measures of work outcomes, clinical status, and medication side effects were collected. Among 107 participants, 63% obtained at least one job. WPFM did not increase job tenure (51.53 and 41.37 total weeks worked for IPS only and IPS plus WPFM, respectively) or affect other work outcomes. Participants on olanzapine experienced increased body mass index, whereas those on risperidone lost weight, but medications did not differentially affect clinical or job outcomes. Clinic-based skills training did not improve work outcomes accruing from IPS. Risperidone, compared with olanzapine, may reduce body mass but has no differential effect on other work or clinical outcomes.

Polycistronic lentiviral vector for "hit and run" reprogramming of adult skin fibroblasts to induced pluripotent stem cells.

PubMed

Chang, Chia-Wei; Lai, Yi-Shin; Pawlik, Kevin M; Liu, Kaimao; Sun, Chiao-Wang; Li, Chao; Schoeb, Trenton R; Townes, Tim M

2009-05-01

We report the derivation of induced pluripotent stem (iPS) cells from adult skin fibroblasts using a single, polycistronic lentiviral vector encoding the reprogramming factors Oct4, Sox2, and Klf4. Porcine teschovirus-1 2A sequences that trigger ribosome skipping were inserted between human cDNAs for these factors, and the polycistron was subcloned downstream of the elongation factor 1 alpha promoter in a self-inactivating (SIN) lentiviral vector containing a loxP site in the truncated 3' long terminal repeat (LTR). Adult skin fibroblasts from a humanized mouse model of sickle cell disease were transduced with this single lentiviral vector, and iPS cell colonies were picked within 30 days. These cells expressed endogenous Oct4, Sox2, Nanog, alkaline phosphatase, stage-specific embryonic antigen-1, and other markers of pluripotency. The iPS cells produced teratomas containing tissue derived from all three germ layers after injection into immunocompromised mice and formed high-level chimeras after injection into murine blastocysts. iPS cell lines with as few as three lentiviral insertions were obtained. Expression of Cre recombinase in these iPS cells resulted in deletion of the lentiviral vector, and sequencing of insertion sites demonstrated that remnant 291-bp SIN LTRs containing a single loxP site did not interrupt coding sequences, promoters, or known regulatory elements. These results suggest that a single, polycistronic "hit and run" vector can safely and effectively reprogram adult dermal fibroblasts into iPS cells.

p53 isoform Δ133p53 promotes efficiency of induced pluripotent stem cells and ensures genomic integrity during reprogramming.

PubMed

Gong, Lu; Pan, Xiao; Chen, Haide; Rao, Lingjun; Zeng, Yelin; Hang, Honghui; Peng, Jinrong; Xiao, Lei; Chen, Jun

2016-11-22

Human induced pluripotent stem (iPS) cells have great potential in regenerative medicine, but this depends on the integrity of their genomes. iPS cells have been found to contain a large number of de novo genetic alterations due to DNA damage response during reprogramming. Thus, to maintain the genetic stability of iPS cells is an important goal in iPS cell technology. DNA damage response can trigger tumor suppressor p53 activation, which ensures genome integrity of reprogramming cells by inducing apoptosis and senescence. p53 isoform Δ133p53 is a p53 target gene and functions to not only antagonize p53 mediated apoptosis, but also promote DNA double-strand break (DSB) repair. Here we report that Δ133p53 is induced in reprogramming. Knockdown of Δ133p53 results 2-fold decrease in reprogramming efficiency, 4-fold increase in chromosomal aberrations, whereas overexpression of Δ133p53 with 4 Yamanaka factors showes 4-fold increase in reprogamming efficiency and 2-fold decrease in chromosomal aberrations, compared to those in iPS cells induced only with 4 Yamanaka factors. Overexpression of Δ133p53 can inhibit cell apoptosis and promote DNA DSB repair foci formation during reprogramming. Our finding demonstrates that the overexpression of Δ133p53 not only enhances reprogramming efficiency, but also results better genetic quality in iPS cells.

Generation of Healthy Mice from Gene-Corrected Disease-Specific Induced Pluripotent Stem Cells

PubMed Central

Rittelmeyer, Ina; Sharma, Amar Deep; Sgodda, Malte; Zaehres, Holm; Bleidißel, Martina; Greber, Boris; Gentile, Luca; Han, Dong Wook; Rudolph, Cornelia; Steinemann, Doris; Schambach, Axel; Ott, Michael; Schöler, Hans R.; Cantz, Tobias

2011-01-01

Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH −/− mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH −/−-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH −/− iPS cell lines, we aggregated FAH −/−-iPS cells with tetraploid embryos and obtained entirely FAH −/−-iPS cell–derived mice that were viable and exhibited the phenotype of the founding FAH −/− mice. Then, we transduced FAH cDNA into the FAH −/−-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell–derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models. PMID:21765802

Mastoparan-Induced Intracellular Ca2+ Fluxes May Regulate Cell-to-Cell Communication in Plants.

PubMed

Tucker, E. B.; Boss, W. F.

1996-06-01

The relationship of Ca2+ and plasmodesmatal closure was examined in staminal hairs of Setcreasea purpurea by microinjecting cells with active mastoparan (Mas-7), inactive mastoparan (Mas-17), active inositol-1,4,5-trisphosphate (IP3), or inactive IP3. Calcium green dextran 10,000 was used to study cellular free Ca2+, and carboxyfluorescein was used to monitor plasmodesmatal closure. When Mas-7 was microinjected into the cytoplasm of cell 1 (the tip cell of a chain of cells), a rapid increase in calcium green dextran-10,000 fluorescence was observed in the cytoplasmic areas on both sides of the plasmodesmata connecting cells 1 and 2 during the same time that the diffusion of carboxyfluorescein through them was blocked. The inhibition of cell-to-cell diffusion was transient, and the closed plasmodesmata reopened within 30 s. The elevated Ca2+ level near plasmodesmata was also transient and returned to base level in about 1.5 min. The transient increase in Ca2+, once initiated in cell 1, repeated with an oscillatory period of 3 min. Elevated Ca2+ and oscillations of Ca2+ were also observed near interconnecting cell walls throughout the chain of cells, indicating that the signal had been transmitted. Previously, we reported that IP3 closed plasmodesmata; now we report that it stimulated Ca2+ and oscillations similar to Mas-7. The effect was specific for similar concentrations of Mas-7 over Mas-17 and active IP3 over inactive IP3. It is important that the Ca2+ channel blocker La3+ eliminated the responses from Mas-7 and IP3, indicating that an influx of Ca2+ was required. These results support the contention that plasmodesmata functioning is regulated via Ca2+ and that IP3 may be an intermediary between the stimulus and Ca2+ elevations.

Mastoparan-Induced Intracellular Ca2+ Fluxes May Regulate Cell-to-Cell Communication in Plants.

PubMed Central

Tucker, E. B.; Boss, W. F.

1996-01-01

The relationship of Ca2+ and plasmodesmatal closure was examined in staminal hairs of Setcreasea purpurea by microinjecting cells with active mastoparan (Mas-7), inactive mastoparan (Mas-17), active inositol-1,4,5-trisphosphate (IP3), or inactive IP3. Calcium green dextran 10,000 was used to study cellular free Ca2+, and carboxyfluorescein was used to monitor plasmodesmatal closure. When Mas-7 was microinjected into the cytoplasm of cell 1 (the tip cell of a chain of cells), a rapid increase in calcium green dextran-10,000 fluorescence was observed in the cytoplasmic areas on both sides of the plasmodesmata connecting cells 1 and 2 during the same time that the diffusion of carboxyfluorescein through them was blocked. The inhibition of cell-to-cell diffusion was transient, and the closed plasmodesmata reopened within 30 s. The elevated Ca2+ level near plasmodesmata was also transient and returned to base level in about 1.5 min. The transient increase in Ca2+, once initiated in cell 1, repeated with an oscillatory period of 3 min. Elevated Ca2+ and oscillations of Ca2+ were also observed near interconnecting cell walls throughout the chain of cells, indicating that the signal had been transmitted. Previously, we reported that IP3 closed plasmodesmata; now we report that it stimulated Ca2+ and oscillations similar to Mas-7. The effect was specific for similar concentrations of Mas-7 over Mas-17 and active IP3 over inactive IP3. It is important that the Ca2+ channel blocker La3+ eliminated the responses from Mas-7 and IP3, indicating that an influx of Ca2+ was required. These results support the contention that plasmodesmata functioning is regulated via Ca2+ and that IP3 may be an intermediary between the stimulus and Ca2+ elevations. PMID:12226302

Mechanism of cisplatin resistance in human urothelial carcinoma cells.

PubMed

Yu, Hui-Min; Wang, Tsing-Cheng

2012-05-01

An isogenic pair of cisplatin-susceptible (NTUB1) and -resistant (NTUB1/P) human urothelial carcinoma cell lines was used to elucidate the mechanism of cisplatin resistance. The significantly lower intracellular platinum (IP) concentration, which resulted from the decreased cisplatin uptake, was found in NTUB1/P cells. The enhancement of IP concentration did not increase the susceptibility of NTUB1/P cells to cisplatin treatment. The reduction of IP concentration as well was unable to enhance the cisplatin-resistance in susceptible NTUB1 cells. This indicated that reduction of IP concentration was not the account for the development of cisplatin resistance here. Instead, the over expression of anti-apoptotic Bcl-2, anti-oxidative heme oxygenase-1 (HO-1) and cell cycle regulator p16INK4 seemed to be more important for the gaining of cisplatin in these human urothelial carcinoma cell. Copyright © 2012 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taguchi, M.; Field, J.B.

Thyrotropin (TSH) and carbachol stimulated in a dose-dependent manner the accumulation of 3H-glycerophosphoinositol (GPI), 3H-inositol monophosphate (IP1), 3H-inositol bisphosphate (IP2) and 3H-inositol trisphosphate (IP3) in primary cultures of dog thyroid cells prelabeled with myo-(2-3H)inositol. TSH, 250 mU/mL, stimulated 3H-IP3 level after a 10-minute incubation while 10 mU/mL TSH increased it during a 60-minute incubation. The effect of carbachol was more rapid and greater than that of TSH. Carbachol, 100 mumol/L, elevated 3H-IP3 after a 2-minute incubation and 3H-IP3 formation was increased by as little as 1 mumol/L carbachol. TSH stimulation was observed only if the cells were deprived of TSHmore » for 5 days before being labeled with 3H-inositol. Prolongation of the labeling period or addition of TSH, (Bu)2cAMP or carbachol during the labeling increased 3H-inositol incorporation into polyphoinositides (PIPs). When the cells were labeled without any other addition, control and TSH-stimulated 3H-IP3 levels increased in parallel with 3H-PIP levels. However, TSH or carbachol-stimulated 3H-IP3 levels did not increase in proportion to 3H-PIPs level when the cells were labeled with TSH or (Bu)2cAMP. Thus, the ratio of 3H-IP3/3H-PIPs (both control and TSH or carbachol-stimulated) decreased in the cells labeled with TSH or (Bu)2cAMP, which might reflect TSH stimulation of 3H-inositol incorporation into PIPs pool(s) that do not participate in hormone-induced hydrolysis of PIPs.« less

An Impact Assessment Model for Distributed Adaptive Security Situation Assessment

DTIC Science & Technology

2005-01-01

the cargo manifest can be either a 56K modem-based TCP/IP connection (the oval labeled internet) or a 40K wireless modem connection ( cell phone ) that...via a UDP connection on the 40K wireless modem ( cell phone ). For each resource, either alternative may be used to achieve the same goal, but some...Manifests Comm-in Comp- power Comm- out JTF Internet (TCP-IP) Cell phone (TCP-IP) Internet (UDP) Cell phone (UDP) Manual Computer 4

Distribution Profile of Inositol 1,4,5-Trisphosphate Receptor/Ca2+ Channels in α and β Cells of Pancreas: Dominant Localization in Secretory Granules and Common Error in Identification of Secretory Granule Membranes.

PubMed

Hur, Yong Suk; Yoo, Seung Hyun

2015-01-01

The α and β cells of pancreatic islet release important hormones in response to intracellular Ca increases that result from Ca releases through the inositol 1,4,5-trisphoshate receptor (IP3R)/Ca channels. Yet no systematic studies on distribution of IP3R/Ca channels have been done, prompting us to investigate the distribution of all 3 IP3R isoforms. Immunogold electron microscopy was performed to determine the presence and the relative concentrations of all 3 IP3R isoforms in 2 major organelles secretory granules (SGs) and the endoplasmic reticulum of α and β cells of rat pancreas. All 3 IP3R isoforms were present in SG membranes of both cells, and the IP3R concentrations in SGs were ∼2-fold higher than those in the endoplasmic reticulum. Moreover, large halos shown in the electron microscope images of insulin-containing SGs of β cells were gap spaces that resulted from separation of granule membranes from the surrounding cytoplasm. These results strongly suggest the important roles of SGs in IP3-induced, Ca-dependent regulatory secretory pathway in pancreas. Moreover, the accurate location of SG membranes of β cells was further confirmed by the location of another integral membrane protein synaptotagmin V and of membrane phospholipid PI(4,5)P2.

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples.

PubMed

Rehimi, Rizwan; Bartusel, Michaela; Solinas, Francesca; Altmüller, Janine; Rada-Iglesias, Alvaro

2017-08-29

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 10 4 - 5 x 10 5 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

MLF1-interacting protein is mainly localized in nucleolus through N-terminal bipartite nuclear localization signal.

PubMed

Suzuki, Hideaki; Arakawa, Yasuhiro; Ito, Masaki; Saito, Shinobu; Takeda, Nobuakira; Yamada, Hisashi; Horiguchi-Yamada, Junko

2007-01-01

The myelodysplasia/myeloid leukemia factor 1-interacting protein (MLF1LP, also called KLIP1 and CENP-50) is reported to localize in both the nucleus and the cytoplasm. To investigate the functions of MLF1IP, its subnuclear localization was studied. MLF1IP was tagged with green fluorescent protein (EGFP). Fibrillarin was tagged with red fluorescent protein (DsRed). EGFP-tagged MLF1IP deletion vectors were also constructed. Plasmid-constructs were transfected into human cervical adenocarcinoma HeLa cells or monkey kidney fibroblast COS-7 cells, and the localization was studied by either confocal fluorescence microscopy or fluorescence microscopy. Ectopically expressed MLF1IP was localized mainly in the nucleolus. In some cells, small dot-like particles of MLF1IP fluorescence were observed in the nucleoplasm. Co-staining of fibrillarin disclosed that MLF1IP was co-localized with fibrillarin in the nucleolus. Deletion mutants of MLF1IP revealed that the N-terminal bipartite nuclear localization signal (NLS) was responsible for nucleolar targeting. MLF1IP was localized mainly in the nucleolus through the N-terminal bipartite NLS and partly in the nucleoplasm featuring small dot-like particles. These findings suggest that MLF1IP may have multi-functions and its different localizations may contribute to carcinogenesis.

Polo-like kinase 1 expression is suppressed by CCAAT/enhancer-binding protein α to mediate colon carcinoma cell differentiation and apoptosis.

PubMed

Dasgupta, Nirmalya; Thakur, Bhupesh Kumar; Ta, Atri; Das, Sayan; Banik, George; Das, Santasabuj

2017-07-01

Human polo-like kinase 1 (PLK1), a highly conserved serine/threonine kinase is a key player in several essential cell-cycle events. PLK1 is considered an oncogene and its overexpression often correlates with poor prognosis of cancers, including colorectal cancer (CRC). However, regulation of PLK1 expression in colorectal cells was never studied earlier and it is currently unknown if PLK1 regulates differentiation and apoptosis of CRC. PLK1 expression was analyzed by real-time PCR and western blotting. Transcriptional regulation was studied by reporter assay, gene knock-down, EMSA and ChIP. PLK1 expression was down-regulated during butyrate-induced differentiation of HT-29 and other CRC cells. Also, PLK1 down-regulation mediated the role of butyrate in CRC differentiation and apoptosis. We report here a novel transcriptional regulation of PLK1 by butyrate. Transcription factors CCAAT/enhancer-binding protein α (C/EBPα) and Oct-1 share an overlapping binding site over the PLK1 promoter. Elevated levels of C/EBPα by butyrate treatment of CRC cells competed out the activator protein Oct-1 from binding to the PLK1 promoter and sequestered it. Binding of C/EBPα was associated with increased deacetylation near the transcription start site (TSS) of the PLK1 promoter, which abrogated transcription through reduced recruitment of RNA polymerase II. We also found a synergistic role between the synthetic PLK1-inhibitor SBE13 and butyrate on the apoptosis of CRC cells. This study offered a novel p53-independent regulation of PLK1 during CRC differentiation and apoptosis. Down-regulation of PLK1 is one of the mechanisms underlying the anti-cancer role of dietary fibre-derived butyrate in CRC. Copyright © 2017 Elsevier B.V. All rights reserved.

Single-cell imaging techniques for the real-time detection of IP₃ in live cells.

PubMed

Nelson, Carl P

2013-01-01

Inositol 1,4,5-trisphosphate (IP(3)) is a ubiquitous second messenger, derived from the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP(2)) by enzymes of the phospholipase C (PLC) family. Binding of IP(3) to its cognate receptor in the endoplasmic reticulum membrane leads to release of Ca(2+) into the cytoplasm, which is involved in the regulation of an array of cellular functions. Traditional techniques for the detection of IP(3) have required the extraction of a large number of cells, with limitations in the time resolution of changes in IP(3) and an inability to obtain detailed information on the dynamics of this second messenger in single cells. Recent progress in this field has led to the development of a number of genetically encoded fluorescent biosensors, which upon recombinant expression are able selectively to detect real-time changes in IP(3) in single live cells. In this chapter, I detail protocols for the expression, visualization (by confocol or fluorescence microscopy), and interpretation of data obtained with such biosensors expressed in mammalian cells.

Cross talk between metabotropic and ionotropic glutamate receptor-mediated signaling in parallel fiber-induced inositol 1,4,5-trisphosphate production in cerebellar Purkinje cells.

PubMed

Okubo, Yohei; Kakizawa, Sho; Hirose, Kenzo; Iino, Masamitsu

2004-10-27

In many excitatory glutamatergic synapses, both ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs) are closely distributed on the postsynaptic membrane. However, the functional significance of the close distribution of the two types of glutamate receptors has not been fully clarified. In this study, we examined the functional interaction between iGluR and mGluR at parallel fiber (PF)--> Purkinje cell synapses in the generation of inositol 1,4,5-trisphosphate (IP3), a key second messenger that regulates many important cellular functions. We visualized local IP3 dynamics in Purkinje cells using the green fluorescent protein-tagged pleckstrin homology domain (GFP-PHD) as a fluorescent IP3 probe. Purkinje cells were transduced with Sindbis virus encoding GFP-PHD and imaged with a two-photon laser scanning microscope. Translocation of GFP-PHD from the plasma membrane to the cytoplasm attributable to an increase in IP3 concentration was observed on PF stimulation in fine dendrites of Purkinje cells. Surprisingly, this PF-induced IP3 production was blocked not only by the group I mGluR antagonist but also by the AMPA receptor (AMPAR) antagonist. The PF-induced IP3 production was blocked by either the inhibition of G-protein activation by GDP-betaS or intracellular Ca2+ buffering by BAPTA. These results show that IP3 production is mediated cooperatively by group I mGluR and AMPAR through G-protein activation and Ca2+ influx at PF--> Purkinje cell synapses, identifying the robust cross talk between iGluR and mGluR for the generation of IP3 signals.

Expression and high glucose-mediated regulation of K+ channel interacting protein 3 (KChIP3) and KV4 channels in retinal Müller glial cells.

PubMed

Chavira-Suárez, Erika; Sandoval, Alejandro; Felix, Ricardo; Lamas, Mónica

2011-01-14

Normal vision depends on the correct function of retinal neurons and glia and it is impaired in the course of diabetic retinopathy. Müller cells, the main glial cells of the retina, suffer morphological and functional alterations during diabetes participating in the pathological retinal dysfunction. Recently, we showed that Müller cells express the pleiotropic protein potassium channel interacting protein 3 (KChIP3), an integral component of the voltage-gated K(+) channels K(V)4. Here, we sought to analyze the role of KChIP3 in the molecular mechanisms underlying hyperglycemia-induced phenotypic changes in the glial elements of the retina. The expression and function of KChIp3 was analyzed in vitro in rat Müller primary cultures grown under control (5.6 mM) or high glucose (25 mM) (diabetic-like) conditions. We show the up-regulation of KChIP3 expression in Müller cell cultures under high glucose conditions and demonstrate a previously unknown interaction between the K(V)4 channel and KChIP3 in Müller cells. We show evidence for the expression of a 4-AP-sensitive transient outward voltage-gated K(+) current and an alteration in the inactivation of the macroscopic outward K(+) currents expressed in high glucose-cultured Müller cells. Our data support the notion that induction of KChIP3 and functional changes of K(V)4 channels in Müller cells could exert a physiological role in the onset of diabetic retinopathy. Copyright © 2010 Elsevier Inc. All rights reserved.

RdgB2 is required for dim-light input into intrinsically photosensitive retinal ganglion cells.

PubMed

Walker, Marquis T; Rupp, Alan; Elsaesser, Rebecca; Güler, Ali D; Sheng, Wenlong; Weng, Shijun; Berson, David M; Hattar, Samer; Montell, Craig

2015-10-15

A subset of retinal ganglion cells is intrinsically photosensitive (ipRGCs) and contributes directly to the pupillary light reflex and circadian photoentrainment under bright-light conditions. ipRGCs are also indirectly activated by light through cellular circuits initiated in rods and cones. A mammalian homologue (RdgB2) of a phosphoinositide transfer/exchange protein that functions in Drosophila phototransduction is expressed in the retinal ganglion cell layer. This raised the possibility that RdgB2 might function in the intrinsic light response in ipRGCs, which depends on a cascade reminiscent of Drosophila phototransduction. Here we found that under high light intensities, RdgB2(-/-) mutant mice showed normal pupillary light responses and circadian photoentrainment. Consistent with this behavioral phenotype, the intrinsic light responses of ipRGCs in RdgB2(-/-) were indistinguishable from wild-type. In contrast, under low-light conditions, RdgB2(-/-) mutants displayed defects in both circadian photoentrainment and the pupillary light response. The RdgB2 protein was not expressed in ipRGCs but was in GABAergic amacrine cells, which provided inhibitory feedback onto bipolar cells. We propose that RdgB2 is required in a cellular circuit that transduces light input from rods to bipolar cells that are coupled to GABAergic amacrine cells and ultimately to ipRGCs, thereby enabling ipRGCs to respond to dim light. © 2015 Walker et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Rebamipide attenuates autoimmune arthritis severity in SKG mice via regulation of B cell and antibody production.

PubMed

Byun, J-K; Moon, S-J; Jhun, J-Y; Kim, E-K; Park, J-S; Youn, J; Min, J-K; Park, S-H; Kim, H-Y; Cho, M-L

2014-10-01

Oxidative stress is involved in the pathophysiology of rheumatoid arthritis (RA). We investigated the therapeutic potential of rebamipide, a gastroprotective agent with a property of reactive oxygen species scavenger, on the development of inflammatory polyarthritis and the pathophysiological mechanisms by which rebamipide might confer anti-arthritic effects in SKG mice, an animal model of RA. Intraperitoneal (i.p.) injection of rebamipide attenuated the severity of clinical and histological arthritis. Rebampide treatment reduced the number of T helper type 1 (Th1), Th2, Th17, inducible T cell co-stimulator (ICOS)(+) follicular helper T (Tfh) transitional type (T2) and mature B cells in the spleen, but increased the number of regulatory T (Treg ), CD19(+) CD1d(high) CD5(high) , CD19(+) CD25(high) forkhead box protein 3 (FoxP3)(+) regulatory B (Breg ) cells, memory B cells, and transitional type 1 (T1) B cells. In addition, flow cytometric analysis revealed significantly decreased populations of FAS(+) GL-7(+) germinal centre B cells and B220(-) CD138(+) plasma cells in the spleens of rebamipide-treated SKG mice compared to controls. Rebamipide decreased germinal centre B cells and reciprocally induced Breg cells in a dose-dependent manner in vitro. Rebamipide-induced Breg cells had more suppressive capacity in relation to T cell proliferation and also inhibited Th17 differentiation from murine CD4(+) T cells. Together, these data show that i.p. administration of rebamipide suppresses arthritis severity by inducing Breg and Treg cells and suppressing Tfh and Th17 cells in a murine model of RA. © 2014 British Society for Immunology.

Human Induced Pluripotent Stem Cell-Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation.

PubMed

Sharmin, Sazia; Taguchi, Atsuhiro; Kaku, Yusuke; Yoshimura, Yasuhiro; Ohmori, Tomoko; Sakuma, Tetsushi; Mukoyama, Masashi; Yamamoto, Takashi; Kurihara, Hidetake; Nishinakamura, Ryuichi

2016-06-01

Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator-like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in vitro These induced human podocytes exhibited apicobasal polarity, with nephrin proteins accumulated close to the basal domain, and possessed primary processes that were connected with slit diaphragm-like structures. Microarray analysis of sorted iPS cell-derived podocytes identified well conserved marker gene expression previously shown in mouse and human podocytes in vivo Furthermore, we developed a novel transplantation method using spacers that release the tension of host kidney capsules, thereby allowing the effective formation of glomeruli from human iPS cell-derived nephron progenitors. The human glomeruli were vascularized with the host mouse endothelial cells, and iPS cell-derived podocytes with numerous cell processes accumulated around the fenestrated endothelial cells. Therefore, the podocytes generated from iPS cells retain the podocyte-specific molecular and structural features, which will be useful for dissecting human glomerular development and diseases. Copyright © 2016 by the American Society of Nephrology.

Inhibition of muscarinic-stimulated polyphosphoinositide hydrolysis and Ca2+ mobilization in cat iris sphincter smooth muscle cells by cAMP-elevating agents.

PubMed

Ding, K H; Husain, S; Akhtar, R A; Isales, C M; Abdel-Latif, A A

1997-09-01

The effects of carbachol (CCh) on inositol 1,4,5-trisphosphate (IP3) production and intracellular calcium ([Ca2+]i) mobilization, and their regulation by cAMP-elevating agents were investigated in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. CCh produced time- and dose-dependent increases in IP3 production; the t1/2 and EC50 values were 68 s and 0.5 microM, respectively. The muscarinic agonist provoked a transient increase in [Ca2+]i which reached maximum within 77 s, and increased [Ca2+]i mobilization in a concentration-dependent manner with an EC50 of 1.4 microM. Thapsigargin, a Ca(2+)-pump inhibitor, caused a rapid rise in [Ca2+]i and subsequent addition of CCh was without effect. Both CCh-induced IP3 production and CCh-induced [Ca2+]i mobilization were more potently antagonized by 4-DAMP, an M3 muscarinic receptor antagonist, than by pirenzepine, an M1 receptor antagonist, suggesting that both responses are mediated through the M3 receptor subtype. Treatment of the cells with U73122, a phospholipase C (PLC) inhibitor, resulted in a concentration-dependent decrease in both CCh-stimulated IP3 production and [Ca2+]i mobilization. These data indicate close correlation between enhanced IP3 production and [Ca2+]i mobilization in these smooth muscle cells and suggest that the CCh-stimulated increase in [Ca2+]i could be mediated through increased IP3 production. Isoproterenol (ISO) inhibited CCh-induced IP3 production (IC50 = 80 nM) and [Ca2+]i mobilization (IC50 = 0.17 microM) in a concentration-dependent manner. Microsomal fractions isolated from SV-CISM-2 cells contained phospholipase C (PLC) which was stimulated by CCh (10 microM) and GTP gamma S (0.1 microM). Pretreatment of the cells with ISO or forskolin, 5 microM each, produced membrane fractions in which CCh-stimulated PLC activity was significantly attenuated. Furthermore, when microsomal fractions isolated from SV-CISM-2 cells were phosphorylated with Protein kinase A (PKA), the CCh- and GTP gamma S-stimulated IP3 production were significantly inhibited. It can be concluded from these studies that in SV-CISM-2 cells, activation of M3 muscarinic receptors results in stimulation of PLC-mediated PIP2 hydrolysis, generating IP3 which mobilizes [Ca2+]i. Furthermore, elevation of cAMP may inhibit IP3 production and [Ca2+]i mobilization through mechanisms involving PKA-dependent phosphorylation of PLC, G-proteins, IP3 receptor and/or IP3 metabolizing enzymes.

'Hearts and bones': the ups and downs of 'plasticity' in stem cell biology.

PubMed

Bonfanti, Paola; Barrandon, Yann; Cossu, Giulio

2012-05-01

More than a decade ago, 'plasticity' suddenly became a 'fashionable' topic with overemphasized implications for regenerative medicine. The concept of 'plasticity' is supported by old transplantation work, at least for embryonic cells, and metaplasia is a classic example of plasticity observed in patients. Nevertheless, the publication of a series of papers showing rare conversion of a given cell type into another unrelated cell raised the possibility of using any unaffected tissue to create at will new cells to replace a different failing tissue or organ. This resulted in disingenuous interpretations and a reason not to fund anymore research on embryonic stem cells (ESc). Moreover, many papers on plasticity were difficult to reproduce and thus questioned; raising issues about plasticity as a technical artefact or a consequence of rare spontaneous cells fusion. More recently, reprogramming adult differentiated cells to a pluripotent state (iPS) became possible, and later, one type of differentiated cell could be directly reprogrammed into another (e.g. fibroblasts into neurons) without reverting to pluripotency. Although the latter results from different and more robust experimental protocols, these phenomena also exemplify 'plasticity'. In this review, we want to place 'plasticity' in a historical perspective still taking into account ethical and political implications. Copyright © 2012 EMBO Molecular Medicine.

[Unresolved issues in the evaluation of research projects involving induced pluripotent stem cells (iPS)].

PubMed

Casado, María; de Lecuona, Itziar

2013-01-01

This paper identifies problems and analyzes those conflicts posed by the evaluation of research projects involving the collection and use of human induced pluripotent stem cells (iPS) in Spain. Current legislation is causing problems of interpretation, circular and unnecessary referrals, legal uncertainty and undue delays. Actually, this situation may cause a lack of control and monitoring, and even some paralysis in regenerative medicine and cell therapy research, that is a priority nowadays. The analysis of the current legislation and its bioethical implications, led us to conclude that the review of iPS research projects cannot be assimilated to the evaluation of research projects that involve human embryonic stem cell (hESC). In this context, our proposal is based on the review by the Research Ethics Committees and the checkout by the Spanish Comission of Guarantees for Donation and Use of Human Cells and Tissues (CGDUCTH) of human iPS cells research projects. Moreover, this article claims for a more transparent research system, by effectively articulating the Registry on Research Projects. Finally, a model of verification protocol (checklist) for checking out biomedical research projects involving human iPS cells is suggested.

Generation of an ICF syndrome model by efficient genome editing of human induced pluripotent stem cells using the CRISPR system.

PubMed

Horii, Takuro; Tamura, Daiki; Morita, Sumiyo; Kimura, Mika; Hatada, Izuho

2013-09-30

Genome manipulation of human induced pluripotent stem (iPS) cells is essential to achieve their full potential as tools for regenerative medicine. To date, however, gene targeting in human pluripotent stem cells (hPSCs) has proven to be extremely difficult. Recently, an efficient genome manipulation technology using the RNA-guided DNase Cas9, the clustered regularly interspaced short palindromic repeats (CRISPR) system, has been developed. Here we report the efficient generation of an iPS cell model for immunodeficiency, centromeric region instability, facial anomalies syndrome (ICF) syndrome using the CRISPR system. We obtained iPS cells with mutations in both alleles of DNA methyltransferase 3B (DNMT3B) in 63% of transfected clones. Our data suggest that the CRISPR system is highly efficient and useful for genome engineering of human iPS cells.

Genetic modification of human mesenchymal stem cells helps to reduce adiposity and improve glucose tolerance in an obese diabetic mouse model.

PubMed

Sen, Sabyasachi; Domingues, Cleyton C; Rouphael, Carol; Chou, Cyril; Kim, Chul; Yadava, Nagendra

2015-12-09

Human mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into fat, muscle, bone and cartilage cells. Exposure of subcutaneous abdominal adipose tissue derived AD-MSCs to high glucose (HG) leads to superoxide accumulation and up-regulation of inflammatory molecules. Our aim was to inquire how HG exposure affects MSCs differentiation and whether the mechanism is reversible. We exposed human adipose tissue derived MSCs to HG (25 mM) and compared it to normal glucose (NG, 5.5 mM) exposed cells at 7, 10 and 14 days. We examined mitochondrial superoxide accumulation (Mitosox-Red), cellular oxygen consumption rate (OCR, Seahorse) and gene expression. HG increased reactive superoxide (ROS) accumulation noted by day 7 both in cytosol and mitochondria. The OCR between the NG and HG exposed groups however did not change until 10 days at which point OCR of HG exposed cells were reduced significantly. We noted that HG exposure upregulated mRNA expression of adipogenic (PPARG, FABP-4, CREBP alpha and beta), inflammatory (IL-6 and TNF alpha) and antioxidant (SOD2 and Catalase) genes. Next, we used AdSOD2 to upregulate SOD2 prior to HG exposure and thereby noted reduction in superoxide generation. SOD2 upregulation helped reduce mRNA over-expression of PPARG, FABP-4, IL-6 and TNFα. In a series of separate experiments, we delivered the eGFP and SOD2 upregulated MSCs (5 days post ex-vivo transduction) and saline intra-peritoneally (IP) to obese diabetic (db/db) mice. We confirmed homing-in of eGFP labeled MSCs, delivered IP, to different inflamed fat pockets, particularly omental fat. Mice receiving SOD2-MSCs showed progressive reduction in body weight and improved glucose tolerance (GTT) at 4 weeks, post MSCs transplantation compared to the GFP-MSC group (control). High glucose evokes superoxide generation, OCR reduction and adipogenic differentiation. Mitochondrial superoxide dismutase upregulation quenches excess superoxide and reduces adipocyte inflammation. Delivery of superoxide dismutase (SOD2) using MSCs as a gene delivery vehicle reduces inflammation and improves glucose tolerance in vivo. Suppression of superoxide production and adipocyte inflammation using mitochondrial superoxide dismutase may be a novel and safe therapeutic tool to combat hyperglycemia mediated effects.

The inositol trisphosphate receptor in the control of autophagy.

PubMed

Criollo, Alfredo; Vicencio, José Miguel; Tasdemir, Ezgi; Maiuri, M Chiara; Lavandero, Sergio; Kroemer, Guido

2007-01-01

The second messenger myo-inositol-1,4,5-trisphosphate (IP(3)) acts on the IP(3) receptor (IP(3)R), an IP(3)-activated Ca(2+) channel of the endoplasmic reticulum (ER). The IP(3)R agonist IP(3) inhibits starvation-induced autophagy. The IP(3)R antagonist xestospongin B induces autophagy in human cells through a pathway that requires the obligate contribution of Beclin-1, Atg5, Atg10, Atg12 and hVps34, yet is inhibited by ER-targeted Bcl-2 or Bcl-XL, two proteins that physically interact with IP(3)R. Autophagy can also be induced by depletion of the IP(3)R by small interfering RNAs. Autophagy induction by IP(3)R blockade cannot be explained by changes in steady state levels of Ca(2+) in the endoplasmic reticulum (ER) and the cytosol. Autophagy induction by IP(3)R blockade is effective in cells lacking the obligate mediator of ER stress IRE1. In contrast, IRE1 is required for autophagy induced by ER stress-inducing agents such a tunicamycin or thapsigargin. These findings suggest that there are several distinct pathways through which autophagy can be initiated at the level of the ER.

Generation of safe and therapeutically effective human induced pluripotent stem cell‐derived hepatocyte‐like cells for regenerative medicine

PubMed Central

Takayama, Kazuo; Akita, Naoki; Mimura, Natsumi; Akahira, Rina; Taniguchi, Yukimasa; Ikeda, Makoto; Sakurai, Fuminori; Ohara, Osamu; Morio, Tomohiro

2017-01-01

Hepatocyte‐like cells (HLCs) differentiated from human induced pluripotent stem (iPS) cells are expected to be applied for regenerative medicine. In this study, we attempted to generate safe and therapeutically effective human iPS‐HLCs for hepatocyte transplantation. First, human iPS‐HLCs were generated from a human leukocyte antigen‐homozygous donor on the assumption that the allogenic transplantation might be carried out. Highly efficient hepatocyte differentiation was performed under a feeder‐free condition using human recombinant laminin 111, laminin 511, and type IV collagen. The percentage of asialoglycoprotein receptor 1‐positive cells was greater than 80%, while the percentage of residual undifferentiated cells was approximately 0.003%. In addition, no teratoma formation was observed even at 16 weeks after human iPS‐HLC transplantation. Furthermore, harmful genetic somatic single‐nucleotide substitutions were not observed during the hepatocyte differentiation process. We also developed a cryopreservation protocol for hepatoblast‐like cells without negatively affecting their hepatocyte differentiation potential by programming the freezing temperature. To evaluate the therapeutic potential of human iPS‐HLCs, these cells (1 × 106 cells/mouse) were intrasplenically transplanted into acute liver injury mice treated with 3 mL/kg CCl4 only once and chronic liver injury mice treated with 0.6 mL/kg CCl4 twice weekly for 8 weeks. By human iPS‐HLC transplantation, the survival rate of the acute liver injury mice was significantly increased and the liver fibrosis level of chronic liver injury mice was significantly decreased. Conclusion: We were able to generate safe and therapeutically effective human iPS‐HLCs for hepatocyte transplantation. (Hepatology Communications 2017;1:1058–1069) PMID:29404442

Effects of antioxidants on the quality and genomic stability of induced pluripotent stem cells

PubMed Central

Luo, Lan; Kawakatsu, Miho; Guo, Chao-Wan; Urata, Yoshishige; Huang, Wen-Jing; Ali, Haytham; Doi, Hanako; Kitajima, Yuriko; Tanaka, Takayuki; Goto, Shinji; Ono, Yusuke; Xin, Hong-Bo; Hamano, Kimikazu; Li, Tao-Sheng

2014-01-01

Effects of antioxidants on the quality and genomic stability of induced pluripotent stem (iPS) cells were investigated with two human iPS cell lines (201B7 and 253G1). Cells used in this study were expanded from a single colony of each cell line with the addition of proprietary antioxidant supplement or homemade antioxidant cocktail in medium, and maintained in parallel for 2 months. The cells grew well in all culture conditions and kept “stemness”. Although antioxidants modestly decreased the levels of intracellular reactive oxygen species, there were no differences in the expression of 53BP1 and pATM, two critical molecules related with DNA damage and repair, under various culture conditions. CGH analysis showed that the events of genetic aberrations were decreased only in the 253G1 iPS cells with the addition of homemade antioxidant cocktail. Long-term culture will be necessary to confirm whether low dose antioxidants improve the quality and genomic stability of iPS cells. PMID:24445363

Xeno-free culture of human pluripotent stem cells on oligopeptide-grafted hydrogels with various molecular designs

PubMed Central

Chen, Yen-Ming; Chen, Li-Hua; Li, Meng-Pei; Li, Hsing-Fen; Higuchi, Akon; Kumar, S. Suresh; Ling, Qing-Dong; Alarfaj, Abdullah A.; Munusamy, Murugan A.; Chang, Yung; Benelli, Giovanni; Murugan, Kadarkarai; Umezawa, Akihiro

2017-01-01

Establishing cultures of human embryonic (ES) and induced pluripotent (iPS) stem cells in xeno-free conditions is essential for producing clinical-grade cells. Development of cell culture biomaterials for human ES and iPS cells is critical for this purpose. We designed several structures of oligopeptide-grafted poly (vinyl alcohol-co-itaconic acid) hydrogels with optimal elasticity, and prepared them in formations of single chain, single chain with joint segment, dual chain with joint segment, and branched-type chain. Oligopeptide sequences were selected from integrin- and glycosaminoglycan-binding domains of the extracellular matrix. The hydrogels grafted with vitronectin-derived oligopeptides having a joint segment or a dual chain, which has a storage modulus of 25 kPa, supported the long-term culture of human ES and iPS cells for over 10 passages. The dual chain and/or joint segment with cell adhesion molecules on the hydrogels facilitated the proliferation and pluripotency of human ES and iPS cells. PMID:28332572

Xeno-free culture of human pluripotent stem cells on oligopeptide-grafted hydrogels with various molecular designs.

PubMed

Chen, Yen-Ming; Chen, Li-Hua; Li, Meng-Pei; Li, Hsing-Fen; Higuchi, Akon; Kumar, S Suresh; Ling, Qing-Dong; Alarfaj, Abdullah A; Munusamy, Murugan A; Chang, Yung; Benelli, Giovanni; Murugan, Kadarkarai; Umezawa, Akihiro

2017-03-23

Establishing cultures of human embryonic (ES) and induced pluripotent (iPS) stem cells in xeno-free conditions is essential for producing clinical-grade cells. Development of cell culture biomaterials for human ES and iPS cells is critical for this purpose. We designed several structures of oligopeptide-grafted poly (vinyl alcohol-co-itaconic acid) hydrogels with optimal elasticity, and prepared them in formations of single chain, single chain with joint segment, dual chain with joint segment, and branched-type chain. Oligopeptide sequences were selected from integrin- and glycosaminoglycan-binding domains of the extracellular matrix. The hydrogels grafted with vitronectin-derived oligopeptides having a joint segment or a dual chain, which has a storage modulus of 25 kPa, supported the long-term culture of human ES and iPS cells for over 10 passages. The dual chain and/or joint segment with cell adhesion molecules on the hydrogels facilitated the proliferation and pluripotency of human ES and iPS cells.

Dependence of Na+ pump current on external monovalent cations and membrane potential in rabbit cardiac Purkinje cells.

PubMed Central

Bielen, F V; Glitsch, H G; Verdonck, F

1991-01-01

1. The effect of membrane potential and various extracellular monovalent cations on the Na+ pump current (Ip) was studied on isolated, single Purkinje cells of the rabbit heart by means of whole-cell recording. 2. Ip was identified as current activated by external K+ or its congeners NH4+ and Tl+. The current was blocked by dihydroouabain (1-5 x 10(-4) M) over the whole range of membrane potentials tested. 3. In Na(+)-containing solution half-maximum Ip activation (K0.5) occurred at 0.4 mM-Tl+, 1.9 mM-K+ and 5.7 mM-NH4+ (holding potential, -20 mV). 4. The pump current (Ip)-voltage (V) relationship of the cells in Na(+)-containing media with K+ or its congeners at the tested concentrations greater than K0.5 displayed a steep positive slope at negative membrane potentials between -120 and -20 mV. Little voltage dependence of Ip was observed at more positive potentials up to +40 mV. At even more positive potentials Ip measured at 2 and 5.4 mM-K+ decreased. 5. Lowering the concentration of K+ or its congeners below the K0.5 value in Na(+)-containing solution induced a region of negative slope of the Ip-V curve at membrane potentials positive to -20 mV. 6. The shape of the Ip-V relationship remained unchanged when the K+ concentration (5.4 mM) of the Na(+)-containing medium was replaced by NH4+ or Tl+ concentrations of similar potency to activate Ip (20 mM-NH4+ or 2 mM-Tl+). 7. In Na(+)-free, choline-containing solution half-maximum Ip activation occurred at 0.13 mM-K+ (holding potential, -20 mV). 8. At negative membrane potentials the positive slope of the Ip-V curve was flatter in Na(+)-free than in Na(+)-containing media. A reduced voltage dependence of Ip persisted, regardless of whether choline ions or Li+ were used as a Na+ substitute. 9. Lowering the K+ concentration of the Na(+)-free, choline-containing solution to 0.05 mM evoked an extended region of negative slope in the Ip-V relationship at membrane potentials between -40 and +60 mV. 10. It is concluded that the apparent affinity of the Na(+)-K+ pump towards K+ in cardiac Purkinje cells depends on both the membrane potential and the extracellular Na+ concentration. 11. The region of negative slope of the Ip-V curve observed in cells which were superfused with media containing low concentrations of K+ or its congeners strongly suggests the existence of at least two voltage-sensitive steps in the cardiac Na(+)-K+ pump cycle.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1665855

Human type II pneumocyte chemotactic responses to CXCR3 activation are mediated by splice variant A.

PubMed

Ji, Rong; Lee, Clement M; Gonzales, Linda W; Yang, Yi; Aksoy, Mark O; Wang, Ping; Brailoiu, Eugen; Dun, Nae; Hurford, Matthew T; Kelsen, Steven G

2008-06-01

Chemokine receptors control several fundamental cellular processes in both hematopoietic and structural cells, including directed cell movement, i.e., chemotaxis, cell differentiation, and proliferation. We have previously demonstrated that CXCR3, the chemokine receptor expressed by Th1/Tc1 inflammatory cells present in the lung, is also expressed by human airway epithelial cells. In airway epithelial cells, activation of CXCR3 induces airway epithelial cell movement and proliferation, processes that underlie lung repair. The present study examined the expression and function of CXCR3 in human alveolar type II pneumocytes, whose destruction causes emphysema. CXCR3 was present in human fetal and adult type II pneumocytes as assessed by immunocytochemistry, immunohistochemistry, and Western blotting. CXCR3-A and -B splice variant mRNA was present constitutively in cultured type II cells, but levels of CXCR3-B greatly exceeded CXCR3-A mRNA. In cultured type II cells, I-TAC, IP-10, and Mig induced chemotaxis. Overexpression of CXCR3-A in the A549 pneumocyte cell line produced robust chemotactic responses to I-TAC and IP-10. In contrast, I-TAC did not induce chemotactic responses in CXCR3-B and mock-transfected cells. Finally, I-TAC increased cytosolic Ca(2+) and activated the extracellular signal-regulated kinase, p38, and phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B kinases only in CXCR3-A-transfected cells. These data indicate that the CXCR3 receptor is expressed by human type II pneumocytes, and the CXCR3-A splice variant mediates chemotactic responses possibly through Ca(2+) activation of both mitogen-activated protein kinase and PI 3-kinase signaling pathways. Expression of CXCR3 in alveolar epithelial cells may be important in pneumocyte repair from injury.

Altered intestinal microbiota and blood T cell phenotype are shared by patients with Crohn's disease and their unaffected siblings.

PubMed

Hedin, Charlotte R; McCarthy, Neil E; Louis, Petra; Farquharson, Freda M; McCartney, Sara; Taylor, Kirstin; Prescott, Natalie J; Murrells, Trevor; Stagg, Andrew J; Whelan, Kevin; Lindsay, James O

2014-10-01

Crohn's disease (CD) is associated with intestinal dysbiosis, altered blood T cell populations, elevated faecal calprotectin (FC) and increased intestinal permeability (IP). CD-associated features present in siblings (increased risk of CD) but not in healthy controls, provide insight into early CD pathogenesis. We aimed to (1) Delineate the genetic, immune and microbiological profile of patients with CD, their siblings and controls and (2) Determine which factors discriminate between groups. Faecal microbiology was analysed by quantitative PCR targeting 16S ribosomal RNA, FC by ELISA, blood T cell phenotype by flow cytometry and IP by differential lactulose-rhamnose absorption in 22 patients with inactive CD, 21 of their healthy siblings and 25 controls. Subject's genotype relative risk was determined by Illumina Immuno BeadChip. Strikingly, siblings shared aspects of intestinal dysbiosis with patients with CD (lower concentrations of Faecalibacterium prausnitzii (p=0.048), Clostridia cluster IV (p=0.003) and Roseburia spp. (p=0.09) compared with controls). As in CD, siblings demonstrated a predominance of memory T cells (p=0.002) and elevated naïve CD4 T cell β7 integrin expression (p=0.01) compared with controls. FC was elevated (>50 μg/g) in 8/21 (38%) siblings compared with 2/25 (8%) controls (p=0.028); whereas IP did not differ between siblings and controls. Discriminant function analysis determined that combinations of these factors significantly discriminated between groups (χ(2)=80.4, df=20, p<0.001). Siblings were separated from controls by immunological and microbiological variables. Healthy siblings of patients with CD manifest immune and microbiological abnormalities associated with CD distinct from their genotype-related risk and provide an excellent model in which to investigate early CD pathogenesis. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Egr-1 mediated cardiac miR-99 family expression diverges physiological hypertrophy from pathological hypertrophy.

PubMed

Ramasamy, Subbiah; Velmurugan, Ganesan; Rekha, Balakrishnan; Anusha, Sivakumar; Shanmugha Rajan, K; Shanmugarajan, Suresh; Ramprasath, Tharmarajan; Gopal, Pandi; Tomar, Dhanendra; Karthik, Karuppusamy V; Verma, Suresh Kumar; Garikipati, Venkata Naga Srikanth; Sudarsan, Rajan

2018-04-01

The physiological cardiac hypertrophy is an adaptive condition without myocyte cell death, while pathological hypertrophy is a maladaptive condition associated with myocyte cell death. This study explores the miRNome of α-2M-induced physiologically hypertrophied cardiomyocytes and the role of miRNA-99 family during cardiac hypertrophy. Physiological and pathological cardiac hypertrophy was induced in H9c2 cardiomyoblast cell lines using α-2M and isoproterenol respectively. Total RNA isolation and small RNA sequencing were executed for physiological hypertrophy model. The differentially expressed miRNAs and its target mRNAs were validated in animal models. Transcription factor binding sites were predicted in the promoter of specific miRNAs and validated by ChIP-PCR. Subsequently, the selected miRNA was functionally characterized by overexpression and silencing. The effects of silencing of upstream regulator and downstream target gene were studied. Analysis of small RNA reads revealed the differential expression of a large set of miRNAs during hypertrophy, of which miR-99 family was highly downregulated upon α-2M treatment. However, this miR-99 family expression was upregulated during pathological hypertrophy and confirmed in animal models. ChIP-PCR confirms the binding of Egr-1 transcription factor to the miR-99 promoter. Further, silencing of Egr-1 decreased the expression of miR-99. The overexpression or silencing of miR-99 diverges the physiological hypertrophy to pathological hypertrophy and vice versa by regulating Akt-1 pathway. Silencing of Akt-1 replicates the effect of overexpression of miR-99. The results proved Egr-1 mediated regulation of miR-99 family that plays a key role in determining the fate of cardiac hypertrophy by regulating Akt-1 signaling. Copyright © 2018 Elsevier Inc. All rights reserved.

p63 regulates glutaminase 2 expression

PubMed Central

Giacobbe, Arianna; Bongiorno-Borbone, Lucilla; Bernassola, Francesca; Terrinoni, Alessandro; Markert, Elke Katrin; Levine, Arnold J.; Feng, Zhaohui; Agostini, Massimilano; Zolla, Lello; Agrò, Alessandro Finazzi; Notterman, Daniel A.; Melino, Gerry; Peschiaroli, Angelo

2013-01-01

The transcription factor p63 is critical for many biological processes, including development and maintenance of epidermal tissues and tumorigenesis. Here, we report that the TAp63 isoforms regulate cell metabolism through the induction of the mitochondrial glutaminase 2 (GLS2) gene both in primary cells and tumor cell lines. By ChIP analysis and luciferase assay, we confirmed that TAp63 binds directly to the p53/p63 consensus DNA binding sequence within the GLS2 promoter region. Given the critical role of p63 in epidermal differentiation, we have investigated the regulation of GLS2 expression during this process. GLS2 and TAp63 expression increases during the in vitro differentiation of primary human keratinocytes, and depletion of GLS2 inhibits skin differentiation both at molecular and cellular levels. We found that GLS2 and TAp63 expression are concomitantly induced in cancer cells exposed to oxidative stresses. siRNA-mediated depletion of GLS2 sensitizes cells to ROS-induced apoptosis, suggesting that the TAp63/GLS2 axis can be functionally important as a cellular antioxidant pathway in the absence of p53. Accordingly, we found that GLS2 is upregulated in colon adenocarcinoma. Altogether, our findings demonstrate that GLS2 is a bona fide TAp63 target gene, and that the TAp63-dependent regulation of GLS2 is important for both physiological and pathological processes. PMID:23574722

Antisense Oligonucleotides Targeting Parasite Inositol 1,4,5-Trisphosphate Receptor Inhibits Mammalian Host Cell Invasion by Trypanosoma cruzi

NASA Astrophysics Data System (ADS)

Hashimoto, Muneaki; Nara, Takeshi; Hirawake, Hiroko; Morales, Jorge; Enomoto, Masahiro; Mikoshiba, Katsuhiko

2014-02-01

Chagas disease is caused by an intracellular parasitic protist, Trypanosoma cruzi. As there are no highly effective drugs against this agent that also demonstrate low toxicity, there is an urgent need for development of new drugs to treat Chagas disease. We have previously demonstrated that the parasite inositol 1,4,5-trisphosphate receptor (TcIP3R) is crucial for invasion of the mammalian host cell by T. cruzi. Here, we report that TcIP3R is a short-lived protein and that its expression is significantly suppressed in trypomastigotes. Treatment of trypomastigotes, an infective stage of T. cruzi, with antisense oligonucleotides specific to TcIP3R deceased TcIP3R protein levels and impaired trypomastigote invasion of host cells. Due to the resulting instability and very low expression level of TcIP3R in trypomastigotes indicates that TcIP3R is a promising target for antisense therapy in Chagas disease.

Polyphosphate induces matrix metalloproteinase-3-mediated proliferation of odontoblast-like cells derived from induced pluripotent stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ozeki, Nobuaki; Hase, Naoko; Yamaguchi, Hideyuki

2015-05-01

Inorganic polyphosphate [Poly(P)] may represent a physiological source of phosphate and has the ability to induce bone differentiation in osteoblasts. We previously reported that cytokine-induced matrix metalloproteinase (MMP)-3 accelerates the proliferation of purified odontoblast-like cells. In this study, MMP-3 small interfering RNA (siRNA) was transfected into odontoblast-like cells derived from induced pluripotent stem cells to investigate whether MMP-3 activity is induced by Poly(P) and/or is associated with cell proliferation and differentiation into odontoblast-like cells. Treatment with Poly(P) led to an increase in both cell proliferation and additional odontoblastic differentiation. Poly(P)-treated cells showed a small but significant increase in dentin sialophosphoproteinmore » (DSPP) and dentin matrix protein-1 (DMP-1) mRNA expression, which are markers of mature odontoblasts. The cells also acquired additional odontoblast-specific properties including adoption of an odontoblastic phenotype typified by high alkaline phosphatase (ALP) activity and a calcification capacity. In addition, Poly(P) induced expression of MMP-3 mRNA and protein, and increased MMP-3 activity. MMP-3 siRNA-mediated disruption of the expression of these effectors potently suppressed the expression of odontoblastic biomarkers ALP, DSPP, and DMP-1, and blocked calcification. Interestingly, upon siRNA-mediated silencing of MMP-3, we noted a potent and significant decrease in cell proliferation. Using specific siRNAs, we revealed that a unique signaling cascade, Poly(P)→MMP-3→DSPP and/or DMP-1, was intimately involved in the proliferation of odontoblast-like cells. - Highlights: • Polyphosphate increases proliferation of iPS cell-derived odontoblast-like cells. • Polyphosphate-induced MMP-3 results in an increase of cell proliferation. • Induced cell proliferation involves MMP-3, DSPP, and/or DMP-1 sequentially. • Induced MMP-3 also results in an increase of odontoblastic differentiation.« less

An integrated global regulatory network of hematopoietic precursor cell self-renewal and differentiation.

PubMed

You, Yanan; Cuevas-Diaz Duran, Raquel; Jiang, Lihua; Dong, Xiaomin; Zong, Shan; Snyder, Michael; Wu, Jia Qian

2018-06-12

Systematic study of the regulatory mechanisms of Hematopoietic Stem Cell and Progenitor Cell (HSPC) self-renewal is fundamentally important for understanding hematopoiesis and for manipulating HSPCs for therapeutic purposes. Previously, we have characterized gene expression and identified important transcription factors (TFs) regulating the switch between self-renewal and differentiation in a multipotent Hematopoietic Progenitor Cell (HPC) line, EML (Erythroid, Myeloid, and Lymphoid) cells. Herein, we report binding maps for additional TFs (SOX4 and STAT3) by using chromatin immunoprecipitation (ChIP)-Sequencing, to address the underlying mechanisms regulating self-renewal properties of lineage-CD34+ subpopulation (Lin-CD34+ EML cells). Furthermore, we applied the Assay for Transposase Accessible Chromatin (ATAC)-Sequencing to globally identify the open chromatin regions associated with TF binding in the self-renewing Lin-CD34+ EML cells. Mass spectrometry (MS) was also used to quantify protein relative expression levels. Finally, by integrating the protein-protein interaction database, we built an expanded transcriptional regulatory and interaction network. We found that MAPK (Mitogen-activated protein kinase) pathway and TGF-β/SMAD signaling pathway components were highly enriched among the binding targets of these TFs in Lin-CD34+ EML cells. The present study integrates regulatory information at multiple levels to paint a more comprehensive picture of the HSPC self-renewal mechanisms.

GenoGAM: genome-wide generalized additive models for ChIP-Seq analysis.

PubMed

Stricker, Georg; Engelhardt, Alexander; Schulz, Daniel; Schmid, Matthias; Tresch, Achim; Gagneur, Julien

2017-08-01

Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) is a widely used approach to study protein-DNA interactions. Often, the quantities of interest are the differential occupancies relative to controls, between genetic backgrounds, treatments, or combinations thereof. Current methods for differential occupancy of ChIP-Seq data rely however on binning or sliding window techniques, for which the choice of the window and bin sizes are subjective. Here, we present GenoGAM (Genome-wide Generalized Additive Model), which brings the well-established and flexible generalized additive models framework to genomic applications using a data parallelism strategy. We model ChIP-Seq read count frequencies as products of smooth functions along chromosomes. Smoothing parameters are objectively estimated from the data by cross-validation, eliminating ad hoc binning and windowing needed by current approaches. GenoGAM provides base-level and region-level significance testing for full factorial designs. Application to a ChIP-Seq dataset in yeast showed increased sensitivity over existing differential occupancy methods while controlling for type I error rate. By analyzing a set of DNA methylation data and illustrating an extension to a peak caller, we further demonstrate the potential of GenoGAM as a generic statistical modeling tool for genome-wide assays. Software is available from Bioconductor: https://www.bioconductor.org/packages/release/bioc/html/GenoGAM.html . gagneur@in.tum.de. Supplementary information is available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

T-box Transcription Regulator Tbr2 Is Essential for the Formation and Maintenance of Opn4/Melanopsin-Expressing Intrinsically Photosensitive Retinal Ganglion Cells

PubMed Central

Li, Hongyan; Zhang, Zhijing; Kiyama, Takae; Panda, Satchidananda; Hattar, Samer; Ribelayga, Christophe P.; Mills, Stephen L.

2014-01-01

Opsin 4 (Opn4)/melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) play a major role in non-image-forming visual system. Although advances have been made in understanding their morphological features and functions, the molecular mechanisms that regulate their formation and survival remain unknown. Previously, we found that mouse T-box brain 2 (Tbr2) (also known as Eomes), a T-box-containing transcription factor, was expressed in a subset of newborn RGCs, suggesting that it is involved in the formation of specific RGC subtypes. In this in vivo study, we used complex mouse genetics, single-cell dye tracing, and behavioral analyses to determine whether Tbr2 regulates ipRGC formation and survival. Our results show the following: (1) Opn4 is expressed exclusively in Tbr2-positive RGCs; (2) no ipRGCs are detected when Tbr2 is genetically ablated before RGC specification; and (3) most ipRGCs are eliminated when Tbr2 is deleted in established ipRGCs. The few remaining ipRGCs display abnormal dendritic morphological features and functions. In addition, some Tbr2-expressing RGCs can activate Opn4 expression on the loss of native ipRGCs, suggesting that Tbr2-expressing RGCs may serve as a reservoir of ipRGCs to regulate the number of ipRGCs and the expression levels of Opn4. PMID:25253855

Stem cells in dentistry--part I: stem cell sources.

PubMed

Egusa, Hiroshi; Sonoyama, Wataru; Nishimura, Masahiro; Atsuta, Ikiru; Akiyama, Kentaro

2012-07-01

Stem cells can self-renew and produce different cell types, thus providing new strategies to regenerate missing tissues and treat diseases. In the field of dentistry, adult mesenchymal stem/stromal cells (MSCs) have been identified in several oral and maxillofacial tissues, which suggests that the oral tissues are a rich source of stem cells, and oral stem and mucosal cells are expected to provide an ideal source for genetically reprogrammed cells such as induced pluripotent stem (iPS) cells. Furthermore, oral tissues are expected to be not only a source but also a therapeutic target for stem cells, as stem cell and tissue engineering therapies in dentistry continue to attract increasing clinical interest. Part I of this review outlines various types of intra- and extra-oral tissue-derived stem cells with regard to clinical availability and applications in dentistry. Additionally, appropriate sources of stem cells for regenerative dentistry are discussed with regard to differentiation capacity, accessibility and possible immunomodulatory properties. Copyright © 2012 Japan Prosthodontic Society. Published by Elsevier Ltd. All rights reserved.

The cooked meat-derived mammary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine promotes invasive behaviour of breast cancer cells.

PubMed

Lauber, Sandra N; Gooderham, Nigel J

2011-01-11

The cooked meat derived genotoxic carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces cancer of the colon, prostate and mammary gland when fed to rats. Epidemiology studies link these tumours to a Western diet and exposure to heterocyclic amines such as PhIP. We have shown that PhIP is also potently estrogenic and have proposed that this hormonal activity contributes to its target site carcinogenicity. We now postulate that the estrogenic properties of PhIP influence metastatic potential. We have used an in vitro assay for cell invasion based upon digestion and migration through a reconstituted basement membrane model. Zymography and immunoblotting were used to confirm PhIP-mediated changes associated with induction of the invasive phenotype. Treatment of the mammary cancer cell lines MCF-7 and T47D with PhIP induces cells to digest and migrate through a reconstituted basement membrane. The response was dose dependent, observed at sub-nanomolar concentrations of PhIP and was inhibited by the antiestrogen ICI 182,780. The PhIP-induced invasive phenotype was associated with expression of cathepsin D, cyclooxygenase-2 and matrix metalloproteinase activity. These findings emphasise the range and potency of the biological activities associated with this cooked meat product and mechanistically support the tissue-specific carcinogenicity of the chemical. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Inositol trisphosphate metabolism in carrot (Daucus carota L. ) cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Memon, A.R.; Rincon, M.; Boss, W.F.

1989-10-01

The metabolism of exogenously added D-myo-(1-{sup 3}H)inositol 1,4,5-trisphosphate (IP{sub 3}) has been examined in microsomal membrane and soluble fractions of carrot cells grown in suspension culture. When ({sup 3}H)IP{sub 3} was added to a microsomal membrane fraction, ({sup 3}H)IP{sub 2} was the primary metabolite consisting of approximately 83% of the total recovered ({sup 3}H) by electrophoresis. ({sup 3}H)IP was only 6% of the ({sup 3}H) recovered, and 10% of the ({sup 3}H)IP{sub 3} was not further metabolized. In contrast, when ({sup 3}H)IP{sub 3} was added to the soluble fraction, approximately equal amounts of ({sup 3}H)IP{sub 2} and ({sup 3}H)IP weremore » recovered. Ca{sup 2+} (100 micromolar) tended to enhance IP{sub 3} dephosphorylation but inhibited the IP{sub 2} dephosphorylation in the soluble fraction by about 20%. MoO{sub 4}{sup 2{minus}} (1 millimolar) inhibited the dephosphorylation of IP{sub 3} by the microsomal fraction and the dephosphorylation of IP{sub 2} by the soluble fraction. MoO{sub 4}{sup 2{minus}}, however, did not inhibit the dephosphorylation of IP{sub 3} by the soluble fraction. Li{sup +} (10 and 50 millimolar) had no effect on IP{sub 3} metabolism in either the soluble or membrane fraction; however, Li{sup +} (50 millimolar) inhibited IP{sub 2} dephosphorylation in the soluble fraction about 25%.« less

Type 1 and 3 inositol trisphosphate receptors are required for extra-embryonic vascular development.

PubMed

Uchida, Keiko; Nakazawa, Maki; Yamagishi, Chihiro; Mikoshiba, Katsuhiko; Yamagishi, Hiroyuki

2016-10-01

The embryonic-maternal interface of the placental labyrinth, allantois, and yolk sac are vital during embryogenesis; however, the precise mechanism underlying the vascularization of these structures remains unknown. Herein we focus on the role of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R), which are intracellular Ca(2+) release channels, in placentation. Double knockout (DKO) of type 1 and 3 IP3Rs (IP3R1 and IP3R3, respectively) in mice resulted in embryonic lethality around embryonic day (E) 11.5. Because IP3R1 and IP3R3 were co-expressed in endothelial cells in the labyrinth, allantois, and yolk sac, we investigated extra-embryonic vascular development in IP3R1- and IP3R3-DKO mice. The formation of chorionic plates and yolk sac vessels seemed dysregulated around the timing of the chorio-allantoic attachment, immediately followed by the disorganization of allantoic vessels, the decreased expression of the spongiotrophoblast cell marker Tpbpa and the growth retardation of the embryos in DKO mice. Fluorescent immunohistochemistry demonstrated downregulation of a vascular endothelial marker, CD31, in labyrinth embryonic vessels and poor elongation of extra-embryonic mesoderm into the labyrinth layer in DKO placenta, whereas the branching of the DKO chorionic trophoblast was initiated. In addition, allantoic and yolk sac vessels in extra-embryonic tissues were less remodeled in DKO mice. In vitro endothelial cord formation and migration activities of cultured vascular endothelial cells derived from human umbilical vein were downregulated under the inhibition of IP3R. Our results suggest that IP3R1 and IP3R3 are required for extra-embryonic vascularization in the placenta, allantois, and yolk sac. This is the first demonstration of the essential role of IP3/IP3Rs signaling in the development of the vasculature at the embryonic-maternal interface. Copyright © 2016 Elsevier Inc. All rights reserved.

Temperature and nucleotide dependence of calcium release by myo-inositol 1,4,5-trisphosphate in cultured vascular smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, J.B.; Smith, L.; Higgins, B.L.

1985-11-25

Inositol 1,4,5-trisphosphate (IP3) rapidly increased UVCaS efflux from a nonmitochondrial organelle in cultured vascular smooth muscle cells that were permeabilized with saponin. A nucleotide, preferably ATP, was essential for IP3-evoked UVCaS release. Two nonhydrolyzable ATP analogues satisfied the nucleotide requirement for IP3-evoked UVCaS release. IP3 strongly stimulated UVCaS efflux at low temperatures (1 to 15 degrees C). Decreasing the temperature from 37 to 4 degrees C inhibited the rate of IP3-stimulated efflux by only about 33%. The failure of such low temperatures to strongly inhibit IP3-induced UVCaS efflux suggests that IP3 activated a CaS channel, rather than a carrier, bymore » a ligand-binding, rather than a metabolic, reaction.« less

Human iPS cell-engineered cardiac tissue sheets with cardiomyocytes and vascular cells for cardiac regeneration

PubMed Central

Masumoto, Hidetoshi; Ikuno, Takeshi; Takeda, Masafumi; Fukushima, Hiroyuki; Marui, Akira; Katayama, Shiori; Shimizu, Tatsuya; Ikeda, Tadashi; Okano, Teruo; Sakata, Ryuzo; Yamashita, Jun K.

2014-01-01

To realize cardiac regeneration using human induced pluripotent stem cells (hiPSCs), strategies for cell preparation, tissue engineering and transplantation must be explored. Here we report a new protocol for the simultaneous induction of cardiomyocytes (CMs) and vascular cells [endothelial cells (ECs)/vascular mural cells (MCs)], and generate entirely hiPSC-engineered cardiovascular cell sheets, which showed advantageous therapeutic effects in infarcted hearts. The protocol adds to a previous differentiation protocol of CMs by using stage-specific supplementation of vascular endothelial cell growth factor for the additional induction of vascular cells. Using this cell sheet technology, we successfully generated physically integrated cardiac tissue sheets (hiPSC-CTSs). HiPSC-CTS transplantation to rat infarcted hearts significantly improved cardiac function. In addition to neovascularization, we confirmed that engrafted human cells mainly consisted of CMs in >40% of transplanted rats four weeks after transplantation. Thus, our HiPSC-CTSs show promise for cardiac regenerative therapy. PMID:25336194

Distribution profile of inositol 1,4,5-trisphosphate receptor isoforms in adrenal chromaffin cells.

PubMed

Huh, Yang Hoon; Yoo, Jie Ae; Bahk, Sook Jin; Yoo, Seung Hyun

2005-05-09

Given the importance of inositol 1,4,5-trisphosphate receptor (IP(3)R)/Ca(2+) channels in the control of intracellular Ca(2+) concentrations, we determined the relative concentrations of the IP(3)R isoforms in subcellular organelles, based on serially sectioned electron micrographs. The endoplasmic reticulum (ER) was estimated to contain 15-20% of each of the three IP(3)R isoforms while secretory granules contained 58-69%. The nucleus contained approximately 15% each of IP(3)R-1 and -2, but 25% of IP(3)R-3, whereas the plasma membrane contained approximately 1% or less of each. These suggested that secretory granules, the nucleus and ER are at the center of IP(3)-dependent intracellular Ca(2+) control mechanisms in chromaffin cells.

Modeling and inversion Matlab algorithms for resistivity, induced polarization and seismic data

NASA Astrophysics Data System (ADS)

Karaoulis, M.; Revil, A.; Minsley, B. J.; Werkema, D. D.

2011-12-01

M. Karaoulis (1), D.D. Werkema (3), A. Revil (1,2), A., B. Minsley (4), (1) Colorado School of Mines, Dept. of Geophysics, Golden, CO, USA. (2) ISTerre, CNRS, UMR 5559, Université de Savoie, Equipe Volcan, Le Bourget du Lac, France. (3) U.S. EPA, ORD, NERL, ESD, CMB, Las Vegas, Nevada, USA . (4) USGS, Federal Center, Lakewood, 10, 80225-0046, CO. Abstract We propose 2D and 3D forward modeling and inversion package for DC resistivity, time domain induced polarization (IP), frequency-domain IP, and seismic refraction data. For the resistivity and IP case, discretization is based on rectangular cells, where each cell has as unknown resistivity in the case of DC modelling, resistivity and chargeability in the time domain IP modelling, and complex resistivity in the spectral IP modelling. The governing partial-differential equations are solved with the finite element method, which can be applied to both real and complex variables that are solved for. For the seismic case, forward modeling is based on solving the eikonal equation using a second-order fast marching method. The wavepaths are materialized by Fresnel volumes rather than by conventional rays. This approach accounts for complicated velocity models and is advantageous because it considers frequency effects on the velocity resolution. The inversion can accommodate data at a single time step, or as a time-lapse dataset if the geophysical data are gathered for monitoring purposes. The aim of time-lapse inversion is to find the change in the velocities or resistivities of each model cell as a function of time. Different time-lapse algorithms can be applied such as independent inversion, difference inversion, 4D inversion, and 4D active time constraint inversion. The forward algorithms are benchmarked against analytical solutions and inversion results are compared with existing ones. The algorithms are packaged as Matlab codes with a simple Graphical User Interface. Although the code is parallelized for multi-core cpus, it is not as fast as machine code. In the case of large datasets, someone should consider transferring parts of the code to C or Fortran through mex files. This code is available through EPA's website on the following link http://www.epa.gov/esd/cmb/GeophysicsWebsite/index.html Although this work was reviewed by EPA and approved for publication, it may not necessarily reflect official Agency policy.

Identification of Epigenetic Changes in Prostate Cancer using Induced Pluripotent Stem Cells

DTIC Science & Technology

2014-04-01

somatic cells in human iPS cells. Nat Cell Bioi, 13: 541, 2011 5. Polo, J. M., Liu, S., Figueroa , M. E. et al.: Cell type of origin influences the...human iPS cells. Nat Cell Bioi 13: 5•1 \\ - 5•19. 18. Poloj:\\ə, Lu S, Figueroa ME, Kulalert W, Eminli S, ct a!. (20 10) Cell type of origin

Potassium Channel Interacting Protein 2 (KChIP2) is not a transcriptional regulator of cardiac electrical remodeling

PubMed Central

Winther, Sine V.; Tuomainen, Tomi; Borup, Rehannah; Tavi, Pasi; Antoons, Gudrun; Thomsen, Morten B.

2016-01-01

The heart-failure relevant Potassium Channel Interacting Protein 2 (KChIP2) augments CaV1.2 and KV4.3. KChIP3 represses CaV1.2 transcription in cardiomyocytes via interaction with regulatory DNA elements. Hence, we tested nuclear presence of KChIP2 and if KChIP2 translocates into the nucleus in a Ca2+ dependent manner. Cardiac biopsies from human heart-failure patients and healthy donor controls showed that nuclear KChIP2 abundance was significantly increased in heart failure; however, this was secondary to a large variation of total KChIP2 content. Administration of ouabain did not increase KChIP2 content in nuclear protein fractions in anesthetized mice. KChIP2 was expressed in cell lines, and Ca2+ ionophores were applied in a concentration- and time-dependent manner. The cell lines had KChIP2-immunoreactive protein in the nucleus in the absence of treatments to modulate intracellular Ca2+ concentration. Neither increasing nor decreasing intracellular Ca2+ concentrations caused translocation of KChIP2. Microarray analysis did not identify relief of transcriptional repression in murine KChIP2−/− heart samples. We conclude that although there is a baseline presence of KChIP2 in the nucleus both in vivo and in vitro, KChIP2 does not directly regulate transcriptional activity. Moreover, the nuclear transport of KChIP2 is not dependent on Ca2+. Thus, KChIP2 does not function as a conventional transcription factor in the heart. PMID:27349185

Hindered cytoplasmic diffusion of inositol trisphosphate restricts its cellular range of action

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dickinson, G. D.; Ellefsen, K. L.; Dawson, S. P.

The range of action of intracellular messengers is determined by their rates of diffusion and degradation. Previous measurements in oocyte cytoplasmic extracts indicated that the Ca 2+-liberating second messenger inositol trisphosphate (IP 3) diffuses with a coefficient (~280 μm 2 s -1) similar to that in water, corresponding to a range of action of ~25 μm. Consequently, IP 3 is generally considered a “global” cellular messenger. We also reexamined this issue by measuring local IP 3-evoked Ca 2+ puffs to monitor IP 3 diffusing from spot photorelease in neuroblastoma cells. Fitting these data by numerical simulations yielded a diffusion coefficientmore » (≤10 μm 2 s -1) about 30-fold slower than that previously reported. Here, we propose that diffusion of IP 3 in mammalian cells is hindered by binding to immobile, functionally inactive receptors that were diluted in oocyte extracts. The predicted range of action of IP 3 (<5 μm) is thus smaller than the size of typical mammalian cells, indicating that IP 3 should better be considered as a local rather than a global cellular messenger.« less

Hindered cytoplasmic diffusion of inositol trisphosphate restricts its cellular range of action

DOE PAGES

Dickinson, G. D.; Ellefsen, K. L.; Dawson, S. P.; ...

2016-11-08

The range of action of intracellular messengers is determined by their rates of diffusion and degradation. Previous measurements in oocyte cytoplasmic extracts indicated that the Ca 2+-liberating second messenger inositol trisphosphate (IP 3) diffuses with a coefficient (~280 μm 2 s -1) similar to that in water, corresponding to a range of action of ~25 μm. Consequently, IP 3 is generally considered a “global” cellular messenger. We also reexamined this issue by measuring local IP 3-evoked Ca 2+ puffs to monitor IP 3 diffusing from spot photorelease in neuroblastoma cells. Fitting these data by numerical simulations yielded a diffusion coefficientmore » (≤10 μm 2 s -1) about 30-fold slower than that previously reported. Here, we propose that diffusion of IP 3 in mammalian cells is hindered by binding to immobile, functionally inactive receptors that were diluted in oocyte extracts. The predicted range of action of IP 3 (<5 μm) is thus smaller than the size of typical mammalian cells, indicating that IP 3 should better be considered as a local rather than a global cellular messenger.« less

Genetic diversity of Malus cultivars and wild relatives in the Chinese National Repository of Apple Germplasm Resources

USDA-ARS?s Scientific Manuscript database

The Research Institute of Pomology (IP), Chinese Academy of Agricultural Sciences (CAAS) in Xingcheng, China, maintains hundreds of apple accessions that originated from around the world. We have used 16 microsatellites to assess the diversity and differentiation of 391 accessions within the IP tha...

A Semantic Differential Evaluation of Attitudinal Outcomes of Introductory Physical Science.

ERIC Educational Resources Information Center

Hecht, Alfred Roland

This study was designed to assess the attitudinal outcomes of Introductory Physical Science (IPS) curriculum materials used in schools. Random samples of 240 students receiving IPS instruction and 240 non-science students were assigned to separate Solomon four-group designs with non-equivalent control groups. Random samples of 60 traditional…

Induced pluripotent stem cells: challenges and opportunities for cancer immunotherapy.

PubMed

Sachamitr, Patty; Hackett, Simon; Fairchild, Paul Jonathan

2014-01-01

Despite recent advances in cancer treatment over the past 30 years, therapeutic options remain limited and do not always offer a cure for malignancy. Given that tumor-associated antigens (TAA) are, by definition, self-proteins, the need to productively engage autoreactive T cells remains at the heart of strategies for cancer immunotherapy. These have traditionally focused on the administration of autologous monocyte-derived dendritic cells (moDC) pulsed with TAA, or the ex vivo expansion and adoptive transfer of tumor-infiltrating lymphocytes (TIL) as a source of TAA-specific cytotoxic T cells (CTL). Although such approaches have shown some efficacy, success has been limited by the poor capacity of moDC to cross present exogenous TAA to the CD8(+) T-cell repertoire and the potential for exhaustion of CTL expanded ex vivo. Recent advances in induced pluripotency offer opportunities to generate patient-specific stem cell lines with the potential to differentiate in vitro into cell types whose properties may help address these issues. Here, we review recent success in the differentiation of NK cells from human induced pluripotent stem (iPS) cells as well as minor subsets of dendritic cells (DCs) with therapeutic potential, including CD141(+)XCR1(+) DC, capable of cross presenting TAA to naïve CD8(+) T cells. Furthermore, we review recent progress in the use of TIL as the starting material for the derivation of iPSC lines, thereby capturing their antigen specificity in a self-renewing stem cell line, from which potentially unlimited numbers of naïve TAA-specific T cells may be differentiated, free of the risks of exhaustion.

Mapping of transcription factor binding regions in mammalian cells by ChIP: Comparison of array- and sequencing-based technologies

PubMed Central

Euskirchen, Ghia M.; Rozowsky, Joel S.; Wei, Chia-Lin; Lee, Wah Heng; Zhang, Zhengdong D.; Hartman, Stephen; Emanuelsson, Olof; Stolc, Viktor; Weissman, Sherman; Gerstein, Mark B.; Ruan, Yijun; Snyder, Michael

2007-01-01

Recent progress in mapping transcription factor (TF) binding regions can largely be credited to chromatin immunoprecipitation (ChIP) technologies. We compared strategies for mapping TF binding regions in mammalian cells using two different ChIP schemes: ChIP with DNA microarray analysis (ChIP-chip) and ChIP with DNA sequencing (ChIP-PET). We first investigated parameters central to obtaining robust ChIP-chip data sets by analyzing STAT1 targets in the ENCODE regions of the human genome, and then compared ChIP-chip to ChIP-PET. We devised methods for scoring and comparing results among various tiling arrays and examined parameters such as DNA microarray format, oligonucleotide length, hybridization conditions, and the use of competitor Cot-1 DNA. The best performance was achieved with high-density oligonucleotide arrays, oligonucleotides ≥50 bases (b), the presence of competitor Cot-1 DNA and hybridizations conducted in microfluidics stations. When target identification was evaluated as a function of array number, 80%–86% of targets were identified with three or more arrays. Comparison of ChIP-chip with ChIP-PET revealed strong agreement for the highest ranked targets with less overlap for the low ranked targets. With advantages and disadvantages unique to each approach, we found that ChIP-chip and ChIP-PET are frequently complementary in their relative abilities to detect STAT1 targets for the lower ranked targets; each method detected validated targets that were missed by the other method. The most comprehensive list of STAT1 binding regions is obtained by merging results from ChIP-chip and ChIP-sequencing. Overall, this study provides information for robust identification, scoring, and validation of TF targets using ChIP-based technologies. PMID:17568005

Central melanopsin projections in the diurnal rodent, Arvicanthis niloticus

PubMed Central

Langel, Jennifer L.; Smale, Laura; Esquiva, Gema; Hannibal, Jens

2015-01-01

The direct effects of photic stimuli on behavior are very different in diurnal and nocturnal species, as light stimulates an increase in activity in the former and a decrease in the latter. Studies of nocturnal mice have implicated a select population of retinal ganglion cells that are intrinsically photosensitive (ipRGCs) in mediation of these acute responses to light. ipRGCs are photosensitive due to the expression of the photopigment melanopsin; these cells use glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP) as neurotransmitters. PACAP is useful for the study of central ipRGC projections because, in the retina, it is found exclusively within melanopsin cells. Little is known about the central projections of ipRGCs in diurnal species. Here, we first characterized these cells in the retina of the diurnal Nile grass rat using immunohistochemistry (IHC). The same basic subtypes of melanopsin cells that have been described in other mammals were present, but nearly 25% of them were displaced, primarily in its superior region. PACAP was present in 87.7% of all melanopsin cells, while 97.4% of PACAP cells contained melanopsin. We then investigated central projections of ipRGCs by examining the distribution of immunoreactive PACAP fibers in intact and enucleated animals. This revealed evidence that these cells project to the suprachiasmatic nucleus, lateral geniculate nucleus (LGN), pretectum, and superior colliculus. This distribution was confirmed with injections of cholera toxin subunit β coupled with Alexa Fluor 488 in one eye and Alexa Fluor 594 in the other, combined with IHC staining of PACAP. These studies also revealed that the ventral and dorsal LGN and the caudal olivary pretectal nucleus receive less innervation from ipRGCs than that reported in nocturnal rodents. Overall, these data suggest that although ipRGCs and their projections are very similar in diurnal and nocturnal rodents, they may not be identical. PMID:26236201

Central melanopsin projections in the diurnal rodent, Arvicanthis niloticus.

PubMed

Langel, Jennifer L; Smale, Laura; Esquiva, Gema; Hannibal, Jens

2015-01-01

The direct effects of photic stimuli on behavior are very different in diurnal and nocturnal species, as light stimulates an increase in activity in the former and a decrease in the latter. Studies of nocturnal mice have implicated a select population of retinal ganglion cells that are intrinsically photosensitive (ipRGCs) in mediation of these acute responses to light. ipRGCs are photosensitive due to the expression of the photopigment melanopsin; these cells use glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP) as neurotransmitters. PACAP is useful for the study of central ipRGC projections because, in the retina, it is found exclusively within melanopsin cells. Little is known about the central projections of ipRGCs in diurnal species. Here, we first characterized these cells in the retina of the diurnal Nile grass rat using immunohistochemistry (IHC). The same basic subtypes of melanopsin cells that have been described in other mammals were present, but nearly 25% of them were displaced, primarily in its superior region. PACAP was present in 87.7% of all melanopsin cells, while 97.4% of PACAP cells contained melanopsin. We then investigated central projections of ipRGCs by examining the distribution of immunoreactive PACAP fibers in intact and enucleated animals. This revealed evidence that these cells project to the suprachiasmatic nucleus, lateral geniculate nucleus (LGN), pretectum, and superior colliculus. This distribution was confirmed with injections of cholera toxin subunit β coupled with Alexa Fluor 488 in one eye and Alexa Fluor 594 in the other, combined with IHC staining of PACAP. These studies also revealed that the ventral and dorsal LGN and the caudal olivary pretectal nucleus receive less innervation from ipRGCs than that reported in nocturnal rodents. Overall, these data suggest that although ipRGCs and their projections are very similar in diurnal and nocturnal rodents, they may not be identical.

Highly Expandable Human iPS Cell-Derived Neural Progenitor Cells (NPC) and Neurons for Central Nervous System Disease Modeling and High-Throughput Screening.

PubMed

Cheng, Chialin; Fass, Daniel M; Folz-Donahue, Kat; MacDonald, Marcy E; Haggarty, Stephen J

2017-01-11

Reprogramming of human somatic cells into induced pluripotent stem (iPS) cells has greatly expanded the set of research tools available to investigate the molecular and cellular mechanisms underlying central nervous system (CNS) disorders. Realizing the promise of iPS cell technology for the identification of novel therapeutic targets and for high-throughput drug screening requires implementation of methods for the large-scale production of defined CNS cell types. Here we describe a protocol for generating stable, highly expandable, iPS cell-derived CNS neural progenitor cells (NPC) using multi-dimensional fluorescence activated cell sorting (FACS) to purify NPC defined by cell surface markers. In addition, we describe a rapid, efficient, and reproducible method for generating excitatory cortical-like neurons from these NPC through inducible expression of the pro-neural transcription factor Neurogenin 2 (iNgn2-NPC). Finally, we describe methodology for the use of iNgn2-NPC for probing human neuroplasticity and mechanisms underlying CNS disorders using high-content, single-cell-level automated microscopy assays. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Alkaline phosphatase and OCT-3/4 as useful markers for predicting susceptibility of human deciduous teeth-derived dental pulp cells to reprogramming factor-induced iPS cells.

PubMed

Inada, Emi; Saitoh, Issei; Kubota, Naoko; Soda, Miki; Matsueda, Kazunari; Murakami, Tomoya; Sawami, Tadashi; Kagoshima, Akiko; Yamasaki, Youichi; Sato, Masahiro

2017-11-01

The aim of the present study was to prove that primary cells enriched with stem cells are more easily reprogrammed to generate induced pluripotent stem (iPS) cells than those with scarce numbers of stem cells. We surveyed the alkaline phosphatase (ALP) activity in five primarily-isolated human deciduous teeth-derived dental pulp cells (HDDPC) with cytochemical staining to examine the possible presence of stem cells. Next, the expression of stemness-specific factors, such as OCT(Octumer-binding transcription factor)3/4, NANOG, SOX2(SRY (sex determining region Y)-box 2), CD90, muscle segment homeodomain homeobox (MSX) 1, and MSX2, was assessed with a reverse transcription polymerase chain reaction method. Finally, these isolated HDDPC were transfected with plasmids carrying genes coding Yamanaka factors to determine whether these cells could be reprogrammed to generate iPS cells. Of the five primarily-isolated HDDPC, two (HDDPC-1 and -5) exhibited higher degrees of ALP activity. OCT-3/4 expression was also prominent in those two lines. Furthermore, these two lines proliferated faster than the other three lines. The transfection of HDDPC with Yamanaka factors resulted in the generation of iPS cells from HDDPC-1 and -5. The number of cells with the stemness property of HDDPC differs among individuals, which suggests that HDDPC showing an increased expression of both ALP and OCT-3/4 can be more easily reprogrammed to generate iPS cells after the forced expression of reprogramming factors. © 2016 John Wiley & Sons Australia, Ltd.

Blocking IP3 signal transduction pathways inhibits melatonin-induced Ca2+ signals and impairs P. falciparum development and proliferation in erythrocytes.

PubMed

Pecenin, Mateus Fila; Borges-Pereira, Lucas; Levano-Garcia, Julio; Budu, Alexandre; Alves, Eduardo; Mikoshiba, Katsuhiko; Thomas, Andrew; Garcia, Celia R S

2018-03-14

Inositol 1,4,5 trisphosphate (IP 3 ) signaling plays a crucial role in a wide range of eukaryotic processes. In Plasmodium falciparum, IP 3 elicits Ca 2+ release from intracellular Ca 2+ stores, even though no IP 3 receptor homolog has been identified to date. The human host hormone melatonin plays a key role in entraining the P. falciparum life cycle in the intraerythrocytic stages, apparently through an IP 3 -dependent Ca 2+ signal. The melatonin-induced cytosolic Ca 2+ ([Ca 2+ ] cyt ) increase and malaria cell cycle can be blocked by the IP 3 receptor blocker 2-aminoethyl diphenylborinate (2-APB). However, 2-APB also inhibits store-operated Ca 2+ entry (SOCE). Therefore, we have used two novel 2-APB derivatives, DPB162-AE and DPB163-AE, which are 100-fold more potent than 2-APB in blocking SOCE in mammalian cells, and appear to act by interfering with clustering of STIM proteins. In the present work we report that DPB162-AE and DPB163-AE block the [Ca 2+ ] cyt rise in response to melatonin in P. falciparum, but only at high concentrations. These compounds also block SOCE in the parasite at similarly high concentrations suggesting that P. falciparum SOCE is not activated in the same way as in mammalian cells. We further find that DPB162-AE and DPB163-AE affect the development of the intraerythrocytic parasites and invasion of new red blood cells. Our efforts to episomally express proteins that compete with native IP 3 receptor like IP 3 -sponge and an IP 3 sensor such as IRIS proved to be lethal to P. falciparum during intraerythrocytic cycle. The present findings point to an important role of IP 3 -induced Ca 2+ release in intraerythrocytic stage of P. falciparum. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

ChIP-PIT: Enhancing the Analysis of ChIP-Seq Data Using Convex-Relaxed Pair-Wise Interaction Tensor Decomposition.

PubMed

Zhu, Lin; Guo, Wei-Li; Deng, Su-Ping; Huang, De-Shuang

2016-01-01

In recent years, thanks to the efforts of individual scientists and research consortiums, a huge amount of chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) experimental data have been accumulated. Instead of investigating them independently, several recent studies have convincingly demonstrated that a wealth of scientific insights can be gained by integrative analysis of these ChIP-seq data. However, when used for the purpose of integrative analysis, a serious drawback of current ChIP-seq technique is that it is still expensive and time-consuming to generate ChIP-seq datasets of high standard. Most researchers are therefore unable to obtain complete ChIP-seq data for several TFs in a wide variety of cell lines, which considerably limits the understanding of transcriptional regulation pattern. In this paper, we propose a novel method called ChIP-PIT to overcome the aforementioned limitation. In ChIP-PIT, ChIP-seq data corresponding to a diverse collection of cell types, TFs and genes are fused together using the three-mode pair-wise interaction tensor (PIT) model, and the prediction of unperformed ChIP-seq experimental results is formulated as a tensor completion problem. Computationally, we propose efficient first-order method based on extensions of coordinate descent method to learn the optimal solution of ChIP-PIT, which makes it particularly suitable for the analysis of massive scale ChIP-seq data. Experimental evaluation the ENCODE data illustrate the usefulness of the proposed model.

Role of Specificity Protein-1 and Activating Protein-2 Transcription Factors in the Regulation of the Gap Junction Protein Beta-2 Gene in the Epididymis of the Rat.

PubMed

Adam, Cécile; Cyr, Daniel G

2016-06-01

In prepubertal rats, connexin 26 (GJB2) is expressed between adjacent columnar cells of the epididymis. At 28 days of age, when columnar cells differentiate into adult epithelial cell types, Gjb2 mRNA levels decrease to barely detectable levels. There is no information on the regulation of GJB2 in the epididymis. The present study characterized regulation of the Gjb2 gene promoter in the epididymis. A single transcription start site at position -3829 bp relative to the ATG was identified. Computational analysis revealed several TFAP2A, SP1, and KLF4 putative binding sites. A 1.5-kb fragment of the Gjb2 promoter was cloned into a vector containing a luciferase reporter gene. Transfection of the construct into immortalized rat caput epididymal (RCE-1) cells indicated that the promoter contained sufficient information to drive expression of the reporter gene. Deletion constructs showed that the basal activity of the promoter resides in the first -230 bp of the transcriptional start site. Two response elements necessary for GJB2 expression were identified: an overlapping TFAP2A/SP1 site (-136 to -126 bp) and an SP1 site (-50 bp). Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays confirmed that SP1 and TFAP2A were bound to the promoter. ChIP analysis of chromatin from young and pubertal rats indicated that TFAP2A and SP1 binding decreased with age. SP1 and TFAP2A knockdown indicated that SP1 is necessary for Gjb2 expression. DNA methylation did not appear to be involved in the regulation of Gjb2 expression. Results indicate that SP1 and TFAP2A regulate Gjb2 promoter activity during epididymal differentiation in rat. © 2016 by the Society for the Study of Reproduction, Inc.

myo-Inositol hexakisphosphate is a major component of an extracellular structure in the parasitic cestode Echinococcus granulosus.

PubMed Central

Irigoín, Florencia; Ferreira, Fernando; Fernández, Cecilia; Sim, Robert B; Díaz, Alvaro

2002-01-01

myo-Inositol hexakisphosphate (IP(6)) is an abundant intracellular component of animal cells. In this study we describe the presence of extracellular IP(6) in the hydatid cyst wall (HCW) of the larval stage of the cestode parasite Echinococcus granulosus. The HCW comprises an inner cellular layer and an outer, acellular (laminated) layer up to 2 mm in thickness that protects the parasite from host immune cells. A compound, subsequently identified as IP(6), was detected in and purified from an HCW extract on the basis of its capacity to inhibit complement activation. The identification of the isolated compound was carried out by a combination of NMR, MS and TLC. The majority of IP(6) in the HCW was found in the acellular layer, with only a small fraction of the compound being extracted from cells. In the laminated layer, IP(6) was present in association with calcium, and accounted for up to 15% of the total dry mass of the HCW. IP(6) was not detected in any other structures or stages of the parasite. Our results imply that IP(6) is secreted by the larval stage of the parasite in a polarized fashion towards the interface with the host. This is the first report of the secretion of IP(6), and the possible implications beyond the biology of E. granulosus are discussed. PMID:11853537

A Positive Feedback Loop between Glial Cells Missing 1 and Human Chorionic Gonadotropin (hCG) Regulates Placental hCGβ Expression and Cell Differentiation

PubMed Central

Cheong, Mei-Leng; Wang, Liang-Jie; Chuang, Pei-Yun; Chang, Ching-Wen; Lee, Yun-Shien; Lo, Hsiao-Fan; Tsai, Ming-Song

2015-01-01

Human chorionic gonadotropin (hCG) is composed of a common α subunit and a placenta-specific β subunit. Importantly, hCG is highly expressed in the differentiated and multinucleated syncytiotrophoblast, which is formed via trophoblast cell fusion and stimulated by cyclic AMP (cAMP). Although the ubiquitous activating protein 2 (AP2) transcription factors TFAP2A and TFAP2C may regulate hCGβ expression, it remains unclear how cAMP stimulates placenta-specific hCGβ gene expression and trophoblastic differentiation. Here we demonstrated that the placental transcription factor glial cells missing 1 (GCM1) binds to a highly conserved promoter region in all six hCGβ paralogues by chromatin immunoprecipitation-on-chip (ChIP-chip) analyses. We further showed that cAMP stimulates GCM1 and the CBP coactivator to activate the hCGβ promoter through a GCM1-binding site (GBS1), which also constitutes a previously identified AP2 site. Given that TFAP2C may compete with GCM1 for GBS1, cAMP enhances the association between the hCGβ promoter and GCM1 but not TFAP2C. Indeed, the hCG-cAMP-protein kinase A (PKA) signaling pathway also stimulates Ser269 and Ser275 phosphorylation of GCM1, which recruits CBP to mediate GCM1 acetylation and stabilization. Consequently, hCG stimulates the expression of GCM1 target genes, including the fusogenic protein syncytin-1, to promote placental cell fusion. Our study reveals a positive feedback loop between GCM1 and hCG regulating placental hCGβ expression and cell differentiation. PMID:26503785

Characteristics of cell lines established from human colorectal carcinoma.

PubMed

Park, J G; Oie, H K; Sugarbaker, P H; Henslee, J G; Chen, T R; Johnson, B E; Gazdar, A

1987-12-15

We have characterized 14 human colorectal carcinoma cell lines established from primary and metastatic sites by us during the years 1982 to 1985. Five lines were established in fully defined ACL-4 medium and 9 in serum supplemented R10 medium. However, after establishment, cultures could be grown interchangeably in either medium. The lines grew as floating cell aggregates in ACL-4 medium, while most demonstrated substrate adherence in R10 medium. The lines had relatively long doubling times and low cloning efficiencies. Twelve were tumorigenic in athymic nude mice when injected s.c., and two grew i.p. as well. Based on culture, xenograft, and ultrastructural morphologies, the 14 lines could be subtyped as follows: 4 were well differentiated; 5 were moderately differentiated; 4 were poorly differentiated; and 1 was a mucinous carcinoma. Membrane associated antigens characteristic for gastrointestinal cells (carcinoembryonic antigen, CA 19-9, and TAG-72 antigens) were expressed by 50-71% of the lines. Lines expressing carcinoembryonic antigen and CA 19-9 actively secreted these antigens into the supernatant fluids while TAG-72 antigen was not secreted. Surprisingly, 5 of 7 of the original tumor samples tested and 13 of 14 cultured lines expressed L-dopa decarboxylase activity, which is a characteristic enzyme marker of neuroendocrine cells and tumors. In addition, one poorly differentiated cell line contained dense core granules, characteristic of endocrine secretion. Preliminary cytogenetic analyses indicated that 9 of 11 lines examined contained double minute chromosomes. In addition, 3 of the 9 lines with double minutes also had homogeneously staining regions. These findings indicate a high incidence of amplification of one or more as yet unidentified genes.

[DAB2IP expression in bladder transitional cell carcinoma and its correlation with clinical outcome].

PubMed

Zhu, Jian-Ning; Wu, Kai-Jie; Guan, Zhen-Feng; Liu, Li-Xia; Ning, Zhong-Yun; Zhou, Jian-Cheng; Wang, Xin-Yang; Fan, Jin-Hai

2014-07-01

To investigate the expression of DAB2IP in bladder transitional cell carcinoma (BTCC) and its correlation with clinical characteristics and prognosis of BTCC patients. Immunohistochemical staining was applied to detect DAB2IP protein level in 79 cases of TCCB tissues and 11 cases of normal bladder tissues, and the relationships of the staining results with pathological grade, stage, lymph node metastasis, gender, age and the 3-year survival rate of the patients were analyzed. The expression of DAB2IP in BTCC tissues was significantly lower than that in normal bladder epithelium, and the expression score and rate of DAB2IP in the high-grade, invasive and metastatic BTCC were significantly lower than those in low-grade, superficial and non-metastatic BTCC (P < 0.05). The 3-year survival rate of the patients with high DAB2IP expression was significantly higher than that of the patients with low DAB2IP expression. DAB2IP may be one of the important inhibitory factors during the occurrence and progression of BTCC.

Characteristics of Human Turbinate-Derived Mesenchymal Stem Cells Are Not Affected by Allergic Condition of Donor

PubMed Central

Hwang, Se Hwan; Cho, Hye Kyung; Park, Sang Hi; Lee, WeonSun; Lee, Hee Jin; Lee, Dong Chang; Park, Sun Hwa; Lim, Mi Hyun; Back, Sang A; Yun, Byeong Gon; Sun, Dong Il

2015-01-01

The characteristics of mesenchymal stem cells (MSCs) derived from human turbinates (hTMSCs) have not been investigated in allergic rhinitis. We evaluated the influence of allergic state of the donor on the characteristics, proliferation, and differentiation potential of hTMSCs, compared with hTMSCs derived from non-allergic patients. hTMSCs were isolated from five non-allergic and five allergic patients. The expression of toll-like receptors (TLRs) in hTMSCs was measured by FACS, and cell proliferation was measured using a cell counting kit. Cytokine secretion was analyzed using multiplex immunoassays. The osteogenic, chondrogenic, and adipogenic differentiation potentials of hTMSCs were evaluated by histology and gene expression analysis. In allergic patients, FACS analysis showed that TLR3 and TLR4 were more highly expressed on the surface of hTMSCs than TLR2 and TLR5. The proliferation of hTMSCs was not influenced by the presence of TLR priming. The expression of IL-6, IL-8, IL-12, IP-10, and RANTES was upregulated after the TLR4 priming. The differentiation potential of hTMSCs was not influenced by TLR priming. These characteristics of hTMSCs were similar to those of hTMSCs from non-allergic patients. We conclude that the allergic condition of the donor does not influence TLR expression, proliferation, or immunomodulatory potential of hTMSCs. PMID:26376485

Epigenetically Modified Bone Marrow Stromal Cells in Silk Scaffolds Promote Craniofacial Bone Repair and Wound Healing.

PubMed

Han, Qianqian; Yang, Pishan; Wu, Yuwei; Meng, Shu; Sui, Lei; Zhang, Lan; Yu, Liming; Tang, Yin; Jiang, Hua; Xuan, Dongying; Kaplan, David L; Kim, Sung Hoon; Tu, Qisheng; Chen, Jake

2015-08-01

Epigenetic regulation of gene expression is a central mechanism that governs cell stemness, determination, commitment, and differentiation. It has been recently found that PHF8, a major H4K20/H3K9 demethylase, plays a critical role in craniofacial and bone development. In this study, we hypothesize that PHF8 promotes osteoblastogenesis by epigenetically regulating the expression of a nuclear matrix protein, special AT-rich sequence-binding protein 2 (SATB2) that plays pivotal roles in skeletal patterning and osteoblast differentiation. Our results showed that expression levels of PHF8 and SATB2 in preosteoblasts and bone marrow stromal cells (BMSCs) increased simultaneously during osteogenic induction. Overexpressing PHF8 in these cells upregulated the expression of SATB2, Runx2, osterix, and bone matrix proteins. Conversely, knockdown of PHF8 reduced the expression of these genes. Furthermore, ChIP assays confirmed that PHF8 specifically bound to the transcription start site (TSS) of the SATB2 promoter, and the expression of H3K9me1 at the TSS region of SATB2 decreased in PHF8 overexpressed group. Implantation of the BMSCs overexpressing PHF8 with silk protein scaffolds promoted bone regeneration in critical-sized defects in mouse calvaria. Taken together, our results demonstrated that PHF8 epigenetically modulates SATB2 activity, triggering BMSCs osteogenic differentiation and facilitating bone formation and regeneration in biodegradable silk scaffolds.

Prevalence of human papillomavirus, Epstein-Barr virus, p21, and p53 expression in sinonasal inverted papilloma, nasal polyp, and hypertrophied turbinate in Hong Kong patients.

PubMed

Sham, C L; To, K F; Chan, Paul K S; Lee, Dennis L Y; Tong, Michael C F; van Hasselt, C Andrew

2012-04-01

The purpose of this study of human papillomavirus (HPV), Epstein-Barr virus (EBV), p21, and p53 in sinonasal inverted papilloma (IP) was to help elucidate its pathogenesis. Seventy-three IPs, 48 nasal polyps, and 85 hypertrophied turbinates were subjected to HPV polymerase chain reaction (PCR) study. Seventy-three IPs, 30 nasal polyps, and 32 hypertrophied turbinates were subjected to EBV in situ hybridization (ISH), p21, and p53 immunohistochemical (IHC) studies. HPV was positive in 3 of 73 IPs (4.1%). All specimens were EBV negative. In all, 99% of IPs showed strong and diffuse p21 nuclear reactivity. Most nasal polyps and hypertrophied turbinates showed weak to moderate immunoreactivity of the basal and parabasal cells. Only focal p53 immunoreactivity of the basal and parabasal cells was found in 19% of IPs and 40% of nasal polyps. HPV prevalence of our IP is low. EBV is not present in IP. High p21 and low p53 expression in IP suggests a non-p53-dependent regulation pathway. Copyright © 2011 Wiley Periodicals, Inc.

Integrity of the LXXLL motif in Stat6 is required for the inhibition of breast cancer cell growth and enhancement of differentiation in the context of progesterone

PubMed Central

2014-01-01

Background Progesterone is essential for the proliferation and differentiation of mammary gland epithelium. Studies of breast cancer cells have demonstrated a biphasic progesterone response consisting of an initial proliferative burst followed by sustained growth arrest. However, the transcriptional factors acting with the progesterone receptor (PR) to mediate the effects of progesterone on mammary cell growth and differentiation remain to be determined. Recently, it was demonstrated that signal transducer and activator of transcription 6 (Stat6) is a cell growth suppressor. Similar to progesterone-bound PR, Stat6 acts by inducing the expression of the G1 cyclin-dependent kinase inhibitors p21 and p27. The possible interaction between Stat6 and progesterone pathways in mammary cells was therefore investigated in the present study. Methods ChIP and luciferase were assayed to determine whether Stat6 induces p21 and p27 expression by recruitment at the proximal Sp1-binding sites of the gene promoters. Immunoprecipitation and Western blotting were performed to investigate the interaction between Stat6 and PR-B. The cellular DNA content and cell cycle distribution in breast cancer cells were analyzed by FACS. Results We found that Stat6 interacts with progesterone-activated PR in T47D cells. Stat6 synergizes with progesterone-bound PR to transactivate the p21 and p27 gene promoters at the proximal Sp1-binding sites. Moreover, Stat6 overexpression and knockdown, respectively, increased or prevented the induction of p21 and p27 gene expression by progesterone. Stat6 knockdown also abolished the inhibitory effects of progesterone on pRB phosphorylation, G1/S cell cycle progression, and cell proliferation. In addition, knockdown of Stat6 expression prevented the induction of breast cell differentiation markers, previously identified as progesterone target genes. Finally, Stat6 gene expression levels increased following progesterone treatment, indicating a positive auto-regulatory loop between PR and Stat6. Conclusions Taken together, these data identify Stat6 as a coactivator of PR mediating the growth-inhibitory and differentiation effects of progesterone on breast cancer cells. PMID:24401087

Inhibitory effects of albuterol and fenoterol on RANTES and IP-10 expression in bronchial epithelial cells.

PubMed

Lam, Ka-Pan; Chu, Yu-Te; Lee, Min-Sheng; Chen, Huan-Nan; Wang, Wei-Li; Tok, Teck-Siang; Chin, Yow-Yue; Chen, Solomon Chih-Cheng; Kuo, Chang-Hung; Hung, Chih-Hsing

2011-06-01

Short-acting β2-adrenoreceptor agonist (SABA) is the major asthma reliever as indicated in the GINA guidelines. Regulated on activation, normal T expressed and secreted (RANTES) is a chemokine that attracts eosinophils, mast cells, and basophils toward site of allergic inflammation. Interferon γ-inducible protein (IP)-10 is a Th1-related chemokine that is also important in asthmatic inflammation and also involved in our immune defense against pathogens. Bronchial epithelial cells are first-line barrier against invasive pathogen and also have immunomodulatory function. However, whether albuterol and fenoterol (two SABAs) have modulatory effects on RANTES and IP-10 expression in bronchial epithelial cells is unknown. The human bronchial epithelial cell lines, BEAS-2B cells, were pre-treated with different concentrations of albuterol, fenoterol or dibutyryl-cAMP (a cyclic AMP analog) before polyinosinic-polycytidylic acid (poly I:C) stimulation. In some condition, BEAS-2B cells were pre-treated with ICI-118551, a selective β2-adrenoreceptor antagonist, 30 min before albuterol or fenoterol treatment. The levels of RANTES and IP-10 were measured by ELISA. Intracellular signaling was investigated using cAMP assay, mitogen-activated protein kinase (MAPK) inhibitor, nuclear factor (NF)-κB inhibitor, and western blot. Albuterol and fenoterol suppressed poly I:C-induced RANTES and IP-10 expression of BEAS-2B cells. ICI-118551 could partly reverse the suppressive effects of albuterol and fenoterol on RANTES and IP-10 expression. Albuterol and fenoterol increased intracellular cAMP levels. Dibutyryl-cAMP conferred the similar effects of albuterol and fenoterol. Western blot revealed that albuterol suppressed p-ERK, p-JNK and pp38, and also their associated kinase expression. Albuterol had no effect on pp65 expression. Albuterol and fenoterol could suppress poly I:C-induced RANTES and IP-10 expression in human bronchial epithelial cells via at least partly the β2-adrenoreceptor-cAMP and the MAPK pathways, implicating that albuterol and fenoterol could exert anti-inflammatory effect and benefit asthmatic patients by suppressing RANTES and IP-10 expression. However, these suppressive effects of albuterol and fenoterol may inhibit the defense against viral infection. © 2010 John Wiley & Sons A/S.

Anti-inflammatory effects of theophylline, cromolyn and salbutamol in a murine model of pleurisy.

PubMed Central

Saleh, T. S.; Calixto, J. B.; Medeiros, Y. S.

1996-01-01

1. The aim of this study was to examine the effect of theophylline, cromolyn and salbutamol, three well-known anti-asthmatic drugs, on the early (4 h) and late (48 h) phases of cell migration and fluid leakage induced by carrageenin in the pleural cavity of mice. 2. In the first set of experiments, animals were pretreated (30 min) with different doses of theophylline (0.5-50 mg kg-1, i.p.), cromolyn (0.02-0.2 mg per pleural cavity) or salbutamol (0.05-50 mg kg-1, i.p.); the total and differential cell content, and also the exudate were analysed 4 h after carrageenin (1%) administration. Afterwards, in order to evaluate the time course effects of these drugs on both phases of the inflammatory reaction, one dose employed in the above protocol was chosen, to pretreat (0.5-24 h) different groups of animals. The studied parameters were evaluated 4 and 48 h after pleurisy induction. 3. Acute administration of theophylline (1-50 mg kg-1, i.p.) cromolyn (0.02-0.2 mg per pleural cavity) and salbutamol (0.5-50 mg kg-1, i.p.), 30 min prior to carrageenin, caused significant inhibition of total cell and fluid leakage in the pleural cavity at 4 h (P < 0.01). All drugs exerted a long-lasting inhibitory effect on both exudation and cell migration (P < 0.01) when administered 0.5-8 h before pleurisy induction. However, the temporal profile of the inhibitory effect induced by these drugs on the first phase of the inflammatory reaction was clearly different. Thus, the inhibitory effect induced by theophylline and cromolyn on exudation was significantly longer (up to 24 h) in comparison to their effects on cell migration (only up to 8 h). In contrast, although salbutamol when administered 30 min before pleurisy induction abolished fluid leakage (P < 0.01), this effect was not sustained in the groups pretreated for 4-8 h. In these latter groups, a significant but much smaller reduction of exudation was observed (P < 0.01), whereas the magnitude of cell migration inhibition did not vary. 4. The second phase (48 h) of the inflammatory reaction induced by carrageenin (1%) was significantly inhibited by cromolyn (0.02 mg per pleural cavity) when this drug was administered 0.5-24 h before pleurisy induction (P < 0.01). Similar results were observed when theophylline (50 mg kg-1, i.p.) was administered 0.5-4 h before the injection of the phlogistic agent (P < 0.01). Treatment of the animals with salbutamol (5 mg kg-1, i.p.), 0.5-24 h before pleurisy induction, did not inhibit either cell migration or fluid leakage. In this condition, a significant increase of these parameters was observed in the group pretreated with salbutamol 8-24 h before pleurisy induction (P < 0.01). 5. These results indicate that theophylline and cromolyn were able to inhibit the early (4 h) and late (48 h) phases of the inflammatory reaction induced by carrageenin in a murine model of pleurisy. Salbutamol was effective only against the early phase. The inhibitory effects of theophylline, cromolyn and salbutamol on the early phase of this inflammatory reaction were long-lasting, although a distinct profile of inhibition was observed among them. These findings confirm and extend previous results described in other models of asthma and support both clinical and experimental evidence suggesting that these anti-asthmatic agents exhibit marked anti-inflammatory properties. PMID:8762112

The Histone Acetyltransferase MOF is a Key Regulator of the Embryonic Stem Cell Core Transcriptional Network

PubMed Central

Li, Xiangzhi; Li, Li; Pandey, Ruchi; Byun, Jung S.; Gardner, Kevin; Qin, Zhaohui; Dou, Yali

2012-01-01

SUMMARY Pluripotent embryonic stem cells (ESCs) maintain self-renewal and the potential for rapid response to differentiation cues. Both ESC features are subject to epigenetic regulation. Here we show that histone acetyltransferase Mof plays an essential role in the maintenance of ESC self-renewal and pluripotency. ESCs with Mof deletion lose characteristic morphology, alkaline phosphatase (AP) staining and differentiation potential. They also have aberrant expression of core transcription factors Nanog, Oct4 and Sox2. Importantly, the phenotypes of Mof null ESCs can be partially suppressed by Nanog overexpression, supporting that Mof functions as an upstream regulator of Nanog in ESCs. Genome-wide ChIP sequencing and transcriptome analyses further demonstrate that Mof is an integral component of ESC core transcription network and Mof primes genes for diverse developmental programs. Mof is also required for Wdr5 recruitment and H3 K4 methylation at key regulatory loci, highlighting complexity and interconnectivity of various chromatin regulators in ESCs. PMID:22862943

miR-14 regulates autophagy during developmental cell death by targeting ip3-kinase 2.

PubMed

Nelson, Charles; Ambros, Victor; Baehrecke, Eric H

2014-11-06

Macroautophagy (autophagy) is a lysosome-dependent degradation process that has been implicated in age-associated diseases. Autophagy is involved in both cell survival and cell death, but little is known about the mechanisms that distinguish its use during these distinct cell fates. Here, we identify the microRNA miR-14 as being both necessary and sufficient for autophagy during developmentally regulated cell death in Drosophila. Loss of miR-14 prevented induction of autophagy during salivary gland cell death, but had no effect on starvation-induced autophagy in the fat body. Moreover, misexpression of miR-14 was sufficient to prematurely induce autophagy in salivary glands, but not in the fat body. Importantly, miR-14 regulates this context-specific autophagy through its target, inositol 1,4,5-trisphosphate kinase 2 (ip3k2), thereby affecting inositol 1,4,5-trisphosphate (IP3) signaling and calcium levels during salivary gland cell death. This study provides in vivo evidence of microRNA regulation of autophagy through modulation of IP3 signaling. Copyright © 2014 Elsevier Inc. All rights reserved.

DNA damage response curtails detrimental replication stress and chromosomal instability induced by the dietary carcinogen PhIP

PubMed Central

Mimmler, Maximilian; Peter, Simon; Kraus, Alexander; Stroh, Svenja; Nikolova, Teodora; Seiwert, Nina; Hasselwander, Solveig; Neitzel, Carina; Haub, Jessica; Monien, Bernhard H.; Nicken, Petra; Steinberg, Pablo; Shay, Jerry W.; Kaina, Bernd; Fahrer, Jörg

2016-01-01

PhIP is an abundant heterocyclic aromatic amine (HCA) and important dietary carcinogen. Following metabolic activation, PhIP causes bulky DNA lesions at the C8-position of guanine. Although C8-PhIP-dG adducts are mutagenic, their interference with the DNA replication machinery and the elicited DNA damage response (DDR) have not yet been studied. Here, we analyzed PhIP-triggered replicative stress and elucidated the role of the apical DDR kinases ATR, ATM and DNA-PKcs in the cellular defense response. First, we demonstrate that PhIP induced C8-PhIP-dG adducts and DNA strand breaks. This stimulated ATR-CHK1 signaling, phosphorylation of histone 2AX and the formation of RPA foci. In proliferating cells, PhIP treatment increased the frequency of stalled replication forks and reduced fork speed. Inhibition of ATR in the presence of PhIP-induced DNA damage strongly promoted the formation of DNA double-strand breaks, activation of the ATM-CHK2 pathway and hyperphosphorylation of RPA. The abrogation of ATR signaling potentiated the cell death response and enhanced chromosomal aberrations after PhIP treatment, while ATM and DNA-PK inhibition had only marginal effects. These results strongly support the notion that ATR plays a key role in the defense against cancer formation induced by PhIP and related HCAs. PMID:27599846

Generation of the SCN1A epilepsy mutation in hiPS cells using the TALEN technique

NASA Astrophysics Data System (ADS)

Chen, Wanjuan; Liu, Jingxin; Zhang, Longmei; Xu, Huijuan; Guo, Xiaogang; Deng, Sihao; Liu, Lipeng; Yu, Daiguan; Chen, Yonglong; Li, Zhiyuan

2014-06-01

Human induced pluripotent stem cells (iPSC) can be used to understand the pathological mechanisms of human disease. These cells are a promising source for cell-replacement therapy. However, such studies require genetically defined conditions. Such genetic manipulations can be performed using the novel Transcription Activator-Like Effector Nucleases (TALENs), which generate site-specific double-strand DNA breaks (DSBs) with high efficiency and precision. Combining the TALEN and iPSC methods, we developed two iPS cell lines by generating the point mutation A5768G in the SCN1A gene, which encodes the voltage-gated sodium channel Nav1.1 α subunit. The engineered iPSC maintained pluripotency and successfully differentiated into neurons with normal functional characteristics. The two cell lines differ exclusively at the epilepsy-susceptibility variant. The ability to robustly introduce disease-causing point mutations in normal hiPS cell lines can be used to generate a human cell model for studying epileptic mechanisms and for drug screening.

Ten years since the discovery of iPS cells: The current state of their clinical application.

PubMed

Aznar, J; Tudela, J

On the 10-year anniversary of the discovery of induced pluripotent stem cells, we review the main results from their various fields of application, the obstacles encountered during experimentation and the potential applications in clinical practice. The efficacy of induced pluripotent cells in clinical experimentation can be equated to that of human embryonic stem cells; however, unlike stem cells, induced pluripotent cells do not involve the severe ethical difficulties entailed by the need to destroy human embryos to obtain them. The finding of these cells, which was in its day a true scientific milestone worthy of a Nobel Prize in Medicine, is currently enveloped by light and shadow: high hopes for regenerative medicine versus the, as of yet, poorly controlled risks of unpredictable reactions, both in the processes of dedifferentiation and subsequent differentiation to the cell strains employed for therapeutic or experimentation goals. Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Medicina Interna (SEMI). All rights reserved.

Comparative epigenetic influence of autologous versus fetal bovine serum on mesenchymal stem cells through in vitro osteogenic and adipogenic differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fani, Nesa; Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran; Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran

Mesenchymal stem cells (MSCs) derived from bone marrow (BM) represents a useful source of adult stem cells for cell therapy and tissue engineering. MSCs are present at a low frequency in the BM; therefore expansion is necessary before performing clinical studies. Fetal bovine serum (FBS) as a nutritional supplement for in vitro culture of MSCs is a suitable additive for human cell culture, but not regarding subsequent use of these cells for clinical treatment of human patients due to the risk of viral and prion transmission as well as xenogeneic immune responses after transplantation. Recently, autologous serum (AS) has beenmore » as a supplement to replace FBS in culture medium. We compared the effect of FBS versus AS on the histone modification pattern of MSCs through in vitro osteogenesis and adipogenesis. Differentiation of stem cells under various serum conditions to a committed state involves global changes in epigenetic patterns that are critically determined by chromatin modifications. Chromatin immunoprecipitation (ChIP) coupled with real-time PCR showed significant changes in the acetylation and methylation patterns in lysine 9 (Lys9) of histone H3 on the regulatory regions of stemness (Nanog, Sox2, Rex1), osteogenic (Runx2, Oc, Sp7) and adipogenic (Ppar-γ, Lpl, adiponectin) marker genes in undifferentiated MSCs, FBS and AS. All epigenetic changes occurred in a serum dependent manner which resulted in higher expression level of stemness genes in undifferentiated MSCs compared to differentiated MSCs and increased expression levels of osteogenic genes in AS compared to FBS. Adipogenic genes showed greater expression in FBS compared to AS. These findings have demonstrated the epigenetic influence of serum culture conditions on differentiation potential of MSCs, which suggest that AS is possibly more efficient serum for osteogenic differentiation of MSCs in cell therapy purposes. - Highlights: • Bone marrow derived MSC could proliferate in AS as well as in FBS. • Different serum conditions influence epigentic patterns of genes. • Osteogenic genes specifically up-regulate in AS in response to differentiation signals.« less

A CI-Independent Form of Replicative Inhibition: Turn Off of Early Replication of Bacteriophage Lambda

PubMed Central

Hayes, Sidney; Horbay, Monique A.; Hayes, Connie

2012-01-01

Several earlier studies have described an unusual exclusion phenotype exhibited by cells with plasmids carrying a portion of the replication region of phage lambda. Cells exhibiting this inhibition phenotype (IP) prevent the plating of homo-immune and hybrid hetero-immune lambdoid phages. We have attempted to define aspects of IP, and show that it is directed to repλ phages. IP was observed in cells with plasmids containing a λ DNA fragment including oop, encoding a short OOP micro RNA, and part of the lambda origin of replication, oriλ, defined by iteron sequences ITN1-4 and an adjacent high AT-rich sequence. Transcription of the intact oop sequence from its promoter, pO is required for IP, as are iterons ITN3–4, but not the high AT-rich portion of oriλ. The results suggest that IP silencing is directed to theta mode replication initiation from an infecting repλ genome, or an induced repλ prophage. Phage mutations suppressing IP, i.e., Sip, map within, or adjacent to cro or in O, or both. Our results for plasmid based IP suggest the hypothesis that there is a natural mechanism for silencing early theta-mode replication initiation, i.e. the buildup of λ genomes with oop + oriλ+ sequence. PMID:22590552

Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins.

PubMed

Fujita, Toshitsugu; Yuno, Miyuki; Fujii, Hodaka

2016-04-01

The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for various biological applications, including genome editing. We developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR to isolate target genomic regions from cells for their biochemical characterization. In this study, we developed 'in vitro enChIP' using recombinant CRISPR ribonucleoproteins (RNPs) to isolate target genomic regions. in vitro enChIP has the great advantage over conventional enChIP of not requiring expression of CRISPR complexes in cells. We first showed that in vitro enChIP using recombinant CRISPR RNPs can be used to isolate target DNA from mixtures of purified DNA in a sequence-specific manner. In addition, we showed that this technology can be used to efficiently isolate target genomic regions, while retaining their intracellular molecular interactions, with negligible contamination from irrelevant genomic regions. Thus, in vitro enChIP technology is of potential use for sequence-specific isolation of DNA, as well as for identification of molecules interacting with genomic regions of interest in vivo in combination with downstream analysis. © 2016 The Authors. Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Melanopsin-expressing retinal ganglion-cell photoreceptors: cellular diversity and role in pattern vision

PubMed Central

Ecker, Jennifer L.; Dumitrescu, Olivia N.; Wong, Kwoon Y.; Alam, Nazia M.; Chen, Shih-Kuo; LeGates, Tara; Renna, Jordan M.; Prusky, Glen T.; Berson, David M.; Hattar, Samer

2010-01-01

Using the photopigment melanopsin, intrinsically photosensitive retinal ganglion cells (ipRGCs) respond directly to light to drive circadian clock resetting and pupillary constriction. We now report that ipRGCs are more abundant and diverse than previously appreciated, project more widely within the brain, and can support spatial visual perception. A Cre-based melanopsin reporter mouse line revealed at least five subtypes of ipRGCs with distinct morphological and physiological characteristics. Collectively, these cells project beyond the known brain targets of ipRGCs to heavily innervate the superior colliculus and dorsal lateral geniculate nucleus, retinotopically-organized nuclei mediating object localization and discrimination. Mice lacking classical rod-cone photoreception, and thus entirely dependent on melanopsin for light detection, were able to discriminate grating stimuli from equiluminant gray, and had measurable visual acuity. Thus, non-classical retinal photoreception occurs within diverse cell types, and influences circuits and functions encompassing luminance as well as spatial information. PMID:20624591

Practical Integration-Free Episomal Methods for Generating Human Induced Pluripotent Stem Cells.

PubMed

Kime, Cody; Rand, Tim A; Ivey, Kathryn N; Srivastava, Deepak; Yamanaka, Shinya; Tomoda, Kiichiro

2015-10-06

The advent of induced pluripotent stem (iPS) cell technology has revolutionized biomedicine and basic research by yielding cells with embryonic stem (ES) cell-like properties. The use of iPS-derived cells for cell-based therapies and modeling of human disease holds great potential. While the initial description of iPS cells involved overexpression of four transcription factors via viral vectors that integrated within genomic DNA, advances in recent years by our group and others have led to safer and higher quality iPS cells with greater efficiency. Here, we describe commonly practiced methods for non-integrating induced pluripotent stem cell generation using nucleofection of episomal reprogramming plasmids. These methods are adapted from recent studies that demonstrate increased hiPS cell reprogramming efficacy with the application of three powerful episomal hiPS cell reprogramming factor vectors and the inclusion of an accessory vector expressing EBNA1. Copyright © 2015 John Wiley & Sons, Inc.

Generation of transgene-free induced pluripotent stem cells with non-viral methods.

PubMed

Wang, Tao; Zhao, Hua-shan; Zhang, Qiu-ling; Xu, Chang-lin; Liu, Chang-bai

2013-03-01

Induced pluripotent stem (iPS) cells were originally generated from mouse fibroblasts by enforced expression of Yamanaka factors (Oct3/4, Sox2, Klf4, and c-Myc). The technique was quickly reproduced with human fibroblasts or mesenchymal stem cells. Although having been showed therapeutic potential in animal models of sickle cell anemia and Parkinson's disease, iPS cells generated by viral methods do not suit all the clinical applications. Various non-viral methods have appeared in recent years for application of iPS cells in cell transplantation therapy. These methods mainly include DNA vector-based approaches, transfection of mRNA, and transduction of reprogramming proteins. This review summarized these non-viral methods and compare the advantages, disadvantages, efficiency, and safety of these methods.

Rapid increase of inositol 1,4,5-trisphosphate in the HeLa cells after hypergravity exposure

NASA Technical Reports Server (NTRS)

Kumei, Yasuhiro; Whitson, Peggy A.; Cintron, Nitza M.; Sato, Atsushige

1990-01-01

The IP3 level in HeLa cells has been elevated through the application in hypergravity in a time-dependent manner. The data obtained for the hydrolytic products of PIP2, IP3, and DG are noted to modulate c-myc gene expression. It is also established that the cAMP accumulation by the IBMX in hypergravity-exposed cells was suppressed relative to the control. In light of IP3 increase and cAMP decrease results, a single GTP-binding protein may play a role in the hypergravity signal transduction of HeLa cells by stimulating PLC while inhibiting adenylate cyclase.

HOXA5 determines cell fate transition and impedes tumor initiation and progression in breast cancer through regulation of E-cadherin and CD24

PubMed Central

Teo, Wei Wen; Merino, Vanessa F.; Cho, Soonweng; Korangath, Preethi; Liang, Xiaohui; Wu, Ren-chin; Neumann, Neil M.; Ewald, Andrew J.; Sukumar, Saraswati

2016-01-01

Loss of HOXA5 expression occurs frequently in breast cancer and correlates with higher pathological grade and poorer disease outcome. However, how HOX proteins drive differentiation in mammalian cells is poorly understood. In this paper, we investigated cellular and molecular consequences of loss of HOXA5 in breast cancer, and the role played by retinoic acid in HOXA5 function. Analysis of global gene expression data from HOXA5-depleted MCF10A breast epithelial cells, followed by validation, pointed to a role for HOXA5 in maintaining several molecular traits typical of the epithelial lineage such as cell-cell adhesion, tight junctions and markers of differentiation. Depleting HOXA5 in immortalized MCF10A or transformed MCF10A-Kras cells reduced their CD24+/CD44lo population, enhanced self-renewal capacity, and reduced expression of E-cadherin (CDH1) and CD24. In the case of MCF10A-Kras, HOXA5 loss increased branching and protrusive morphology in Matrigel, all features suggestive of epithelial to basal transition. Further, orthotopically implanted xenografts of MCF10A-Kras-scr grew as well-differentiated pseudo-luminal carcinomas, while MCF10A-Kras-shHOXA5 cells formed aggressive, poorly differentiated carcinomas. Conversely, ectopic expression of HOXA5 in aggressive SUM149 or SUM159 breast cancer cells reversed the cellular and molecular alterations observed in the HOXA5-depleted cells. Retinoic acid is a known upstream regulator of HOXA5 expression. HOXA5 depletion in MCF10A cells engineered to express doxycycline-induced shHOXA5 slowed transition of cells from a less differentiated CD24−/CD44+ to the more differentiated CD24+/CD44+ state. This transition was promoted by retinal treatment which upregulated endogenous HOXA5 expression, and caused re-expression of, Occludin, and claudin-7 (CLDN7). Expression of CDH1 and CD24 was transcriptionally upregulated by direct binding of HOXA5 to their promoter sequences as demonstrated by luciferase and ChIP analyses. Thus, loss of HOXA5 in mammary cells leads to loss of epithelial traits, an increase in stemness and cell plasticity, and the acquisition of more aggressive phenotypes. PMID:27157614

Lack of galectin-3 up-regulates IgA expression by peritoneal B1 lymphocytes during B cell differentiation.

PubMed

Oliveira, Felipe L; Bernardes, Emerson S; Brand, Camila; dos Santos, Sofia N; Cabanel, Mariana P; Arcanjo, Kátia D; Brito, José M; Borojevic, Radovan; Chammas, Roger; El-Cheikh, Márcia C

2016-02-01

Galectin-3 is a β-galactoside-binding protein with an inhibitory role in B cell differentiation into plasma cells in distinct lymphoid tissues. We use a model of chronic schistosomiasis, a well-characterized experimental disease hallmarked by polyclonal B cell activation, in order to investigate the role of galectin-3 in controlling IgA production through peritoneal B1 cells. Chronically infected, galectin-3-deficient mice (Lgals3(-/-)) display peritoneal fluid hypercellularity, increased numbers of atypical peritoneal IgM(+)/IgA(+) B1a and B1b lymphocytes and histological disturbances in plasma cell niches when compared with Lgals3(+/+) mice. Similar to our infection model, peritoneal B1 cells from uninfected Lgals3(-/-) mice show enhanced switching to IgA after in vitro treatment with interleukin-5 plus transforming growth factor-β (IL-5 + TGF-β1). A higher number of IgA(+) B1a lymphocytes was found in the peritoneal cavity of Lgals3(-/-)-uninfected mice at 1 week after i.p. injection of IL-5 + TGF-β1; this correlates with the increased levels of secreted IgA detected in the peritoneal fluid of these mice after cytokine treatment. Interestingly, a higher number of degranulated mast cells is present in the peritoneal cavity of uninfected and Schistosoma mansoni-infected Lgals3(-/-) mice, indicating that, at least in part, mast cells account for the enhanced differentiation of B1 into IgA-producing B cells found in the absence of galectin-3. Thus, a novel role is revealed for galectin-3 in controlling the expression of surface IgA by peritoneal B1 lymphocytes; this might have important implications for manipulating the mucosal immune response.

Selective dopamine receptor 4 activation mediates the hippocampal neuronal calcium response via IP3 and ryanodine receptors.

PubMed

Wang, Ya-Li; Wang, Jian-Gang; Guo, Fang-Li; Gao, Xia-Huan; Zhao, Dan-Dan; Zhang, Lin; Wang, Jian-Zhi; Lu, Cheng-Biao

2017-09-01

Intracellular calcium is a key factor in most cellular processes, including cell growth, differentiation, proliferation and neurotransmitter release. Dopamine (DA) mediates synaptic transmission by regulating the intracellular calcium content. It is not clear, however, which specific subunit of the DA receptor contributes to DA modulation of intracellular calcium content changes. Through the traditional technique of Fura-2 calcium imaging, this study demonstrated that the DA can induce transient calcium in cultured hippocampal neurons and that this response can be mimicked by a selective dopamine receptor 4 (DR4) agonist PD168077 (PD). PD-induced calcium transience can be blocked by a calcium chelator, such as BAPTA-AM, or by pre-treatment of neurons with thapsigargin, a IP 3 receptor antagonist, or a micromolar concentration of ryanodine, a ryanodine receptor (RyR) antagonist. However PD-induced calcium transience cannot be blocked by pre-treatment of neurons with a free-calcium medium or a cocktail of NMDA receptor, L-type calcium channel and alpha7 nicotinic acetylcholine receptor blockers. These results indicate that the calcium response induced by DR4 activation is mainly through activation of IP 3 receptor in internal stores, which is likely to contribute to the DA modulation of synaptic transmission and cognitive function. Copyright © 2017. Published by Elsevier B.V.

Active medulloblastoma enhancers reveal subgroup-specific cellular origins

PubMed Central

Lin, Charles Y.; Erkek, Serap; Tong, Yiai; Yin, Linlin; Federation, Alexander J.; Zapatka, Marc; Haldipur, Parthiv; Kawauchi, Daisuke; Risch, Thomas; Warnatz, Hans-Jörg; Worst, Barbara C.; Ju, Bensheng; Orr, Brent A.; Zeid, Rhamy; Polaski, Donald R.; Segura-Wang, Maia; Waszak, Sebastian M.; Jones, David T.W.; Kool, Marcel; Hovestadt, Volker; Buchhalter, Ivo; Sieber, Laura; Johann, Pascal; Chavez, Lukas; Gröschel, Stefan; Ryzhova, Marina; Korshunov, Andrey; Chen, Wenbiao; Chizhikov, Victor V.; Millen, Kathleen J.; Amstislavskiy, Vyacheslav; Lehrach, Hans; Yaspo, Marie-Laure; Eils, Roland; Lichter, Peter; Korbel, Jan O.; Pfister, Stefan M.; Bradner, James E.; Northcott, Paul A.

2016-01-01

Summary Medulloblastoma is a highly malignant paediatric brain tumour, often inflicting devastating consequences on the developing child. Genomic studies have revealed four distinct molecular subgroups with divergent biology and clinical behaviour. An understanding of the regulatory circuitry governing the transcriptional landscapes of medulloblastoma subgroups, and how this relates to their respective developmental origins, is lacking. Using H3K27ac and BRD4 ChIP-Seq, coupled with tissue-matched DNA methylation and transcriptome data, we describe the active cis-regulatory landscape across 28 primary medulloblastoma specimens. Analysis of differentially regulated enhancers and super-enhancers reinforced inter-subgroup heterogeneity and revealed novel, clinically relevant insights into medulloblastoma biology. Computational reconstruction of core regulatory circuitry identified a master set of transcription factors, validated by ChIP-Seq, that are responsible for subgroup divergence and implicate candidate cells-of-origin for Group 4. Our integrated analysis of enhancer elements in a large series of primary tumour samples reveals insights into cis-regulatory architecture, unrecognized dependencies, and cellular origins. PMID:26814967

IP-FCM measures physiologic protein-protein interactions modulated by signal transduction and small-molecule drug inhibition.

PubMed

Smith, Stephen E P; Bida, Anya T; Davis, Tessa R; Sicotte, Hugues; Patterson, Steven E; Gil, Diana; Schrum, Adam G

2012-01-01

Protein-protein interactions (PPI) mediate the formation of intermolecular networks that control biological signaling. For this reason, PPIs are of outstanding interest in pharmacology, as they display high specificity and may represent a vast pool of potentially druggable targets. However, the study of physiologic PPIs can be limited by conventional assays that often have large sample requirements and relatively low sensitivity. Here, we build on a novel method, immunoprecipitation detected by flow cytometry (IP-FCM), to assess PPI modulation during either signal transduction or pharmacologic inhibition by two different classes of small-molecule compounds. First, we showed that IP-FCM can detect statistically significant differences in samples possessing a defined PPI change as low as 10%. This sensitivity allowed IP-FCM to detect a PPI that increases transiently during T cell signaling, the antigen-inducible interaction between ZAP70 and the T cell antigen receptor (TCR)/CD3 complex. In contrast, IP-FCM detected no ZAP70 recruitment when T cells were stimulated with antigen in the presence of the src-family kinase inhibitor, PP2. Further, we tested whether IP-FCM possessed sufficient sensitivity to detect the effect of a second, rare class of compounds called SMIPPI (small-molecule inhibitor of PPI). We found that the first-generation non-optimized SMIPPI, Ro-26-4550, inhibited the IL-2:CD25 interaction detected by IP-FCM. This inhibition was detectable using either a recombinant CD25-Fc chimera or physiologic full-length CD25 captured from T cell lysates. Thus, we demonstrate that IP-FCM is a sensitive tool for measuring physiologic PPIs that are modulated by signal transduction and pharmacologic inhibition.

Evolution of a Novel Antiviral Immune-Signaling Interaction by Partial-Gene Duplication

PubMed Central

Korithoski, Bryan; Kolaczkowski, Oralia; Mukherjee, Krishanu; Kola, Reema; Earl, Chandra; Kolaczkowski, Bryan

2015-01-01

The RIG-like receptors (RLRs) are related proteins that identify viral RNA in the cytoplasm and activate cellular immune responses, primarily through direct protein-protein interactions with the signal transducer, IPS1. Although it has been well established that the RLRs, RIG-I and MDA5, activate IPS1 through binding between the twin caspase activation and recruitment domains (CARDs) on the RLR and a homologous CARD on IPS1, it is less clear which specific RLR CARD(s) are required for this interaction, and almost nothing is known about how the RLR-IPS1 interaction evolved. In contrast to what has been observed in the presence of immune-modulating K63-linked polyubiquitin, here we show that—in the absence of ubiquitin—it is the first CARD domain of human RIG-I and MDA5 (CARD1) that binds directly to IPS1 CARD, and not the second (CARD2). Although the RLRs originated in the earliest animals, both the IPS1 gene and the twin-CARD domain architecture of RIG-I and MDA5 arose much later in the deuterostome lineage, probably through a series of tandem partial-gene duplication events facilitated by tight clustering of RLRs and IPS1 in the ancestral deuterostome genome. Functional differentiation of RIG-I CARD1 and CARD2 appears to have occurred early during this proliferation of RLR and related CARDs, potentially driven by adaptive coevolution between RIG-I CARD domains and IPS1 CARD. However, functional differentiation of MDA5 CARD1 and CARD2 occurred later. These results fit a general model in which duplications of protein-protein interaction domains into novel gene contexts could facilitate the expansion of signaling networks and suggest a potentially important role for functionally-linked gene clusters in generating novel immune-signaling pathways. PMID:26356745

Human Induced Pluripotent Stem Cell–Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation

PubMed Central

Sharmin, Sazia; Taguchi, Atsuhiro; Kaku, Yusuke; Yoshimura, Yasuhiro; Ohmori, Tomoko; Sakuma, Tetsushi; Mukoyama, Masashi; Yamamoto, Takashi; Kurihara, Hidetake

2016-01-01

Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator–like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in vitro. These induced human podocytes exhibited apicobasal polarity, with nephrin proteins accumulated close to the basal domain, and possessed primary processes that were connected with slit diaphragm–like structures. Microarray analysis of sorted iPS cell–derived podocytes identified well conserved marker gene expression previously shown in mouse and human podocytes in vivo. Furthermore, we developed a novel transplantation method using spacers that release the tension of host kidney capsules, thereby allowing the effective formation of glomeruli from human iPS cell–derived nephron progenitors. The human glomeruli were vascularized with the host mouse endothelial cells, and iPS cell–derived podocytes with numerous cell processes accumulated around the fenestrated endothelial cells. Therefore, the podocytes generated from iPS cells retain the podocyte-specific molecular and structural features, which will be useful for dissecting human glomerular development and diseases. PMID:26586691

Network analysis of ChIP-Seq data reveals key genes in prostate cancer.

PubMed

Zhang, Yu; Huang, Zhen; Zhu, Zhiqiang; Liu, Jianwei; Zheng, Xin; Zhang, Yuhai

2014-09-03

Prostate cancer (PC) is the second most common cancer among men in the United States, and it imposes a considerable threat to human health. A deep understanding of its underlying molecular mechanisms is the premise for developing effective targeted therapies. Recently, deep transcriptional sequencing has been used as an effective genomic assay to obtain insights into diseases and may be helpful in the study of PC. In present study, ChIP-Seq data for PC and normal samples were compared, and differential peaks identified, based upon fold changes (with P-values calculated with t-tests). Annotations of these peaks were performed. Protein-protein interaction (PPI) network analysis was performed with BioGRID and constructed with Cytoscape, following which the highly connected genes were screened. We obtained a total of 5,570 differential peaks, including 3,726 differentially enriched peaks in tumor samples and 1,844 differentially enriched peaks in normal samples. There were eight significant regions of the peaks. The intergenic region possessed the highest score (51%), followed by intronic (31%) and exonic (11%) regions. The analysis revealed the top 35 highly connected genes, which comprised 33 differential genes (such as YWHAQ, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein and θ polypeptide) from ChIP-Seq data and 2 differential genes retrieved from the PPI network: UBA52 (ubiquitin A-52 residue ribosomal protein fusion product (1) and SUMO2 (SMT3 suppressor of mif two 3 homolog (2) . Our findings regarding potential PC-related genes increase the understanding of PC and provides direction for future research.

Immunopotentiator from Pantoea agglomerans 1 (IP-PA1) Promotes Murine Hair Growth and Human Dermal Papilla Cell Gene Expression.

PubMed

Wakame, Koji; Okawa, Hiroshi; Komatsu, Ken-Ich; Nakata, Akifumi; Sato, Keisuke; Ingawa, Hiroyuki; Kohchi, Chie; Nishizawa, Takashi; Soma, Gen-Ichiro

2016-07-01

The lipopolysaccharide (LPS)-like compound derived from Pantoea agglomerans (immunopotentiator from Pantoea agglomerans 1 (IP-PA1)) has been used not only as dietary supplement or cosmetic for humans, but also by Japanese veterinarians as an anti-tumor, anti-allergy, "keep a fine coat of fur" and hair growth-promoting functional food for dogs and cats. In the present study, we focused on the hair growth-promoting effects of IP-PA1 on a hair-shaved animal model and its mechanism of action. We also investigated its potential on gene expression after stimulating human dermal papilla cells with IP-PA1. The hair on the back of a C3H/HeN mouse was shaved and IP-PA1 was orally administered or applied to the skin. The status of hair growth was observed and recorded for 14 days. Skin was collected and histological tissue examination was performed with respect to hair growth status using hematoxylin and eosin staining. After IP-PA1 administration (2 and 10 μg/ml) to human dermal papilla cell culture system for 24 h, fibroblast growth factor-7 (FGF-7) and vascular endothelial growth factor (VEGF) mRNA expression were measured using real-time polymerase chain reaction (PCR) analysis. IP-PA1, when given orally, showed a tendency to promote hair growth in mice. In addition, skin application also significantly promoted hair growth, while histopathological examinations further demonstrated hair elongation from dermal papilla cells. In the human dermal papilla cell culture system, significant FGF-7 and VEGF mRNA expressions were observed (p<0.05). An underlying mechanism of gene expression by which IP-PA1 promotes hair growth was suggested to be different from that of medicine and traditional hair tonics, such as minoxidil and adenosine. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

The expression of asparaginyl endopeptidase promotes growth potential in epithelial ovarian cancer.

PubMed

Zhu, Qinyi; Tang, Meiling; Wang, Xipeng

2017-04-03

Epithelial ovarian cancer (EOC) is the most common and lethal cancer-related death among females in the world. Asparaginyl endopeptidase (AEP) is a member of C13 family peptidases and expressed in the extracellular matrix and tumor cells. The aim of this article is to explore the function of asparaginyl endopeptidase in epithelial ovarian cancer. The expression of AEP was examined in 20 EOC samples, 3 EOC metastasis samples, 6 fallopian tube metastasis samples, 4 peritoneum metastasis samples and 20 benign ovarian tumor samples by immunohistochemistry. The expression of AEP was also evaluated in serum and ascites of EOC patients by elisa. And we used a lentiviral vector to overexpress AEP in human epithelial ovarian cancer cell lines SKOV3ip and detected the function of AEP-SKOV3ip cells both in vitro and in vivo. The growth of AEP-SKOV3ip cells was observed by MTT, migration and tube formation assays in vitro. Additionally, the subcutaneous mice model was used to identify the tumor growth and metastasis in vivo. Mice tumors were stained for CD31 to determine the microvessel density (MVD). We demonstrated that AEP was highly expressed in the EOC patient tissues and ascites. The AEP transfected SKOV3ip cells could both promote tumor growth in vitro and in vivo. The MVD in AEP-SKOV3ip group was higher than that in NC-SKOV3ip group. Therefore, our results demonstrated that AEP could induce EOC growth and progressionboth in vitro and in vivo.

Blood-derived integration-free iPS cell line UKBi011-A from a diagnosed male Alzheimer's disease patient with APOE ɛ4/ɛ4 genotype.

PubMed

Peitz, Michael; Bechler, Tamara; Thiele, Catrin Cornelia; Veltel, Monika; Bloschies, Melanie; Fliessbach, Klaus; Ramirez, Alfredo; Brüstle, Oliver

2018-04-23

Alzheimer's disease (AD) is most the frequent neurodegenerative disease, and the APOE ε4 allele is the most prominent risk factor for late-onset AD. Here, we present an iPSC line generated from peripheral blood cells of a male AD patient employing Sendai virus vectors encoding the transcription factors OCT4, SOX2, KLF4 and c-MYC. The characterized iPSC line expresses typical human pluripotency markers and shows differentiation into all three germ layers, complete reprogramming vector clearance, a normal SNP genotype and maintenance of the APOE ε4/ε4 allele. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Stem cells in pharmaceutical biotechnology.

PubMed

Zuba-Surma, Ewa K; Józkowicz, Alicja; Dulak, Józef

2011-11-01

Multiple populations of stem cells have been indicated to potentially participate in regeneration of injured organs. Especially, embryonic stem cells (ESC) and recently inducible pluripotent stem cells (iPS) receive a marked attention from scientists and clinicians for regenerative medicine because of their high proliferative and differentiation capacities. Despite that ESC and iPS cells are expected to give rise into multiple regenerative applications when their side effects are overcame during appropriate preparation procedures, in fact their most recent application of human ESC may, however, reside in their use as a tool in drug development and disease modeling. This review focuses on the applications of stem cells in pharmaceutical biotechnology. We discuss possible relevance of pluripotent cell stem populations in developing physiological models for any human tissue cell type useful for pharmacological, metabolic and toxicity evaluation necessary in the earliest steps of drug development. The present models applied for preclinical drug testing consist of primary cells or immortalized cell lines that show limitations in terms of accessibility or relevance to their in vivo counterparts. The availability of renewable human cells with functional similarities to their in vivo counterparts is the first landmark for a new generation of cell-based assays. We discuss the approaches for using stem cells as valuable physiological targets of drug activity which may increase the strength of target validation and efficacy potentially resulting in introducing new safer remedies into clinical trials and the marketplace. Moreover, we discuss the possible applications of stem cells for elucidating mechanisms of disease pathogenesis. The knowledge about the mechanisms governing the development and progression of multitude disorders which would come from the cellular models established based on stem cells, may give rise to new therapeutical strategies for such diseases. All together, the applications of various cell types derived from patient specific pluripotent stem cells may lead to targeted drug and cellular therapies for certain individuals.

Optically triggering spatiotemporally confined GPCR activity in a cell and programming neurite initiation and extension

PubMed Central

Karunarathne, W. K. Ajith; Giri, Lopamudra; Kalyanaraman, Vani; Gautam, N.

2013-01-01

G-protein–coupled receptor (GPCR) activity gradients evoke important cell behavior but there is a dearth of methods to induce such asymmetric signaling in a cell. Here we achieved reversible, rapidly switchable patterns of spatiotemporally restricted GPCR activity in a single cell. We recruited properties of nonrhodopsin opsins—rapid deactivation, distinct spectral tuning, and resistance to bleaching—to activate native Gi, Gq, or Gs signaling in selected regions of a cell. Optical inputs were designed to spatiotemporally control levels of second messengers, IP3, phosphatidylinositol (3,4,5)-triphosphate, and cAMP in a cell. Spectrally selective imaging was accomplished to simultaneously monitor optically evoked molecular and cellular response dynamics. We show that localized optical activation of an opsin-based trigger can induce neurite initiation, phosphatidylinositol (3,4,5)-triphosphate increase, and actin remodeling. Serial optical inputs to neurite tips can refashion early neuron differentiation. Methods here can be widely applied to program GPCR-mediated cell behaviors. PMID:23479634

Induced adult stem (iAS) cells and induced transit amplifying progenitor (iTAP) cells-a possible alternative to induced pluripotent stem (iPS) cells?

PubMed

Heng, Boon Chin; Richards, Mark; Ge, Zigang; Shu, Yimin

2010-02-01

The successful derivation of iPSC lines effectively demonstrates that it is possible to reset the 'developmental clock' of somatic cells all the way back to the initial embryonic state. Hence, it is plausible that this clock may instead be turned back half-way to a less immature developmental stage that is more directly applicable to clinical therapeutic applications or for in vitro pharmacology/toxicology screening assays. Such a suitable developmental state is postulated to be either the putative transit amplifying progenitor stage or adult stem cell stage. It is hypothetically possible to reprogram mature and terminally differentiated somatic cells back to the adult stem cell or transit amplifying progenitor stage, in a manner similar to the derivation of iPSC. It is proposed that the terminology 'Induced Adult Stem Cells' (iASC) or 'Induced Transit Amplifying Progenitor Cells' (iTAPC) be used to described such reprogrammed somatic cells. Of particular interest, is the possibility of resetting the developmental clock of mature differentiated somatic cells of the mesenchymal lineage, explanted from adipose tissue, bone marrow and cartilage. The putative adult stem cell sub-population from which these cells are derived, commonly referred to as 'mesenchymal stem cells', are highly versatile and hold much therapeutic promise in regenerative medicine, as attested to by numerous human clinical trials and animal studies. Perhaps it may be appropriate to term such reprogrammed cells as 'Induced Mesenchymal Stem Cells' (iMSC) or as 'Induced Mesenchumal Progenitor Cells' (iMPC). Given that cells from the same organ/tissue will share some commonalities in gene expression, we hypothesize that the generation of iASC or iTAPC would be more efficient as compared to iPSC generation, since a common epigenetic program must exist between the reprogrammed cells, adult stem cell or progenitor cell types and terminally differentiated cell types from the same organ/tissue.

Cistrome of the aldosterone-activated mineralocorticoid receptor in human renal cells.

PubMed

Le Billan, Florian; Khan, Junaid A; Lamribet, Khadija; Viengchareun, Say; Bouligand, Jérôme; Fagart, Jérôme; Lombès, Marc

2015-09-01

Aldosterone exerts its effects mainly by activating the mineralocorticoid receptor (MR), a transcription factor that regulates gene expression through complex and dynamic interactions with coregulators and transcriptional machinery, leading to fine-tuned control of vectorial ionic transport in the distal nephron. To identify genome-wide aldosterone-regulated MR targets in human renal cells, we set up a chromatin immunoprecipitation (ChIP) assay by using a specific anti-MR antibody in a differentiated human renal cell line expressing green fluorescent protein (GFP)-MR. This approach, coupled with high-throughput sequencing, allowed identification of 974 genomic MR targets. Computational analysis identified an MR response element (MRE) including single or multiple half-sites and palindromic motifs in which the AGtACAgxatGTtCt sequence was the most prevalent motif. Most genomic MR-binding sites (MBSs) are located >10 kb from the transcriptional start sites of target genes (84%). Specific aldosterone-induced recruitment of MR on the first most relevant genomic sequences was further validated by ChIP-quantitative (q)PCR and correlated with concomitant and positive aldosterone-activated transcriptional regulation of the corresponding gene, as assayed by RT-qPCR. It was notable that most MBSs lacked MREs but harbored DNA recognition motifs for other transcription factors (FOX, EGR1, AP1, PAX5) suggesting functional interaction. This work provides new insights into aldosterone MR-mediated renal signaling and opens relevant perspectives for mineralocorticoid-related pathophysiology. © FASEB.

Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1

PubMed Central

Solanes, Paola; Heuzé, Mélina L; Maurin, Mathieu; Bretou, Marine; Lautenschlaeger, Franziska; Maiuri, Paolo; Terriac, Emmanuel; Thoulouze, Maria-Isabel; Launay, Pierre; Piel, Matthieu; Vargas, Pablo; Lennon-Duménil, Ana-Maria

2015-01-01

Dendritic cells (DCs) patrol the interstitial space of peripheral tissues. The mechanisms that regulate their migration in such constrained environment remain unknown. We here investigated the role of calcium in immature DCs migrating in confinement. We found that they displayed calcium oscillations that were independent of extracellular calcium and more frequently observed in DCs undergoing strong speed fluctuations. In these cells, calcium spikes were associated with fast motility phases. IP3 receptors (IP3Rs) channels, which allow calcium release from the endoplasmic reticulum, were identified as required for immature DCs to migrate at fast speed. The IP3R1 isoform was further shown to specifically regulate the locomotion persistence of immature DCs, that is, their capacity to maintain directional migration. This function of IP3R1 results from its ability to control the phosphorylation levels of myosin II regulatory light chain (MLC) and the back/front polarization of the motor protein. We propose that by upholding myosin II activity, constitutive calcium release from the ER through IP3R1 maintains DC polarity during migration in confinement, facilitating the exploration of their environment. PMID:25637353

Mini-dystrophin Expression Down-regulates IP3-mediated Calcium Release Events in Resting Dystrophin-deficient Muscle Cells

PubMed Central

Balghi, Haouaria; Sebille, Stéphane; Mondin, Ludivine; Cantereau, Anne; Constantin, Bruno; Raymond, Guy; Cognard, Christian

2006-01-01

We present here evidence for the enhancement, at rest, of an inositol 1,4,5-trisphosphate (IP3)–mediated calcium signaling pathway in myotubes from dystrophin-deficient cell lines (SolC1(−)) as compared to a cell line from the same origin but transfected with mini-dystrophin (SolD(+)). With confocal microscopy, the number of sites discharging calcium (release site density [RSD]) was quantified and found more elevated in SolC1(−) than in SolD(+) myotubes. Variations of membrane potential had no significant effect on this difference, and higher resting [Ca2+]i in SolC1(−) (Marchand, E., B. Constantin, H. Balghi, M.C. Claudepierre, A. Cantereau, C. Magaud, A. Mouzou, G. Raymond, S. Braun, and C. Cognard. 2004. Exp. Cell Res. 297:363–379) cannot explain alone higher RSD. The exposure with SR Ca2+ channel inhibitors (ryanodine and 2-APB) and phospholipase C inhibitor (U73122) significantly reduced RSD in both cell types but with a stronger effect in dystrophin-deficient SolC1(−) myotubes. Immunocytochemistry allowed us to localize ryanodine receptors (RyRs) as well as IP3 receptors (IP3Rs), IP3R-1 and IP3R-2 isoforms, indicating the presence of both RyRs-dependent and IP3-dependent release systems in both cells. We previously reported evidence for the enhancement, through a Gi protein, of the IP3-mediated calcium signaling pathway in SolC1(−) as compared to SolD(+) myotubes during a high K+ stimulation (Balghi, H., S. Sebille, B. Constantin, S. Patri, V. Thoreau, L. Mondin, E. Mok, A. Kitzis, G. Raymond, and C. Cognard. 2006. J. Gen. Physiol. 127:171–182). Here we show that, at rest, these regulation mechanisms are also involved in the modulation of calcium release activities. The enhancement of resting release activity may participate in the calcium overload observed in dystrophin-deficient myotubes, and our findings support the hypothesis of the regulatory role of mini-dystrophin on intracellular signaling. PMID:16847098

Differential Sox10 Genomic Occupancy in Myelinating Glia

PubMed Central

Lopez-Anido, Camila; Sun, Guannan; Koenning, Matthias; Srinivasan, Rajini; Hung, Holly A.; Emery, Ben; Keles, Sunduz; Svaren, John

2015-01-01

Myelin is formed by specialized myelinating glia: oligodendrocytes and Schwann cells in the central and peripheral nervous systems, respectively. While there are distinct developmental aspects and regulatory pathways in these two cell types, myelination in both systems requires the transcriptional activator Sox10. Sox10 interacts with cell type-specific transcription factors at some loci to induce myelin gene expression, but it is largely unknown how Sox10 transcriptional networks globally compare between oligodendrocytes and Schwann cells. We used in vivo ChIP-Seq analysis of spinal cord and peripheral nerve (sciatic nerve) to identify unique and shared Sox10 binding sites and assess their correlation with active enhancers and transcriptional profiles in oligodendrocytes and Schwann cells. Sox10 binding sites overlap with active enhancers and critical cell type-specific regulators of myelination, such as Olig2 and Myrf in oligodendrocytes, and Egr2/Krox20 in Schwann cells. Sox10 sites also associate with genes critical for myelination in both oligodendrocytes and Schwann cells, and are found within super-enhancers previously defined in brain. In Schwann cells, Sox10 sites contain binding motifs of putative partners in the Sp/Klf, Tead, and nuclear receptor protein families. Specifically, siRNA analysis of nuclear receptors Nr2f1 and Nr2f2 revealed downregulation of myelin genes Mbp and Ndrg1 in primary Schwann cells. Our analysis highlights different mechanisms that establish cell type-specific genomic occupancy of Sox10, which reflects the unique characteristics of oligodendrocyte and Schwann cell differentiation. PMID:25974668

Electroactive 3D materials for cardiac tissue engineering

NASA Astrophysics Data System (ADS)

Gelmi, Amy; Zhang, Jiabin; Cieslar-Pobuda, Artur; Ljunngren, Monika K.; Los, Marek Jan; Rafat, Mehrdad; Jager, Edwin W. H.

2015-04-01

By-pass surgery and heart transplantation are traditionally used to restore the heart's functionality after a myocardial Infarction (MI or heart attack) that results in scar tissue formation and impaired cardiac function. However, both procedures are associated with serious post-surgical complications. Therefore, new strategies to help re-establish heart functionality are necessary. Tissue engineering and stem cell therapy are the promising approaches that are being explored for the treatment of MI. The stem cell niche is extremely important for the proliferation and differentiation of stem cells and tissue regeneration. For the introduction of stem cells into the host tissue an artificial carrier such as a scaffold is preferred as direct injection of stem cells has resulted in fast stem cell death. Such scaffold will provide the proper microenvironment that can be altered electronically to provide temporal stimulation to the cells. We have developed an electroactive polymer (EAP) scaffold for cardiac tissue engineering. The EAP scaffold mimics the extracellular matrix and provides a 3D microenvironment that can be easily tuned during fabrication, such as controllable fibre dimensions, alignment, and coating. In addition, the scaffold can provide electrical and electromechanical stimulation to the stem cells which are important external stimuli to stem cell differentiation. We tested the initial biocompatibility of these scaffolds using cardiac progenitor cells (CPCs), and continued onto more sensitive induced pluripotent stem cells (iPS). We present the fabrication and characterisation of these electroactive fibres as well as the response of increasingly sensitive cell types to the scaffolds.

ChIP-seq and ChIP-exo profiling of Pol II, H2A.Z, and H3K4me3 in human K562 cells.

PubMed

Mchaourab, Zenab F; Perreault, Andrea A; Venters, Bryan J

2018-03-06

The human K562 chronic myeloid leukemia cell line has long served as an experimental paradigm for functional genomic studies. To systematically and functionally annotate the human genome, the ENCODE consortium generated hundreds of functional genomic data sets, such as chromatin immunoprecipitation coupled to sequencing (ChIP-seq). While ChIP-seq analyses have provided tremendous insights into gene regulation, spatiotemporal insights were limited by a resolution of several hundred base pairs. ChIP-exonuclease (ChIP-exo) is a refined version of ChIP-seq that overcomes this limitation by providing higher precision mapping of protein-DNA interactions. To study the interplay of transcription initiation and chromatin, we profiled the genome-wide locations for RNA polymerase II (Pol II), the histone variant H2A.Z, and the histone modification H3K4me3 using ChIP-seq and ChIP-exo. In this Data Descriptor, we present detailed information on parallel experimental design, data generation, quality control analysis, and data validation. We discuss how these data lay the foundation for future analysis to understand the relationship between the occupancy of Pol II and nucleosome positions at near base pair resolution.

Induced pluripotent stem cells in hematology: current and future applications

PubMed Central

Focosi, D; Amabile, G; Di Ruscio, A; Quaranta, P; Tenen, D G; Pistello, M

2014-01-01

Reprogramming somatic cells into induced pluripotent stem (iPS) cells is nowadays approaching effectiveness and clinical grade. Potential uses of this technology include predictive toxicology, drug screening, pathogenetic studies and transplantation. Here, we review the basis of current iPS cell technology and potential applications in hematology, ranging from disease modeling of congenital and acquired hemopathies to hematopoietic stem and other blood cell transplantation. PMID:24813079

[Stem cell research and science and technology policy in Japan].

PubMed

Yashiro, Yoshimi

2011-12-01

In this paper I review the present condition of the regeneration medicine research using pluripotency and a somatic stem cell, and I describe the subject of the science and technology policy in Japan towards realization of regeneration medicine. The Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) supported research promotion by the prompt action in 2007 when establishment of the iPS cell was reported by Shinya Yamanaka. Although the hospitable support of the Japanese government to an iPS cell is continued still now, there are some problems in respect of the support to other stem cell researches, and industrialization of regeneration medicine. In order to win a place in highly competitive area of investigation, MEXT needs to change policy so that funds may be widely supplied also to stem cell researches other than iPS cell research.

Differentiation of RPE cells from integration-free iPS cells and their cell biological characterization.

PubMed

Hazim, Roni A; Karumbayaram, Saravanan; Jiang, Mei; Dimashkie, Anupama; Lopes, Vanda S; Li, Douran; Burgess, Barry L; Vijayaraj, Preethi; Alva-Ornelas, Jackelyn A; Zack, Jerome A; Kohn, Donald B; Gomperts, Brigitte N; Pyle, April D; Lowry, William E; Williams, David S

2017-10-02

Dysfunction of the retinal pigment epithelium (RPE) is implicated in numerous forms of retinal degeneration. The readily accessible environment of the eye makes it particularly suitable for the transplantation of RPE cells, which can now be derived from autologous induced pluripotent stem cells (iPSCs), to treat retinal degeneration. For RPE transplantation to become feasible in the clinic, patient-specific somatic cells should be reprogrammed to iPSCs without the introduction of reprogramming genes into the genome of the host cell, and then subsequently differentiated into RPE cells that are well characterized for safety and functionality prior to transplantation. We have reprogrammed human dermal fibroblasts to iPSCs using nonintegrating RNA, and differentiated the iPSCs toward an RPE fate (iPSC-RPE), under Good Manufacturing Practice (GMP)-compatible conditions. Using highly sensitive assays for cell polarity, structure, organelle trafficking, and function, we found that iPSC-RPE cells in culture exhibited key characteristics of native RPE. Importantly, we demonstrate for the first time with any stem cell-derived RPE cell that live cells are able to support dynamic organelle transport. This highly sensitive test is critical for RPE cells intended for transplantation, since defects in intracellular motility have been shown to promote RPE pathogenesis akin to that found in macular degeneration. To test their capabilities for in-vivo transplantation, we injected the iPSC-RPE cells into the subretinal space of a mouse model of retinal degeneration, and demonstrated that the transplanted cells are capable of rescuing lost RPE function. This report documents the successful generation, under GMP-compatible conditions, of human iPSC-RPE cells that possess specific characteristics of healthy RPE. The report adds to a growing literature on the utility of human iPSC-RPE cells for cell culture investigations on pathogenicity and for therapeutic transplantation, by corroborating findings of others, and providing important new information on essential RPE cell biological properties.

Sulforaphane inhibits the interferon-γ-induced expression of MIG, IP-10 and I-TAC in INS‑1 pancreatic β-cells through the downregulation of IRF-1, STAT-1 and PKB.

PubMed

Park, Yu-Kyoung; Ramalingam, Mahesh; Kim, Shin; Jang, Byeong-Churl; Park, Jong Wook

2017-09-01

Sulforaphane (SFN) is a dietary isothiocyanate abundantly available in cruciferous vegetables and has been shown to possess anti-inflammatory and immunomodulatory activities. Chemokines are important mediators of inflammation and immune responses due to their ability to recruit and activate macrophages and leukocytes. To date, little is known about the SFN-mediated regulation of chemokine expression in pancreatic β-cells. In this study, we investigated the inhibitory effects and mechanisms of SFN on the interferon-γ (IFN-γ)-induced expression of a subset of chemokines, including monokine induced by IFN-γ (MIG), IFN-inducible protein of 10 kDa (IP-10) and IFN-inducible T‑cell alpha chemoattractant (I-TAC), in INS‑1 cells, a rat pancreatic β-cell line. Notably, IFN-γ treatment led to an increase in the mRNA expression levels of MIG, IP-10 and I-TAC in the INS‑1 cells. However, SFN strongly blocked the mRNA expressions of MIG, IP-10 and I-TAC induced by IFN-γ in INS‑1 cells. On the mechanistic level, SFN significanlty decreased not only the mRNA expression levels of interferon regulatory factor-1 (IRF-1), but also the phosphorylation levels of signal transducer and activator of transcription-1 (STAT-1) and protein kinase B (PKB) which were induced by IFN-γ in the INS‑1 cells. Pharmacological inhibition experiments further revealed that treatment with JAK inhibitor I weakly inhibited the IFN-γ-induced expression of IP-10, whereas it strongly suppressed the IFN-γ-induced expression of MIG and I-TAC in the INS‑1 cells. Moreover, treatment with LY294002, a PI3K/PKB inhibitor, was able to slightly repress IFN‑γ‑induced expressions of MIG and I-TAC, but not IP-10, in INS‑1 cells. Importantly, the IFN-γ-induced increase in the expression levels of MIG, IP-10 and I-TAC in the INS-1 cells was strongly inhibited by SFN, but not by other natural substances, such as curcumin, sanguinarine, resveratrol, triptolide and epigallocatechin gallate (EGCG), suggesting the specificity of SFN in downregulating the levels of these chemokines. To the best of our knowledge, these results collectively demonstrate for the first time that SFN strongly inhibits the IFN-γ-induced expression of MIG, IP-10 and I-TAC in INS‑1 cells and this inhibition is, at least in part, mediated through the reduced expression and phosphorylation levels of IRF-1, STAT-1 and PKB.

The chemokine receptor CXCR3 and its splice variant are expressed in human airway epithelial cells.

PubMed

Kelsen, Steven G; Aksoy, Mark O; Yang, Yi; Shahabuddin, Syed; Litvin, Judith; Safadi, Fayez; Rogers, Thomas J

2004-09-01

Activation of the chemokine receptor CXCR3 by its cognate ligands induces several differentiated cellular responses important to the growth and migration of a variety of hematopoietic and structural cells. In the human respiratory tract, human airway epithelial cells (HAEC) release the CXCR3 ligands Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11. Simultaneous expression of CXCR3 by HAEC would have important implications for the processes of airway inflammation and repair. Accordingly, in the present study we sought to determine whether HAEC also express the classic CXCR3 chemokine receptor CXCR3-A and its splice variant CXCR3-B and hence may respond in autocrine fashion to its ligands. We found that cultured HAEC (16-HBE and tracheocytes) constitutively expressed CXCR3 mRNA and protein. CXCR3 mRNA levels assessed by expression array were approximately 35% of beta-actin expression. In contrast, CCR3, CCR4, CCR5, CCR8, and CX3CR1 were <5% beta-actin. Both CXCR3-A and -B were expressed. Furthermore, tracheocytes freshly harvested by bronchoscopy stained positively for CXCR3 by immunofluorescence microscopy, and 68% of cytokeratin-positive tracheocytes (i.e., the epithelial cell population) were positive for CXCR3 by flow cytometry. In 16-HBE cells, CXCR3 receptor density was approximately 78,000 receptors/cell when assessed by competitive displacement of 125I-labeled IP-10/CXCL10. Finally, CXCR3 ligands induced chemotactic responses and actin reorganization in 16-HBE cells. These findings indicate constitutive expression by HAEC of a functional CXC chemokine receptor, CXCR3. Our data suggest the possibility that autocrine activation of CXCR3 expressed by HAEC may contribute to airway inflammation and remodeling in obstructive lung disease by regulating HAEC migration.

Endopolysaccharides from Ganoderma resinaceum, Phlebia rufa, and Trametes versicolor affect differently the proliferation rate of HepG2 cells.

PubMed

Silva, Amélia M; Miranda, Andreia; Fernandes, Elisabete; Santos, Susana; Fraga, Irene; Santos, Dario L; Dias, Albino A; Bezerra, Rui M

2013-03-01

Fungi have been used for medicinal purposes for long time by Asian countries, being a putative source of powerful new phytopharmaceuticals such as polysaccharides. The aim of this study was to extract endopolysaccharides (IPS) from Ganoderma resinaceum, Phlebia rufa, and Trametes versicolor, grown under submerged culture, to compare crude IPS production, total carbohydrate, and protein yield, and to study the effect of these IPS on HepG2 cells proliferation rate. Total biomass produced by G. resinaceum, P. rufa, and T. versicolor was (in gram per liter) 3.32 ± 0.80, 5.42 ± 0.58, and 4.2 ± 1.29 and the IPS yield (as the biomass percent) was 9.9 ± 0.05, 29.0 ± 6.3, and 9.1 ± 3.1 %, respectively. Characterization of IPS has shown different proportion between total sugar and protein being, on average 6.04, 10.74, and 22.62, for G. resinaceum, T. versicolor, and P. rufa, respectively. The IPS effect, at 50, 100, and 200 μg mL(-1) on HepG2 cell growth and viability was negligible for G. resinaceum and P. rufa but, in the case of T. versicolor, 200 μg mL(-1) of IPS evoked 40 % reduction on cell growth. The results suggest that the intracellular polysaccharides from T. versicolor are a potential source for bioactive molecules with anti-proliferative properties.

Preparation of Low-Input and Ligation-Free ChIP-seq Libraries Using Template-Switching Technology.

PubMed

Bolduc, Nathalie; Lehman, Alisa P; Farmer, Andrew

2016-10-10

Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) has become the gold standard for mapping of transcription factors and histone modifications throughout the genome. However, for ChIP experiments involving few cells or targeting low-abundance transcription factors, the small amount of DNA recovered makes ligation of adapters very challenging. In this unit, we describe a ChIP-seq workflow that can be applied to small cell numbers, including a robust single-tube and ligation-free method for preparation of sequencing libraries from sub-nanogram amounts of ChIP DNA. An example ChIP protocol is first presented, resulting in selective enrichment of DNA-binding proteins and cross-linked DNA fragments immobilized on beads via an antibody bridge. This is followed by a protocol for fast and easy cross-linking reversal and DNA recovery. Finally, we describe a fast, ligation-free library preparation protocol, featuring DNA SMART technology, resulting in samples ready for Illumina sequencing. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Induced pluripotent stem cells: advances to applications

PubMed Central

Nelson, Timothy J; Martinez-Fernandez, Almudena; Yamada, Satsuki; Ikeda, Yasuhiro; Perez-Terzic, Carmen; Terzic, Andre

2010-01-01

Induced pluripotent stem cell (iPS) technology has enriched the armamentarium of regenerative medicine by introducing autologous pluripotent progenitor pools bioengineered from ordinary somatic tissue. Through nuclear reprogramming, patient-specific iPS cells have been derived and validated. Optimizing iPS-based methodology will ensure robust applications across discovery science, offering opportunities for the development of personalized diagnostics and targeted therapeutics. Here, we highlight the process of nuclear reprogramming of somatic tissues that, when forced to ectopically express stemness factors, are converted into bona fide pluripotent stem cells. Bioengineered stem cells acquire the genuine ability to generate replacement tissues for a wide-spectrum of diseased conditions, and have so far demonstrated therapeutic benefit upon transplantation in model systems of sickle cell anemia, Parkinson’s disease, hemophilia A, and ischemic heart disease. The field of regenerative medicine is therefore primed to adopt and incorporate iPS cell-based advancements as a next generation stem cell platforms. PMID:21165156

cDNA cloning and characterization of a novel gene encoding the MLF1-interacting protein MLF1IP.

PubMed

Hanissian, Silva H; Akbar, Umar; Teng, Bin; Janjetovic, Zorica; Hoffmann, Anne; Hitzler, Johann K; Iscove, Norman; Hamre, Kristin; Du, Xiaoping; Tong, Yiai; Mukatira, Suraj; Robertson, Jon H; Morris, Stephan W

2004-04-29

Myelodysplasia/acute myeloid leukemia (MDS/AML) is characterized by a t(3;5)(q25.1;q34) chromosomal translocation that forms a fusion gene between nucleophosmin (NPM) and MDS/myeloid leukemia factor 1 (MLF1). We identified a novel protein, MLF1-interacting protein (MLF1IP), that specifically associates with MLF1 by yeast two-hybrid analysis and in pulldown assays, and colocalizes with it in both the nuclei and cytoplasm of cells. The MLF1IP gene locus is at chromosome 4q35.1 and is composed of 14 exons spanning 75.8 kb of genomic DNA. The MLF1IP cDNA encodes a 46-kDa protein that contains two bipartite and two classical nuclear localization signals, two nuclear receptor-binding motifs (LXXLL), two leucine zippers, two PEST residues and several potential phosphorylation sites. MLF1IP transcripts are expressed in a variety of tissues (e.g. fetal liver, bone marrow, thymus and testis). MLF1IP appears to be a lineage-specific gene whose expression is confined exclusively to the CFU-E erythroid precursor cells, but not in mature erythrocytes. These observations, together with previous data demonstrating a role for MLF1 in suppressing red cell maturation, suggest a possible role for MLF1IP and MLF1 deregulation in the genesis of erythroleukemias.

The PLC/IP3R/PKC Pathway is Required for Ethanol-enhanced GABA Release

PubMed Central

Kelm, M. Katherine; Weinberg, Richard J.; Criswell, Hugh E.; Breese, George R.

2010-01-01

Summary Research on the actions of ethanol at the GABAergic synapse has traditionally focused on postsynaptic mechanisms, but recent data demonstrate that ethanol also increases both evoked and spontaneous GABA release in many brain regions. Using whole-cell voltage-clamp recordings, we previously showed that ethanol increases spontaneous GABA release at the rat interneuron-Purkinje cell synapse. This presynaptic ethanol effect is dependent on calcium release from internal stores, possibly through activation of inositol 1,4,5-trisphosphate receptors (IP3Rs). After confirming that ethanol targets vesicular GABA release, in the present study we used electron microscopic immunohistochemistry to demonstrate that IP3Rs are located in presynaptic terminals of cerebellar interneurons. Activation of IP3Rs requires binding of IP3, generated through activation of phospholipase C (PLC). We find that the PLC antagonist edelfosine prevents ethanol from increasing spontaneous GABA release. Diacylglycerol generated by PLC and calcium released by activation of the IP3R activate protein kinase C (PKC). Ethanol-enhanced GABA release was blocked by two PKC antagonists, chelerythrine and calphostin C. When a membrane impermeable PKC antagonist, PKC (19-36), was delivered intracellularly to the postsynaptic neuron, ethanol continued to increase spontaneous GABA release. Overall, these results suggest that activation of the PLC/IP3R/PKC pathway is necessary for ethanol to increase spontaneous GABA release from presynaptic terminals onto Purkinje cells. PMID:20206640

The crystal structure of mammalian inositol 1,3,4,5,6-pentakisphosphate 2-kinase reveals a new zinc-binding site and key features for protein function

PubMed Central

Franco-Echevarría, Elsa; Sanz-Aparicio, Julia; Brearley, Charles A.; González-Rubio, Juana M.; González, Beatriz

2017-01-01

Inositol 1,3,4,5,6-pentakisphosphate 2-kinases (IP5 2-Ks) are part of a family of enzymes in charge of synthesizing inositol hexakisphosphate (IP6) in eukaryotic cells. This protein and its product IP6 present many roles in cells, participating in mRNA export, embryonic development, and apoptosis. We reported previously that the full-length IP5 2-K from Arabidopsis thaliana is a zinc metallo-enzyme, including two separated lobes (the N- and C-lobes). We have also shown conformational changes in IP5 2-K and have identified the residues involved in substrate recognition and catalysis. However, the specific features of mammalian IP5 2-Ks remain unknown. To this end, we report here the first structure for a murine IP5 2-K in complex with ATP/IP5 or IP6. Our structural findings indicated that the general folding in N- and C-lobes is conserved with A. thaliana IP5 2-K. A helical scaffold in the C-lobe constitutes the inositol phosphate-binding site, which, along with the participation of the N-lobe, endows high specificity to this protein. However, we also noted large structural differences between the orthologues from these two eukaryotic kingdoms. These differences include a novel zinc-binding site and regions unique to the mammalian IP5 2-K, as an unexpected basic patch on the protein surface. In conclusion, our findings have uncovered distinct features of a mammalian IP5 2-K and set the stage for investigations into protein-protein or protein-RNA interactions important for IP5 2-K function and activity. PMID:28450399

Mercury-arc photolysis: a method for examining second messenger regulation of endothelial cell monolayer integrity.

PubMed

Patton, W F; Alexander, J S; Dodge, A B; Patton, R J; Hechtman, H B; Shepro, D

1991-07-01

Cell-cell apposition in bovine pulmonary endothelial cell monolayers was modulated by inducing transient increases in intracellular adenosine 3':5'-cyclic monophosphate (cAMP) and 1,4,5-inositol triphosphate (IP3). This was accomplished by mercury-arc flash photolysis of o-nitrobenzyl derivatives of the second messengers (caged compounds). Second messenger release by the mercury-arc lamp was determined by radioimmunoassay of cAMP to have a t1/2 of approximately 8 min. Each second messenger induced the phosphorylation of a distinct subset of cytoskeletal proteins; however, both IP3 and cAMP increased vimentin phosphorylation. Actin isoform patterns were not altered by the second messengers. Intracellular pulses of IP3 in pulmonary endothelial cells caused disruption of endothelial monolayer integrity as determined by phase-contrast microscopy and by visualization of actin stress fibers with rhodamine-phalloidin. Intracellular pulses of cAMP increased cell-cell contact, cell surface area, and apposition. IP3 appeared to have its greatest effect on the actin peripheral band. In silicone rubber contractility assays this agent caused contraction of pulmonary microvascular endothelial cells as visualized by an increase in wrinkles beneath the cells. On the other hand, cAMP appeared to effect both the peripheral band and centralized actin domains. Caged cAMP caused relaxation of endothelial cells as visualized by a disappearance of wrinkles beneath the cells.

Global Mapping of Cell Type–Specific Open Chromatin by FAIRE-seq Reveals the Regulatory Role of the NFI Family in Adipocyte Differentiation

PubMed Central

Yu, Jing; Hirose-Yotsuya, Lisa; Take, Kazumi; Sun, Wei; Iwabu, Masato; Okada-Iwabu, Miki; Fujita, Takanori; Aoyama, Tomohisa; Tsutsumi, Shuichi; Ueki, Kohjiro; Kodama, Tatsuhiko; Sakai, Juro; Aburatani, Hiroyuki; Kadowaki, Takashi

2011-01-01

Identification of regulatory elements within the genome is crucial for understanding the mechanisms that govern cell type–specific gene expression. We generated genome-wide maps of open chromatin sites in 3T3-L1 adipocytes (on day 0 and day 8 of differentiation) and NIH-3T3 fibroblasts using formaldehyde-assisted isolation of regulatory elements coupled with high-throughput sequencing (FAIRE-seq). FAIRE peaks at the promoter were associated with active transcription and histone modifications of H3K4me3 and H3K27ac. Non-promoter FAIRE peaks were characterized by H3K4me1+/me3-, the signature of enhancers, and were largely located in distal regions. The non-promoter FAIRE peaks showed dynamic change during differentiation, while the promoter FAIRE peaks were relatively constant. Functionally, the adipocyte- and preadipocyte-specific non-promoter FAIRE peaks were, respectively, associated with genes up-regulated and down-regulated by differentiation. Genes highly up-regulated during differentiation were associated with multiple clustered adipocyte-specific FAIRE peaks. Among the adipocyte-specific FAIRE peaks, 45.3% and 11.7% overlapped binding sites for, respectively, PPARγ and C/EBPα, the master regulators of adipocyte differentiation. Computational motif analyses of the adipocyte-specific FAIRE peaks revealed enrichment of a binding motif for nuclear family I (NFI) transcription factors. Indeed, ChIP assay showed that NFI occupy the adipocyte-specific FAIRE peaks and/or the PPARγ binding sites near PPARγ, C/EBPα, and aP2 genes. Overexpression of NFIA in 3T3-L1 cells resulted in robust induction of these genes and lipid droplet formation without differentiation stimulus. Overexpression of dominant-negative NFIA or siRNA–mediated knockdown of NFIA or NFIB significantly suppressed both induction of genes and lipid accumulation during differentiation, suggesting a physiological function of these factors in the adipogenic program. Together, our study demonstrates the utility of FAIRE-seq in providing a global view of cell type–specific regulatory elements in the genome and in identifying transcriptional regulators of adipocyte differentiation. PMID:22028663

A dual role for the anti-apoptotic Bcl-2 protein in cancer: mitochondria versus endoplasmic reticulum.

PubMed

Akl, Haidar; Vervloessem, Tamara; Kiviluoto, Santeri; Bittremieux, Mart; Parys, Jan B; De Smedt, Humbert; Bultynck, Geert

2014-10-01

Anti-apoptotic Bcl-2 contributes to cancer formation and progression by promoting the survival of altered cells. Hence, it is a prime target for novel specific anti-cancer therapeutics. In addition to its canonical anti-apoptotic role, Bcl-2 has an inhibitory effect on cell-cycle progression. Bcl-2 acts at two different intracellular compartments, the mitochondria and the endoplasmic reticulum (ER). At the mitochondria, Bcl-2 via its hydrophobic cleft scaffolds the Bcl-2-homology (BH) domain 3 (BH3) of pro-apoptotic Bcl-2-family members. Small molecules (like BH3 mimetics) can disrupt this interaction, resulting in apoptotic cell death in cancer cells. At the ER, Bcl-2 modulates Ca(2+) signaling, thereby promoting proliferation while increasing resistance to apoptosis. Bcl-2 at the ER acts via its N-terminal BH4 domain, which directly binds and inhibits the inositol 1,4,5-trisphosphate receptor (IP3R), the main intracellular Ca(2+)-release channel. Tools targeting the BH4 domain of Bcl-2 reverse Bcl-2's inhibitory action on IP3Rs and trigger pro-apoptotic Ca(2+) signaling in cancer B-cells, including chronic lymphocytic leukemia (CLL) cells and diffuse large B-cell lymphoma (DLBCL) cells. The sensitivity of DLBCL cells to BH4-domain targeting tools strongly correlated with the expression levels of the IP3R2 channel, the IP3R isoform with the highest affinity for IP3. Interestingly, bio-informatic analysis of a database of primary CLL patient cells also revealed a transcriptional upregulation of IP3R2. Finally, this review proposes a model, in which cancer cell survival depends on Bcl-2 at the mitochondria and/or the ER. This dependence likely will have an impact on their responses to BH3-mimetic drugs and BH4-domain targeting tools. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau. Copyright © 2014 Elsevier B.V. All rights reserved.

The B-MYB Transcriptional Network Guides Cell Cycle Progression and Fate Decisions to Sustain Self-Renewal and the Identity of Pluripotent Stem Cells

PubMed Central

Zhan, Ming; Riordon, Daniel R.; Yan, Bin; Tarasova, Yelena S.; Bruweleit, Sarah; Tarasov, Kirill V.; Li, Ronald A.; Wersto, Robert P.; Boheler, Kenneth R.

2012-01-01

Embryonic stem cells (ESCs) are pluripotent and have unlimited self-renewal capacity. Although pluripotency and differentiation have been examined extensively, the mechanisms responsible for self-renewal are poorly understood and are believed to involve an unusual cell cycle, epigenetic regulators and pluripotency-promoting transcription factors. Here we show that B-MYB, a cell cycle regulated phosphoprotein and transcription factor critical to the formation of inner cell mass, is central to the transcriptional and co-regulatory networks that sustain normal cell cycle progression and self-renewal properties of ESCs. Phenotypically, B-MYB is robustly expressed in ESCs and induced pluripotent stem cells (iPSCs), and it is present predominantly in a hypo-phosphorylated state. Knockdown of B-MYB results in functional cell cycle abnormalities that involve S, G2 and M phases, and reduced expression of critical cell cycle regulators like ccnb1 and plk1. By conducting gene expression profiling on control and B-MYB deficient cells, ChIP-chip experiments, and integrative computational analyses, we unraveled a highly complex B-MYB-mediated transcriptional network that guides ESC self-renewal. The network encompasses critical regulators of all cell cycle phases and epigenetic regulators, pluripotency transcription factors, and differentiation determinants. B-MYB along with E2F1 and c-MYC preferentially co-regulate cell cycle target genes. B-MYB also co-targets genes regulated by OCT4, SOX2 and NANOG that are significantly associated with stem cell differentiation, embryonic development, and epigenetic control. Moreover, loss of B-MYB leads to a breakdown of the transcriptional hierarchy present in ESCs. These results coupled with functional studies demonstrate that B-MYB not only controls and accelerates cell cycle progression in ESCs it contributes to fate decisions and maintenance of pluripotent stem cell identity. PMID:22936984

The B-MYB transcriptional network guides cell cycle progression and fate decisions to sustain self-renewal and the identity of pluripotent stem cells.

PubMed

Zhan, Ming; Riordon, Daniel R; Yan, Bin; Tarasova, Yelena S; Bruweleit, Sarah; Tarasov, Kirill V; Li, Ronald A; Wersto, Robert P; Boheler, Kenneth R

2012-01-01

Embryonic stem cells (ESCs) are pluripotent and have unlimited self-renewal capacity. Although pluripotency and differentiation have been examined extensively, the mechanisms responsible for self-renewal are poorly understood and are believed to involve an unusual cell cycle, epigenetic regulators and pluripotency-promoting transcription factors. Here we show that B-MYB, a cell cycle regulated phosphoprotein and transcription factor critical to the formation of inner cell mass, is central to the transcriptional and co-regulatory networks that sustain normal cell cycle progression and self-renewal properties of ESCs. Phenotypically, B-MYB is robustly expressed in ESCs and induced pluripotent stem cells (iPSCs), and it is present predominantly in a hypo-phosphorylated state. Knockdown of B-MYB results in functional cell cycle abnormalities that involve S, G2 and M phases, and reduced expression of critical cell cycle regulators like ccnb1 and plk1. By conducting gene expression profiling on control and B-MYB deficient cells, ChIP-chip experiments, and integrative computational analyses, we unraveled a highly complex B-MYB-mediated transcriptional network that guides ESC self-renewal. The network encompasses critical regulators of all cell cycle phases and epigenetic regulators, pluripotency transcription factors, and differentiation determinants. B-MYB along with E2F1 and c-MYC preferentially co-regulate cell cycle target genes. B-MYB also co-targets genes regulated by OCT4, SOX2 and NANOG that are significantly associated with stem cell differentiation, embryonic development, and epigenetic control. Moreover, loss of B-MYB leads to a breakdown of the transcriptional hierarchy present in ESCs. These results coupled with functional studies demonstrate that B-MYB not only controls and accelerates cell cycle progression in ESCs it contributes to fate decisions and maintenance of pluripotent stem cell identity.

Generating Porcine Chimeras Using Inner Cell Mass Cells and Parthenogenetic Preimplantation Embryos

PubMed Central

Nakano, Kazuaki; Watanabe, Masahito; Matsunari, Hitomi; Matsuda, Taisuke; Honda, Kasumi; Maehara, Miki; Kanai, Takahiro; Hayashida, Gota; Kobayashi, Mirina; Kuramoto, Momoko; Arai, Yoshikazu; Umeyama, Kazuhiro; Fujishiro, Shuh-hei; Mizukami, Yoshihisa; Nagaya, Masaki; Hanazono, Yutaka; Nagashima, Hiroshi

2013-01-01

Background The development and validation of stem cell therapies using induced pluripotent stem (iPS) cells can be optimized through translational research using pigs as large animal models, because pigs have the closest characteristics to humans among non-primate animals. As the recent investigations have been heading for establishment of the human iPS cells with naïve type characteristics, it is an indispensable challenge to develop naïve type porcine iPS cells. The pluripotency of the porcine iPS cells can be evaluated using their abilities to form chimeras. Here, we describe a simple aggregation method using parthenogenetic host embryos that offers a reliable and effective means of determining the chimera formation ability of pluripotent porcine cells. Methodology/Significant Principal Findings In this study, we show that a high yield of chimeric blastocysts can be achieved by aggregating the inner cell mass (ICM) from porcine blastocysts with parthenogenetic porcine embryos. ICMs cultured with morulae or 4–8 cell-stage parthenogenetic embryos derived from in vitro-matured (IVM) oocytes can aggregate to form chimeric blastocysts that can develop into chimeric fetuses after transfer. The rate of production of chimeric blastocysts after aggregation with host morulae (20/24, 83.3%) was similar to that after the injection of ICMs into morulae (24/29, 82.8%). We also found that 4–8 cell-stage embryos could be used; chimeric blastocysts were produced with a similar efficiency (17/26, 65.4%). After transfer into recipients, these blastocysts yielded chimeric fetuses at frequencies of 36.0% and 13.6%, respectively. Conclusion/Significance Our findings indicate that the aggregation method using parthenogenetic morulae or 4–8 cell-stage embryos offers a highly reproducible approach for producing chimeric fetuses from porcine pluripotent cells. This method provides a practical and highly accurate system for evaluating pluripotency of undifferentiated cells, such as iPS cells, based on their ability to form chimeras. PMID:23626746

Genome-wide survey by ChIP-seq reveals YY1 regulation of lincRNAs in skeletal myogenesis

PubMed Central

Lu, Leina; Sun, Kun; Chen, Xiaona; Zhao, Yu; Wang, Lijun; Zhou, Liang; Sun, Hao; Wang, Huating

2013-01-01

Skeletal muscle differentiation is orchestrated by a network of transcription factors, epigenetic regulators, and non-coding RNAs. The transcription factor Yin Yang 1 (YY1) silences multiple target genes in myoblasts (MBs) by recruiting Ezh2 (Enhancer of Zeste Homologue2). To elucidate genome-wide YY1 binding in MBs, we performed chromatin immunoprecipitation (ChIP)-seq and found 1820 specific binding sites in MBs with a large portion residing in intergenic regions. Detailed analysis demonstrated that YY1 acts as an activator for many loci in addition to its known repressor function. No significant co-occupancy was found between YY1 and Ezh2, suggesting an additional Ezh2-independent function for YY1 in MBs. Further analysis of intergenic binding sites showed that YY1 potentially regulates dozens of large intergenic non-coding RNAs (lincRNAs), whose function in myogenesis is underexplored. We characterized a novel muscle-associated lincRNA (Yam-1) that is positively regulated by YY1. Yam-1 is downregulated upon differentiation and acts as an inhibitor of myogenesis. We demonstrated that Yam-1 functions through in cis regulation of miR-715, which in turn targets Wnt7b. Our findings not only provide the first genome-wide picture of YY1 association in muscle cells, but also uncover the functional role of lincRNA Yam-1. PMID:23942234

Monitoring nitric oxide (NO) in rat locus coeruleus: differential effects of NO synthase inhibitors.

PubMed

Desvignes, C; Robert, F; Vachette, C; Chouvet, G; Cespuglio, R; Renaud, B; Lambás-Señas, L

1997-04-14

A porphyrinic microsensor combined with in vivo voltammetry was used to monitor extracellular nitric oxide (NO) in the locus coeruleus (LC) of anaesthetized rats. Administration of N omega-nitro-L-arginine p-nitro-anilide (100 mg/kg, i.p) or 7-nitro indazole (30 mg/kg, i.p.), which both inhibit preferentially neuronal NO synthase (NOS), induced a marked decrease in the NO oxidation peak height. On the other hand, N omega-nitro-L-arginine methyl ester (L-NAME) (200 mg/kg, i.p.), a less selective NOS inhibitor, failed to decrease the NO signal. Moreover, intra LC administration of NMDA, known to activate LC noradrenergic neurones, increased the NO signal. This study demonstrates the usefulness of in vivo voltammetry to monitor basal levels of NO and their changes in the LC. Differential effects of NOS inhibitors show that their central activity need to be assessed through in situ measurement of NO before using these inhibitors as neuropharmacological tools.

CD73-Generated Adenosine Is Critical for Immune Regulation during Toxoplasma gondii Infection

PubMed Central

Mahamed, Deeqa A.; Toussaint, Leon E.

2014-01-01

As an obligate intracellular pathogen, the apicomplexan parasite Toxoplasma gondii evades immune system-mediated clearance by undergoing stage differentiation to persist indefinitely in susceptible hosts. Previously, we found that mice deficient in the ectoenzyme CD73, which generates adenosine in the extracellular matrix, were resistant to chronic toxoplasmosis after oral infection with T. gondii. Resistance in CD73 knockout mice was due to a delay in parasite differentiation in the central nervous system (CNS). To further clarify the role of CD73 and extracellular adenosine in T. gondii pathogenesis, we infected wild-type (WT) and CD73−/− mice with T. gondii cysts systemically by the intraperitoneal (i.p.) route. In contrast to oral infection, i.p. infected CD73−/− mice were highly susceptible to immune-mediated pathology, with significantly increased infiltration of neutrophils and T cells into the peritoneal cavity. Administration of the broad-spectrum adenosine receptor agonist 5′-N-ethylcarboxamidoadenosine (NECA) protected CD73−/− mice against T. gondii-induced immunopathology, suggesting that the absence of CD73-generated adenosine led to the increased susceptibility in these mice. Peritoneal exudate cells from infected CD73−/− mice produced higher levels of the inflammatory mediators nitric oxide, tumor necrosis factor alpha (TNF-α), and interleukin-1β (IL-1β), without enhanced parasite killing or clearance. Bone marrow chimeras established that CD73 expression in both hematopoietic and nonhematopoietic compartments contributes to limiting T. gondii-induced immunopathology. In addition, mice deficient in the adenosine receptor A2A were more susceptible to immunopathology during intraperitoneal infection with T. gondii than WT mice. Thus, extracellular adenosine is a key immune regulator that limits collateral tissue damage due to an intracellular pathogen and promotes host survival. PMID:25452548

Epigenetically Modified Bone Marrow Stromal Cells in Silk Scaffolds Promote Craniofacial Bone Repair and Wound Healing

PubMed Central

Han, Qianqian; Yang, Pishan; Wu, Yuwei; Meng, Shu; Sui, Lei; Zhang, Lan; Yu, Liming; Tang, Yin; Jiang, Hua; Xuan, Dongying; Kaplan, David L.; Kim, Sung Hoon

2015-01-01

Epigenetic regulation of gene expression is a central mechanism that governs cell stemness, determination, commitment, and differentiation. It has been recently found that PHF8, a major H4K20/H3K9 demethylase, plays a critical role in craniofacial and bone development. In this study, we hypothesize that PHF8 promotes osteoblastogenesis by epigenetically regulating the expression of a nuclear matrix protein, special AT-rich sequence-binding protein 2 (SATB2) that plays pivotal roles in skeletal patterning and osteoblast differentiation. Our results showed that expression levels of PHF8 and SATB2 in preosteoblasts and bone marrow stromal cells (BMSCs) increased simultaneously during osteogenic induction. Overexpressing PHF8 in these cells upregulated the expression of SATB2, Runx2, osterix, and bone matrix proteins. Conversely, knockdown of PHF8 reduced the expression of these genes. Furthermore, ChIP assays confirmed that PHF8 specifically bound to the transcription start site (TSS) of the SATB2 promoter, and the expression of H3K9me1 at the TSS region of SATB2 decreased in PHF8 overexpressed group. Implantation of the BMSCs overexpressing PHF8 with silk protein scaffolds promoted bone regeneration in critical-sized defects in mouse calvaria. Taken together, our results demonstrated that PHF8 epigenetically modulates SATB2 activity, triggering BMSCs osteogenic differentiation and facilitating bone formation and regeneration in biodegradable silk scaffolds. PMID:25923143

The Bcl-2 homolog Nrz inhibits binding of IP3 to its receptor to control calcium signaling during zebrafish epiboly.

PubMed

Bonneau, Benjamin; Nougarède, Adrien; Prudent, Julien; Popgeorgiev, Nikolay; Peyriéras, Nadine; Rimokh, Ruth; Gillet, Germain

2014-02-11

Members of the Bcl-2 protein family regulate mitochondrial membrane permeability and also localize to the endoplasmic reticulum where they control Ca(2+) homeostasis by interacting with inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs). In zebrafish, Bcl-2-like 10 (Nrz) is required for Ca(2+) signaling during epiboly and gastrulation. We characterized the mechanism by which Nrz controls IP3-mediated Ca(2+) release during this process. We showed that Nrz was phosphorylated during early epiboly, and that in embryos in which Nrz was knocked down, reconstitution with Nrz bearing mutations designed to prevent its phosphorylation disrupted cyclic Ca(2+) transients and the assembly of the actin-myosin ring and led to epiboly arrest. In cultured cells, wild-type Nrz, but not Nrz with phosphomimetic mutations, interacted with the IP3 binding domain of IP3R1, inhibited binding of IP3 to IP3R1, and prevented histamine-induced increases in cytosolic Ca(2+). Collectively, these data suggest that Nrz phosphorylation is necessary for the generation of IP3-mediated Ca(2+) transients and the formation of circumferential actin-myosin cables required for epiboly. Thus, in addition to their role in apoptosis, by tightly regulating Ca(2+) signaling, Bcl-2 family members participate in the cellular events associated with early vertebrate development, including cytoskeletal dynamics and cell movement.

Unanticipated region- and cell-specific downregulation of individual KChIP auxiliary subunit isotypes in Kv4.2 knock-out mouse brain.

PubMed

Menegola, Milena; Trimmer, James S

2006-11-22

Kv4 family voltage-gated potassium channel alpha subunits and Kv channel-interacting protein (KChIP) and dipeptidyl aminopeptidase-like protein subunits comprise somatodendritic A-type channels in mammalian neurons. Recently, a mouse was generated with a targeted deletion of Kv4.2, a Kv4 alpha subunit expressed in many but not all mammalian brain neurons. Kv4.2-/- mice are grossly indistinguishable from wild-type (WT) littermates. Here we used immunohistochemistry to analyze expression of component Kv4 and KChIP subunits of A-type channels in WT and Kv4.2-/- brains. We found that the expression level, and cellular and subcellular distribution of the other prominent brain Kv4 family member Kv4.3, was indistinguishable between WT and Kv4.2-/- samples. However, we found unanticipated regional and cell-specific decreases in expression of KChIPs. The degree of altered expression of individual KChIP isoforms in different regions and neurons precisely follows the level of Kv4.2 normally found at those sites and presumably their extent of association of these KChIPs with Kv4.2. The dramatic effects of Kv4.2 deletion on KChIP expression suggest that, in addition to previously characterized effects of KChIPs on the functional properties, trafficking, and turnover rate of Kv4 channels, Kv4:KChIP association may confer reciprocal Kv4.2-dependent effects on KChIPs. The impact of Kv4.2 deletion on KChIP expression also supports the major role of KChIPs as auxiliary subunits of Kv4 channels.

The mRNA and Proteins Expression Levels Analysis of TC-1 Cells Immune Response to H9N2 Avian Influenza Virus.

PubMed

Liu, Jiyuan; Li, Ning; Meng, Dan; Hao, Mengchan; Wei, Liangmeng; Chai, Tongjie

2016-01-01

Since 1994, the H9N2 avian influenza virus (AIV) has spread widely in mainland China, causing great economic losses to the poultry industry there. Subsequently, it was found that the H9N2 AIV had the ability to infect mammals, which gave rise to great panic. In order to investigate the immune response of a host infected with H9N2 AIV, TC-1 cells were set as a model in this research. Quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay methods were used to study the expression changes of pattern recognition receptors (PRRs), inflammatory cytokines, and chemokines in AIV-infected TC-1 cells. Our research found that TC-1 cells had similar susceptibility to both CK/SD/w3 (A/Chicken/Shandong/W3/2012) and CK/SD/w4 (A/Chicken/Shandong/W4/2012) H9N2 isolates, while the CK/SD/w3 isolate had a stronger capability of replication in the TC-1 cells. At the same time, the expression of PRRs (melanoma differentiation-associated gene 5, MDA-5), cytokines [interleukin (IL)-1β and IL-6], and chemokines [regulated on activation, normal T cell expressed and secreted (RANTES) and interferon-γ-induced protein-10 kDa (IP-10)] were significantly up-regulated. These results indicated that MDA-5, IL-1β, IL-6, RANTES, and IP-10 might play important roles in the host immune response to H9N2 AIV infection. This study provided useful information for further understanding the interaction between H9N2 virus infection and host immunity, and had certain guiding significance for the prevention and treatment of this disease.

Formation of Polymer Networks for Fast In-Plane Switching of Liquid Crystals at Low Temperatures

NASA Astrophysics Data System (ADS)

Yu, Byeong-Hun; Song, Dong Han; Kim, Ki-Han; Wok Park, Byung; Choi, Sun-Wook; Park, Sung Il; Kang, Sung Gu; Yoon, Jeong Hwan; Kim, Byeong Koo; Yoon, Tae-Hoon

2013-09-01

We formed a polymer structure to enable fast in-plane switching of liquid crystals at low temperatures. The problem of the inevitable slow response at low temperatures was reduced by the formation of in-cell polymer networks in in-plane switching (IPS) cells. The electro-optic characteristics of polymer-networked IPS cells were measured at temperatures ranging from -10 to 20 °C. The turn-on and turn-off times of an IPS cell were reduced by 44.5 and 47.2% at -10 °C by the formation of polymer networks. We believe that the proposed technology can be applied to emerging display devices such as mobile phones and automotive displays that may be used at low temperatures.

Cell Penetrating Peptides and Cationic Antibacterial Peptides

PubMed Central

Rodriguez Plaza, Jonathan G.; Morales-Nava, Rosmarbel; Diener, Christian; Schreiber, Gabriele; Gonzalez, Zyanya D.; Lara Ortiz, Maria Teresa; Ortega Blake, Ivan; Pantoja, Omar; Volkmer, Rudolf; Klipp, Edda; Herrmann, Andreas; Del Rio, Gabriel

2014-01-01

Cell penetrating peptides (CPP) and cationic antibacterial peptides (CAP) have similar physicochemical properties and yet it is not understood how such similar peptides display different activities. To address this question, we used Iztli peptide 1 (IP-1) because it has both CPP and CAP activities. Combining experimental and computational modeling of the internalization of IP-1, we show it is not internalized by receptor-mediated endocytosis, yet it permeates into many different cell types, including fungi and human cells. We also show that IP-1 makes pores in the presence of high electrical potential at the membrane, such as those found in bacteria and mitochondria. These results provide the basis to understand the functional redundancy of CPPs and CAPs. PMID:24706763

Modeling Human Neurological and Neurodegenerative Diseases: From Induced Pluripotent Stem Cells to Neuronal Differentiation and Its Applications in Neurotrauma.

PubMed

Bahmad, Hisham; Hadadeh, Ola; Chamaa, Farah; Cheaito, Katia; Darwish, Batoul; Makkawi, Ahmad-Kareem; Abou-Kheir, Wassim

2017-01-01

With the help of several inducing factors, somatic cells can be reprogrammed to become induced pluripotent stem cell (iPSCs) lines. The success is in obtaining iPSCs almost identical to embryonic stem cells (ESCs), therefore various approaches have been tested and ultimately several ones have succeeded. The importance of these cells is in how they serve as models to unveil the molecular pathways and mechanisms underlying several human diseases, and also in its potential roles in the development of regenerative medicine. They further aid in the development of regenerative medicine, autologous cell therapy and drug or toxicity screening. Here, we provide a comprehensive overview of the recent development in the field of iPSCs research, specifically for modeling human neurological and neurodegenerative diseases, and its applications in neurotrauma. These are mainly characterized by progressive functional or structural neuronal loss rendering them extremely challenging to manage. Many of these diseases, including Parkinson's disease (PD), Huntington's disease (HD), Amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD) have been explored in vitro . The main purpose is to generate patient-specific iPS cell lines from the somatic cells that carry mutations or genetic instabilities for the aim of studying their differentiation potential and behavior. This new technology will pave the way for future development in the field of stem cell research anticipating its use in clinical settings and in regenerative medicine in order to treat various human diseases, including neurological and neurodegenerative diseases.

Differential regulation of insulin-like growth factor-I receptor gene expression by wild type and mutant androgen receptor in prostate cancer cells.

PubMed

Schayek, Hagit; Seti, Hila; Greenberg, Norman M; Sun, Shihua; Werner, Haim; Plymate, Stephen R

2010-07-29

The progression of prostate cancer from an organ-confined, androgen-sensitive disease to a metastatic one is associated with dysregulation of androgen receptor (AR)-regulated target genes and with a decrease in insulin-like growth factor-I receptor (IGF-IR) expression. To investigate the differential effects of wild type (wt) and mutant AR on IGF-IR levels we employed a series of isogenic prostate-derived cell lines and human xenografts. We show that basal and phosphorylated IGF-IR levels progressively decreased as prostate cancer cells became more tumorigenic and metastatic. In addition, we show that wt, but not mutant, AR along with dihydrotestosterone treatment increased IGF-IR promoter activity and endogenous IGF-IR levels. ChIP analysis show enhanced AR binding to the IGF-IR promoter in AR-overexpressing cells. Finally, wt AR-overexpressing cells display an enhanced proliferation rate. In summary, we provide evidence that activated wt AR enhances IGF-IR transcription in prostate cancer cells via a mechanism that involves AR binding to the IGF-IR promoter. AR mutations alter the ability of the mutated protein to regulate IGF-IR expression. Our results suggest that prostate cancer progression is associated with a decrease in IGF-IR expression that could be the result of impaired ability of AR to stimulate IGF-IR gene expression. 2010 Elsevier Ireland Ltd. All rights reserved.

Differential regulation of insulin-like growth factor-I receptor gene expression by wild type and mutant androgen receptor in prostate cancer cells

PubMed Central

Schayek, Hagit; Seti, Hila; Greenberg, Norman M.; Sun, Shihua; Werner, Haim; Plymate, Stephen R.

2010-01-01

The progression of prostate cancer from an organ-confined, androgen-sensitive disease to a metastatic one is associated with dysregulation of androgen receptor (AR)-regulated target genes and with a decrease in insulin-like growth factor-I receptor (IGF-IR) expression. To investigate the differential effects of wild type (wt) and mutant AR on IGF-IR levels we employed a series of isogenic prostate-derived cell lines and human xenografts. We show that basal and phosphorylated IGF-IR levels progressively decreased as prostate cancer cells became more tumorigenic and metastatic. In addition, we show that wt, but not mutant, AR along with dihydrotestosterone treatment increased IGF-IR promoter activity and endogenous IGF-IR levels. ChIP analysis show enhanced AR binding to the IGF-IR promoter in AR-overexpressing cells. Finally, wt AR-overexpressing cells display an enhanced proliferation rate. In summary, we provide evidence that activated wt AR enhances IGF-IR transcription in prostate cancer cells via a mechanism that involves AR binding to the IGF-IR promoter. AR mutations alter the ability of the mutated protein to regulate IGF-IR expression. Our results suggest that prostate cancer progression is associated with a decrease in IGF-IR expression that could be the result of impaired ability of AR to stimulate IGF-IR gene expression. PMID:20417685

UVB light upregulates prostaglandin synthases and prostaglandin receptors in mouse keratinocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Black, Adrienne T.; Gray, Joshua P.; Shakarjian, Michael P.

Prostaglandins belong to a class of cyclic lipid-derived mediators synthesized from arachidonic acid via COX-1, COX-2 and various prostaglandin synthases. Members of this family include prostaglandins such as PGE{sub 2}, PGF{sub 2{alpha}}, PGD{sub 2} and PGI{sub 2} (prostacyclin) as well as thromboxane. In the present studies we analyzed the effects of UVB on prostaglandin production and prostaglandin synthase expression in primary cultures of undifferentiated and calcium-differentiated mouse keratinocytes. Both cell types were found to constitutively synthesize PGE{sub 2}, PGD{sub 2} and the PGD{sub 2} metabolite PGJ{sub 2}. Twenty-four hours after treatment with UVB (25 mJ/cm{sup 2}), production of PGE{sub 2}more » and PGJ{sub 2} increased, while PGD{sub 2} production decreased. This was associated with increased expression of COX-2 mRNA and protein. UVB (2.5-25 mJ/cm{sup 2}) also caused marked increases in mRNA expression for the prostanoid synthases PGDS, mPGES-1, mPGES-2, PGFS and PGIS, as well as expression of receptors for PGE{sub 2} (EP1 and EP2), PGD{sub 2} (DP and CRTH2) and prostacyclin (IP). UVB was more effective in inducing COX-2 and DP in differentiated cells and EP1 and IP in undifferentiated cells. UVB readily activated keratinocyte PI-3-kinase (PI3K)/Akt, JNK and p38 MAP signaling pathways which are known to regulate COX-2 expression. While inhibition of PI3K suppressed UVB-induced mPGES-1 and CRTH2 expression, JNK inhibition suppressed mPGES-1, PGIS, EP2 and CRTH2, and p38 kinase inhibition only suppressed EP1 and EP2. These data indicate that UVB modulates expression of prostaglandin synthases and receptors by distinct mechanisms. Moreover, both the capacity of keratinocytes to generate prostaglandins and their ability to respond to these lipid mediators are stimulated by exposure to UVB.« less

Epigenetic regulation of Atoh1 guides hair cell development in the mammalian cochlea.

PubMed

Stojanova, Zlatka P; Kwan, Tao; Segil, Neil

2015-10-15

In the developing cochlea, sensory hair cell differentiation depends on the regulated expression of the bHLH transcription factor Atoh1. In mammals, if hair cells die they do not regenerate, leading to permanent deafness. By contrast, in non-mammalian vertebrates robust regeneration occurs through upregulation of Atoh1 in the surviving supporting cells that surround hair cells, leading to functional recovery. Investigation of crucial transcriptional events in the developing organ of Corti, including those involving Atoh1, has been hampered by limited accessibility to purified populations of the small number of cells present in the inner ear. We used µChIP and qPCR assays of FACS-purified cells to track changes in the epigenetic status of the Atoh1 locus during sensory epithelia development in the mouse. Dynamic changes in the histone modifications H3K4me3/H3K27me3, H3K9ac and H3K9me3 reveal a progression from poised, to active, to repressive marks, correlating with the onset of Atoh1 expression and its subsequent silencing during the perinatal (P1 to P6) period. Inhibition of acetylation blocked the increase in Atoh1 mRNA in nascent hair cells, as well as ongoing hair cell differentiation during embryonic organ of Corti development ex vivo. These results reveal an epigenetic mechanism of Atoh1 regulation underlying hair cell differentiation and subsequent maturation. Interestingly, the H3K4me3/H3K27me3 bivalent chromatin structure observed in progenitors persists at the Atoh1 locus in perinatal supporting cells, suggesting an explanation for the latent capacity of these cells to transdifferentiate into hair cells, and highlighting their potential as therapeutic targets in hair cell regeneration. © 2015. Published by The Company of Biologists Ltd.

Enhancer regions show high histone H3.3 turnover that changes during differentiation

PubMed Central

Deaton, Aimee M; Gómez-Rodríguez, Mariluz; Mieczkowski, Jakub; Tolstorukov, Michael Y; Kundu, Sharmistha; Sadreyev, Ruslan I; Jansen, Lars ET; Kingston, Robert E

2016-01-01

The organization of DNA into chromatin is dynamic; nucleosomes are frequently displaced to facilitate the ability of regulatory proteins to access specific DNA elements. To gain insight into nucleosome dynamics, and to follow how dynamics change during differentiation, we used a technique called time-ChIP to quantitatively assess histone H3.3 turnover genome-wide during differentiation of mouse ESCs. We found that, without prior assumptions, high turnover could be used to identify regions involved in gene regulation. High turnover was seen at enhancers, as observed previously, with particularly high turnover at super-enhancers. In contrast, regions associated with the repressive Polycomb-Group showed low turnover in ESCs. Turnover correlated with DNA accessibility. Upon differentiation, numerous changes in H3.3 turnover rates were observed, the majority of which occurred at enhancers. Thus, time-ChIP measurement of histone turnover shows that active enhancers are unusually dynamic in ESCs and changes in highly dynamic nucleosomes predominate at enhancers during differentiation. DOI: http://dx.doi.org/10.7554/eLife.15316.001 PMID:27304074

Immunohistochemical localization of DPP10 in rat brain supports the existence of a Kv4/KChIP/DPPL ternary complex in neurons.

PubMed

Wang, Wan-Chen; Cheng, Chau-Fu; Tsaur, Meei-Ling

2015-03-01

Subthreshold A-type K(+) currents (ISA s) have been recorded from the cell bodies of hippocampal and neocortical interneurons as well as neocortical pyramidal neurons. Kv4 channels are responsible for the somatodendritic ISA s. It has been proposed that neuronal Kv4 channels are ternary complexes including pore-forming Kv4 subunits, K(+) channel-interacting proteins (KChIPs), and dipeptidyl peptidase-like proteins (DPPLs). However, colocalization evidence was still lacking. The distribution of DPP10 mRNA in rodent brain has been reported but its protein localization remains unknown. In this study, we generated a DPP10 antibody to label DPP10 protein in adult rat brain by immunohistochemistry. Absent from glia, DPP10 proteins appear mainly in the cell bodies of DPP10(+) neurons, not only at the plasma membrane but also in the cytoplasm. At least 6.4% of inhibitory interneurons in the hippocampus coexpressed Kv4.3, KChIP1, and DPP10, with the highest density in the CA1 strata alveus/oriens/pyramidale and the dentate hilus. Colocalization of Kv4.3/KChIP1/DPP10 was also detected in at least 6.9% of inhibitory interneurons scattered throughout the neocortex. Both hippocampal and neocortical Kv4.3/KChIP1/DPP10(+) inhibitory interneurons expressed parvalbumin or somatostatin, but not calbindin or calretinin. Furthermore, we found colocalization of Kv4.2/Kv4.3/KChIP3/DPP10 in neocortical layer 5 pyramidal neurons and olfactory bulb mitral cells. Together, although DPP10 is also expressed in some brain neurons lacking Kv4 (such as parvalbumin- and somatostatin-positive Golgi cells in the cerebellum), colocalization of DPP10 with Kv4 and KChIP at the plasma membrane of ISA -expressing neuron somata supports the existence of Kv4/KChIP/DPPL ternary complex in vivo. © 2014 Wiley Periodicals, Inc.

Local application of IGFBP5 protein enhanced periodontal tissue regeneration via increasing the migration, cell proliferation and osteo/dentinogenic differentiation of mesenchymal stem cells in an inflammatory niche.

PubMed

Han, Nannan; Zhang, Fengqiu; Li, Guoqing; Zhang, Xiuli; Lin, Xiao; Yang, Haoqing; Wang, Lijun; Cao, Yangyang; Du, Juan; Fan, Zhipeng

2017-09-29

Periodontitis is a widespread infectious disease ultimately resulting in tooth loss. The number of mesenchymal stem cells (MSCs) in patients with periodontitis is decreased, and MSC functions are impaired. Rescuing the impaired function of MSCs in periodontitis is the key for treatment, especially in a manner independent of exogenous MSCs. Our previous study found that overexpressed insulin-like growth factor binding protein 5 (IGFBP5) could promote exogenous MSC-mediated periodontal tissue regeneration. Here, we investigate the role of IGFBP5 protein in MSCs and periodontal tissue regeneration independent of exogenous MSCs in an inflammatory niche. TNFα was used to mimic the inflammatory niche. Lentiviral IGFBP5 shRNA was used to silence IGFBP5 and recombinant human IGFBP5 protein (rhIGFBP5) was used to stimulate the periodontal ligament stem cells (PDLSCs) and bone marrow stem cells (BMSCs). The effects of IGFBP5 on PDLSCs were evaluated using the scratch-simulated wound migration, Transwell chemotaxis, alkaline phosphatase (ALP) activity, Alizarin red staining, Cell Counting Kit-8, Western blot, Real-time PCR, Co-IP and ChIP assays. The swine model of periodontitis was used to investigate the functions of IGFBP5 for periodontal regeneration and its anti-inflammation effect. We discovered that 0.5 ng/ml rhIGFBP5 protein enhanced the migration, chemotaxis, osteo/dentinogenic differentiation and cell proliferation of MSCs under the inflammatory condition. Moreover, 0.5 ng/ml rhIGFBP5 application could rescue the impaired functions of IGFBP5-silenced-MSCs in the inflammatory niche. Furthermore, local injection of rhIGFBP5 could promote periodontal tissue regeneration and relieve the local inflammation in a minipig model of periodontitis. Mechanistically, we found that BCOR negatively regulated the expression of IGFBP5 in MSCs. BCOR formed a protein complex with histone demethylase KDM6B and raised histone K27 methylation in the IGFBP5 promoter. This study revealed that rhIGFBP5 could activate the functions of MSCs in an inflammatory niche, provided insight into the mechanism underlying the activated capacities of MSCs, and identified IGFBP5 as a potential cytokine for improving tissue regeneration and periodontitis treatment independent of exogenous MSCs and its potential application in dental clinic.

Serum from dengue virus-infected patients with and without plasma leakage differentially affects endothelial cells barrier function in vitro

PubMed Central

Baimukanova, Gyulnar; Lanteri, Marion Christine; Keating, Sheila Marie; Moraes Ferreira, Frederico; Heitman, John; Pannuti, Cláudio Sérgio; Pati, Shibani; Romano, Camila Malta; Cerdeira Sabino, Ester

2017-01-01

Background Although most of cases of dengue infections are asymptomatic or mild symptomatic some individuals present warning signs progressing to severe dengue in which plasma leakage is a hallmark. Methodology/Principal findings The present study used Electric Cell-substrate Impedance Sensing (ECIS®) which allows for electrical monitoring of cellular barrier function measuring changes in Transendothelial Electric Resistance (TEER) to investigate the parameters associated with dengue induced leakage. Three groups of individuals were tested: dengue-positives with plasma leakage (leakage), dengue-positives without plasma leakage (no leakage), and dengue-negatives (control). Data show that TEER values of human umbilical vein endothelial cells (HUVECs) was significantly lower after incubation with serum from subjects of the leakage group in comparison to the no leakage or control groups. The serum levels of CXCL1, EGF, eotaxin, IFN-γ, sCD40L, and platelets were significantly decreased in the leakage group, while IL-10, IL-6, and IP-10 levels were significantly increased. We also found a strong correlation between TEER values and augmented levels of IP-10, GM-CSF, IL-1α, and IL-8, as well as decreased levels of CXCL1 and platelets. Conclusions/Significance The present work shows that the magnitude of the immune response contributes to the adverse plasma leakage outcomes in patients and that serum components are important mediators of changes in endothelial homeostasis during dengue infections. In particular, the increased levels of IP-10 and the decreased levels of CXCL1 and platelets seem to play a significant role in the disruption of vascular endothelium associated with leakage outcomes after DENV infection. These findings may have important implications for both diagnostic and therapeutic approaches to predict and mitigate vascular permeabilization in those experiencing the most severe clinical disease outcomes after dengue infection. PMID:28586397

Determining the Roles of Inositol Trisphosphate Receptors in Neurodegeneration: Interdisciplinary Perspectives on a Complex Topic.

PubMed

Takada, Silvia Honda; Ikebara, Juliane Midori; de Sousa, Erica; Cardoso, Débora Sterzeck; Resende, Rodrigo Ribeiro; Ulrich, Henning; Rückl, Martin; Rüdiger, Sten; Kihara, Alexandre Hiroaki

2017-11-01

It is well known that calcium (Ca 2+ ) is involved in the triggering of neuronal death. Ca 2+ cytosolic levels are regulated by Ca 2+ release from internal stores located in organelles, such as the endoplasmic reticulum. Indeed, Ca 2+ transit from distinct cell compartments follows complex dynamics that are mediated by specific receptors, notably inositol trisphosphate receptors (IP3Rs). Ca 2+ release by IP3Rs plays essential roles in several neurological disorders; however, details of these processes are poorly understood. Moreover, recent studies have shown that subcellular location, molecular identity, and density of IP3Rs profoundly affect Ca 2+ transit in neurons. Therefore, regulation of IP3R gene products in specific cellular vicinities seems to be crucial in a wide range of cellular processes from neuroprotection to neurodegeneration. In this regard, microRNAs seem to govern not only IP3Rs translation levels but also subcellular accumulation. Combining new data from molecular cell biology with mathematical modelling, we were able to summarize the state of the art on this topic. In addition to presenting how Ca 2+ dynamics mediated by IP3R activation follow a stochastic regimen, we integrated a theoretical approach in an easy-to-apply, cell biology-coherent fashion. Following the presented premises and in contrast to previously tested hypotheses, Ca 2+ released by IP3Rs may play different roles in specific neurological diseases, including Alzheimer's disease and Parkinson's disease.

IP3R1 deficiency in the cerebellum/brainstem causes basal ganglia-independent dystonia by triggering tonic Purkinje cell firings in mice

PubMed Central

Hisatsune, Chihiro; Miyamoto, Hiroyuki; Hirono, Moritoshi; Yamaguchi, Naohide; Sugawara, Takeyuki; Ogawa, Naoko; Ebisui, Etsuko; Ohshima, Toshio; Yamada, Masahisa; Hensch, Takao K.; Hattori, Mitsuharu; Mikoshiba, Katsuhiko

2013-01-01

The type 1 inositol 1,4,5- trisphosphate receptor (IP3R1) is a Ca2+ channel on the endoplasmic reticulum and is a predominant isoform in the brain among the three types of IP3Rs. Mice lacking IP3R1 show seizure-like behavior; however the cellular and neural circuit mechanism by which IP3R1 deletion causes the abnormal movements is unknown. Here, we found that the conditional knockout mice lacking IP3R1 specifically in the cerebellum and brainstem experience dystonia and show that cerebellar Purkinje cell (PC) firing patterns were coupled to specific dystonic movements. Recordings in freely behaving mice revealed epochs of low and high frequency PC complex spikes linked to body extension and rigidity, respectively. Remarkably, dystonic symptoms were independent of the basal ganglia, and could be rescued by inactivation of the cerebellum, inferior olive or in the absence of PCs. These findings implicate IP3R1-dependent PC firing patterns in cerebellum in motor coordination and the expression of dystonia through the olivo-cerebellar pathway. PMID:24109434

Transdifferentiation and reprogramming: Overview of the processes, their similarities and differences.

PubMed

Cieślar-Pobuda, Artur; Knoflach, Viktoria; Ringh, Mikael V; Stark, Joachim; Likus, Wirginia; Siemianowicz, Krzysztof; Ghavami, Saeid; Hudecki, Andrzej; Green, Jason L; Łos, Marek J

2017-07-01

Reprogramming, or generation of induced pluripotent stem (iPS) cells (functionally similar to embryonic stem cells or ES cells) by the use of transcription factors (typically: Oct3/4, Sox2, c-Myc, Klf4) called "Yamanaka factors" (OSKM), has revolutionized regenerative medicine. However, factors used to induce stemness are also overexpressed in cancer. Both, ES cells and iPS cells cause teratoma formation when injected to tissues. This raises a safety concern for therapies based on iPS derivates. Transdifferentiation (lineage reprogramming, or -conversion), is a process in which one mature, specialized cell type changes into another without entering a pluripotent state. This process involves an ectopic expression of transcription factors and/or other stimuli. Unlike in the case of reprogramming, tissues obtained by this method do not carry the risk of subsequent teratomagenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

A comprehensive comparison of tools for differential ChIP-seq analysis

PubMed Central

Steinhauser, Sebastian; Kurzawa, Nils; Eils, Roland

2016-01-01

ChIP-seq has become a widely adopted genomic assay in recent years to determine binding sites for transcription factors or enrichments for specific histone modifications. Beside detection of enriched or bound regions, an important question is to determine differences between conditions. While this is a common analysis for gene expression, for which a large number of computational approaches have been validated, the same question for ChIP-seq is particularly challenging owing to the complexity of ChIP-seq data in terms of noisiness and variability. Many different tools have been developed and published in recent years. However, a comprehensive comparison and review of these tools is still missing. Here, we have reviewed 14 tools, which have been developed to determine differential enrichment between two conditions. They differ in their algorithmic setups, and also in the range of applicability. Hence, we have benchmarked these tools on real data sets for transcription factors and histone modifications, as well as on simulated data sets to quantitatively evaluate their performance. Overall, there is a great variety in the type of signal detected by these tools with a surprisingly low level of agreement. Depending on the type of analysis performed, the choice of method will crucially impact the outcome. PMID:26764273

Distinct Representations of Magnitude and Spatial Position within Parietal Cortex during Number-Space Mapping.

PubMed

Kanayet, Frank J; Mattarella-Micke, Andrew; Kohler, Peter J; Norcia, Anthony M; McCandliss, Bruce D; McClelland, James L

2018-02-01

Mapping numbers onto space is foundational to mathematical cognition. These cognitive operations are often conceptualized in the context of a "mental number line" and involve multiple brain regions in or near the intraparietal sulcus (IPS) that have been implicated both in numeral and spatial cognition. Here we examine possible differentiation of function within these brain areas in relating numbers to spatial positions. By isolating the planning phase of a number line task and introducing spatiotopic mapping tools from fMRI into mental number line task research, we are able to focus our analysis on the neural activity of areas in anterior IPS (aIPS) previously associated with number processing and on spatiotopically organized areas in and around posterior IPS (pIPS), while participants prepare to place a number on a number line. Our results support the view that the nonpositional magnitude of a numerical symbol is coded in aIPS, whereas the position of a number in space is coded in posterior areas of IPS. By focusing on the planning phase, we are able to isolate activation related to the cognitive, rather than the sensory-motor, aspects of the task. Also, to allow the separation of spatial position from magnitude, we tested both a standard positive number line (0 to 100) and a zero-centered mixed number line (-100 to 100). We found evidence of a functional dissociation between aIPS and pIPS: Activity in aIPS was associated with a landmark distance effect not modulated by spatial position, whereas activity in pIPS revealed a contralateral preference effect.

Nuclear adaptor Ldb1 regulates a transcriptional program essential for the maintenance of hematopoietic stem cells

PubMed Central

Li, LiQi; Jothi, Raja; Cui, Kairong; Lee, Jan Y; Cohen, Tsadok; Gorivodsky, Marat; Tzchori, Itai; Zhao, Yangu; Hayes, Sandra M; Bresnick, Emery H; Zhao, Keji; Westphal, Heiner; Love, Paul E

2013-01-01

The nuclear adaptor Ldb1 functions as a core component of multiprotein transcription complexes that regulate differentiation in diverse cell types. In the hematopoietic lineage, Ldb1 forms a complex with the non–DNA-binding adaptor Lmo2 and the transcription factors E2A, Scl and GATA-1 (or GATA-2). Here we demonstrate a critical and continuous requirement for Ldb1 in the maintenance of both fetal and adult mouse hematopoietic stem cells (HSCs). Deletion of Ldb1 in hematopoietic progenitors resulted in the downregulation of many transcripts required for HSC maintenance. Genome-wide profiling by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) identified Ldb1 complex–binding sites at highly conserved regions in the promoters of genes involved in HSC maintenance. Our results identify a central role for Ldb1 in regulating the transcriptional program responsible for the maintenance of HSCs. PMID:21186366

Nuclear adaptor Ldb1 regulates a transcriptional program essential for the maintenance of hematopoietic stem cells.

PubMed

Li, LiQi; Jothi, Raja; Cui, Kairong; Lee, Jan Y; Cohen, Tsadok; Gorivodsky, Marat; Tzchori, Itai; Zhao, Yangu; Hayes, Sandra M; Bresnick, Emery H; Zhao, Keji; Westphal, Heiner; Love, Paul E

2011-02-01

The nuclear adaptor Ldb1 functions as a core component of multiprotein transcription complexes that regulate differentiation in diverse cell types. In the hematopoietic lineage, Ldb1 forms a complex with the non-DNA-binding adaptor Lmo2 and the transcription factors E2A, Scl and GATA-1 (or GATA-2). Here we demonstrate a critical and continuous requirement for Ldb1 in the maintenance of both fetal and adult mouse hematopoietic stem cells (HSCs). Deletion of Ldb1 in hematopoietic progenitors resulted in the downregulation of many transcripts required for HSC maintenance. Genome-wide profiling by chromatin immunoprecipitation followed by sequencing (ChIP-Seq) identified Ldb1 complex-binding sites at highly conserved regions in the promoters of genes involved in HSC maintenance. Our results identify a central role for Ldb1 in regulating the transcriptional program responsible for the maintenance of HSCs.

Measuring Sister Chromatid Cohesion Protein Genome Occupancy in Drosophila melanogaster by ChIP-seq.

PubMed

Dorsett, Dale; Misulovin, Ziva

2017-01-01

This chapter presents methods to conduct and analyze genome-wide chromatin immunoprecipitation of the cohesin complex and the Nipped-B cohesin loading factor in Drosophila cells using high-throughput DNA sequencing (ChIP-seq). Procedures for isolation of chromatin, immunoprecipitation, and construction of sequencing libraries for the Ion Torrent Proton high throughput sequencer are detailed, and computational methods to calculate occupancy as input-normalized fold-enrichment are described. The results obtained by ChIP-seq are compared to those obtained by ChIP-chip (genomic ChIP using tiling microarrays), and the effects of sequencing depth on the accuracy are analyzed. ChIP-seq provides similar sensitivity and reproducibility as ChIP-chip, and identifies the same broad regions of occupancy. The locations of enrichment peaks, however, can differ between ChIP-chip and ChIP-seq, and low sequencing depth can splinter broad regions of occupancy into distinct peaks.

Science spin: iPS cell research in the news.

PubMed

Caulfield, T; Rachul, C

2011-05-01

Big scientific developments have always been spun to meet particular social agendas. We have seen it in the context of global warming, nuclear power, and genetically modified organisms. But few stories illustrate the phenomenon of spin as well as the reaction, and concomitant media coverage, that surrounded the November 2007 announcement regarding the reprogramming of skin cells to produce cells with qualities comparable to those of human embryonic stem cells (hESCs) known as induced pluripotent stem (iPS) cells.

DREAM Mediated Regulation of GCM1 in the Human Placental Trophoblast

PubMed Central

Baczyk, Dora; Kibschull, Mark; Mellstrom, Britt; Levytska, Khrystyna; Rivas, Marcos; Drewlo, Sascha; Lye, Stephen J.; Naranjo, Jose R.; Kingdom, John C. P.

2013-01-01

The trophoblast transcription factor glial cell missing-1 (GCM1) regulates differentiation of placental cytotrophoblasts into the syncytiotrophoblast layer in contact with maternal blood. Reduced placental expression of GCM1 and abnormal syncytiotrophoblast structure are features of hypertensive disorder of pregnancy – preeclampsia. In-silico techniques identified the calcium-regulated transcriptional repressor – DREAM (Downstream Regulatory Element Antagonist Modulator) - as a candidate for GCM1 gene expression. Our objective was to determine if DREAM represses GCM1 regulated syncytiotrophoblast formation. EMSA and ChIP assays revealed a direct interaction between DREAM and the GCM1 promoter. siRNA-mediated DREAM silencing in cell culture and placental explant models significantly up-regulated GCM1 expression and reduced cytotrophoblast proliferation. DREAM calcium dependency was verified using ionomycin. Furthermore, the increased DREAM protein expression in preeclamptic placental villi was predominantly nuclear, coinciding with an overall increase in sumolylated DREAM and correlating inversely with GCM1 levels. In conclusion, our data reveal a calcium-regulated pathway whereby GCM1-directed villous trophoblast differentiation is repressed by DREAM. This pathway may be relevant to disease prevention via calcium-supplementation. PMID:23300953

Impact of α-targeted radiation therapy on gene expression in a pre-clinical model for disseminated peritoneal disease when combined with paclitaxel.

PubMed

Yong, Kwon Joong; Milenic, Diane E; Baidoo, Kwamena E; Brechbiel, Martin W

2014-01-01

To better understand the molecular basis of the enhanced cell killing effected by the combined modality of paclitaxel and ²¹²Pb-trastuzumab (Pac/²¹²Pb-trastuzumab), gene expression in LS-174T i.p. xenografts was investigated 24 h after treatment. Employing a real time quantitative PCR array (qRT-PCR array), 84 DNA damage response genes were quantified. Differentially expressed genes following therapy with Pac/²¹²Pb-trastuzumab included those involved in apoptosis (BRCA1, CIDEA, GADD45α, GADD45γ, GML, IP6K3, PCBP4, PPP1R15A, RAD21, and p73), cell cycle (BRCA1, CHK1, CHK2, GADD45α, GML, GTSE1, NBN, PCBP4, PPP1R15A, RAD9A, and SESN1), and damaged DNA repair (ATRX, BTG2, EXO1, FEN1, IGHMBP2, OGG1, MSH2, MUTYH, NBN, PRKDC, RAD21, and p73). This report demonstrates that the increased stressful growth arrest conditions induced by the Pac/²¹²Pb-trastuzumab treatment suppresses cell proliferation through the regulation of genes which are involved in apoptosis and damaged DNA repair including single and double strand DNA breaks. Furthermore, the study demonstrates that ²¹²Pb-trastuzumab potentiation of cell killing efficacy results from the perturbation of genes related to the mitotic spindle checkpoint and BASC (BRCA1-associated genome surveillance complex), suggesting cross-talk between DNA damage repair and the spindle damage response.

Interferon-gamma inducible protein 10 (IP10) induced cisplatin resistance of HCC after liver transplantation through ER stress signaling pathway

PubMed Central

Geng, Wei; Lo, Chung-Mau; Ng, Kevin T.P.; Ling, Chang-Chun; Qi, Xiang; Li, Chang-Xian; Zhai, Yuan; Liu, Xiao-Bing; Ma, Yuen-Yuen; Man, Kwan

2015-01-01

Tumor recurrence remains an obstacle after liver surgery, especially in living donor liver transplantation (LDLT) for patients with hepatocellular carcinoma (HCC). The acute-phase liver graft injury might potentially induce poor response to chemotherapy in recurrent HCC after liver transplantation. We here intended to explore the mechanism and to identify a therapeutic target to overcome such chemoresistance. The associations among graft injury, overexpression of IP10 and multidrug resistant genes were investigated in a rat liver transplantation model, and further validated in clinical cohort. The role of IP10 on HCC cell proliferation and tumor growth under chemotherapy was studied both in vitro and in vivo. The underlying mechanism was revealed by detecting the activation of endoplasmic reticulum (ER) stress signaling pathways. Moreover, the effect of IP10 neutralizing antibody sensitizing cisplatin treatment was further explored. In rat liver transplantation model, significant up-regulation of IP10 associated with multidrug resistant genes was found in small-for-size liver graft. Clinically, high expression of circulating IP10 was significant correlated with tumor recurrence in HCC patients underwent LDLT. Overexpression of IP10 promoted HCC cell proliferation and tumor growth under cisplatin treatment by activation of ATF6/Grp78 signaling. IP10 neutralizing antibody sensitized cisplatin treatment in nude mice. The overexpression of IP10, which induced by liver graft injury, may lead to cisplatin resistance via ATF6/Grp78 ER stress signaling pathway. IP10 neutralizing antibody could be a potential adjuvant therapy to sensitize cisplatin treatment. PMID:26336986

Interleukin-18 enhances IL-18R/Nox1 binding, and mediates TRAF3IP2-dependent smooth muscle cell migration. Inhibition by simvastatin

PubMed Central

Valente, Anthony J.; Yoshida, Tadashi; Izadpanah, Reza; Delafontaine, Patrice; Siebenlist, Ulrich; Chandrasekar, Bysani

2013-01-01

We investigated the role of TRAF3 interacting protein 2 (TRAF3IP2), a redox-sensitive adapter protein and an upstream regulator of IKK and JNK in interleukin (IL)-18 induced smooth muscle cell migration, and the mechanism of its inhibition by simvastatin. The pleiotropic cytokine IL-18 induced human coronary artery SMC migration through the induction of TRAF3IP2. IL-18 induced Nox1-dependent ROS generation, TRAF3IP2 expression, and IKK/NF-κB and JNK/AP-1 activation. IL-18 induced its own expression and that of its receptor subunit IL-18Rα. Using co-IP/IB and GST pull-down assays, we show for the first time that the subunits of the IL-18R heterodimer physically associate with Nox1 under basal conditions, and IL-18 appears to enhance their binding. Importantly, the HMG-coA reductase inhibitor simvastatin attenuated IL-18-induced TRAF3IP2 induction. These inhibitory effects were reversed by mevalonate and geranylgeranylpyrophosphate (GGPP), but not by farnesylpyrophosphate (FPP). Interestingly, simvastatin, GGPP, FPP, or Rac1 inhibition did not modulate ectopically expressed TRAF3IP2. The promigratory effects of IL-18 are mediated through TRAF3IP2 in a redox-sensitive manner, and this may involve IL-18R/Nox1 physical association. Further, Simvastatin inhibits inducible, but not ectopically-expressed TRAF3IP2. Targeting TRAF3IP2 may blunt progression of hyperplastic vascular diseases in vivo. PMID:23541442

Engraftment of donor mesenchymal stem cells in chimeric BXSB includes vascular endothelial cells and hepatocytes.

PubMed

Jones, Olcay Y; Gok, Faysal; Rushing, Elisabeth J; Horkayne-Szakaly, Iren; Ahmed, Atif A

2011-01-01

Somatic tissue engraftment was studied in BXSB mice treated with mesenchymal stem cell transplantation. Hosts were conditioned with nonlethal radiation prior to introducing donor cells from major histocompatibility complex-matched green fluorescent protein transgenic mice. Transplant protocols differed for route of injection, ie, intravenous (i.v.) versus intraperitoneal (i.p.), and source of mesenchymal stem cells, ie, unfractionated bone marrow cells, ex vivo expanded mesenchymal stem cells, or bone chips. Tissue chimerism was determined after short (10-12 weeks) or long (62 weeks) posttransplant follow-up by immunohistochemistry for green fluorescent protein. Engraftment of endothelial cells was seen in several organs including liver sinusoidal cells in i.v. treated mice with ex vivo expanded mesenchymal stem cells or with unfractionated bone marrow cells. Periportal engraftment of liver hepatocytes, but not engraftment of endothelial cells, was found in mice injected i.p. with bone chips. Engraftment of adipocytes was a common denominator in both i.v. and i.p. routes and occurred during early phases post-transplant. Disease control was more robust in mice that received both i.v. bone marrow and i.p. bone chips compared to mice that received i.v. bone marrow alone. Thus, the data support potential use of mesenchymal stem cell transplant for treatment of severe lupus. Future studies are needed to optimize transplant conditions and tailor protocols that may in part be guided by fat and endothelial biomarkers. Furthermore, the role of liver chimerism in disease control and the nature of cellular communication among donor hematopoietic and mesenchymal stem cells in a chimeric host merit further investigation.

The histone H2A deubiquitinase Usp16 regulates hematopoiesis and hematopoietic stem cell function.

PubMed

Gu, Yue; Jones, Amanda E; Yang, Wei; Liu, Shanrun; Dai, Qian; Liu, Yudong; Swindle, C Scott; Zhou, Dewang; Zhang, Zhuo; Ryan, Thomas M; Townes, Tim M; Klug, Christopher A; Chen, Dongquan; Wang, Hengbin

2016-01-05

Epigenetic mechanisms play important regulatory roles in hematopoiesis and hematopoietic stem cell (HSC) function. Subunits of polycomb repressive complex 1 (PRC1), the major histone H2A ubiquitin ligase, are critical for both normal and pathological hematopoiesis; however, it is unclear which of the several counteracting H2A deubiquitinases functions along with PRC1 to control H2A ubiquitination (ubH2A) level and regulates hematopoiesis in vivo. Here we investigated the function of Usp16 in mouse hematopoiesis. Conditional deletion of Usp16 in bone marrow resulted in a significant increase of global ubH2A level and lethality. Usp16 deletion did not change HSC number but was associated with a dramatic reduction of mature and progenitor cell populations, revealing a role in governing HSC lineage commitment. ChIP- and RNA-sequencing studies in HSC and progenitor cells revealed that Usp16 bound to many important hematopoietic regulators and that Usp16 deletion altered the expression of genes in transcription/chromosome organization, immune response, hematopoietic/lymphoid organ development, and myeloid/leukocyte differentiation. The altered gene expression was partly rescued by knockdown of PRC1 subunits, suggesting that Usp16 and PRC1 counterbalance each other to regulate cellular ubH2A level and gene expression in the hematopoietic system. We further discovered that knocking down Cdkn1a (p21cip1), a Usp16 target and regulated gene, rescued the altered cell cycle profile and differentiation defect of Usp16-deleted HSCs. Collectively, these studies identified Usp16 as one of the histone H2A deubiquitinases, which coordinates with the H2A ubiquitin ligase PRC1 to regulate hematopoiesis, and revealed cell cycle regulation by Usp16 as key for HSC differentiation.

Localization and socialization: Experimental insights into the functional architecture of IP3 receptors

NASA Astrophysics Data System (ADS)

Diambra, Luis; Marchant, Jonathan S.

2009-09-01

Inositol 1,4,5-trisphosphate (IP3)-evoked Ca2+ signals display great spatiotemporal malleability. This malleability depends on diversity in both the cellular organization and in situ functionality of IP3 receptors (IP3Rs) that regulate Ca2+ release from the endoplasmic reticulum (ER). Recent experimental data imply that these considerations are not independent, such that—as with other ion channels—the local organization of IP3Rs impacts their functionality, and reciprocally IP3R activity impacts their organization within native ER membranes. Here, we (i) review experimental data that lead to our understanding of the "functional architecture" of IP3Rs within the ER, (ii) propose an updated terminology to span the organizational hierarchy of IP3Rs observed in intact cells, and (iii) speculate on the physiological significance of IP3R socialization in Ca2+ dynamics, and consequently the emerging need for modeling studies to move beyond gridded, planar, and static simulations of IP3R clustering even over short experimental timescales.

Overexpression of hypoxia-inducible factor 1 alpha improves immunomodulation by dental mesenchymal stem cells.

PubMed

Martinez, Victor G; Ontoria-Oviedo, Imelda; Ricardo, Carolina P; Harding, Sian E; Sacedon, Rosa; Varas, Alberto; Zapata, Agustin; Sepulveda, Pilar; Vicente, Angeles

2017-09-29

Human dental mesenchymal stem cells (MSCs) are considered as highly accessible and attractive MSCs for use in regenerative medicine, yet some of their features are not as well characterized as other MSCs. Hypoxia-preconditioning and hypoxia-inducible factor 1 (HIF-1) alpha overexpression significantly improves MSC therapeutics, but the mechanisms involved are not fully understood. In the present study, we characterize immunomodulatory properties of dental MSCs and determine changes in their ability to modulate adaptive and innate immune populations after HIF-1 alpha overexpression. Human dental MSCs were stably transduced with green fluorescent protein (GFP-MSCs) or GFP-HIF-1 alpha lentivirus vectors (HIF-MSCs). A hypoxic-like metabolic profile was confirmed by mitochondrial and glycolysis stress test. Capacity of HIF-MSCs to modulate T-cell activation, dendritic cell differentiation, monocyte migration, and polarizations towards macrophages and natural killer (NK) cell lytic activity was assessed by a number of functional assays in co-cultures. The expression of relevant factors were determined by polymerase chain reaction (PCR) analysis and enzyme-linked immunosorbent assay (ELISA). While HIF-1 alpha overexpression did not modify the inhibition of T-cell activation by MSCs, HIF-MSCs impaired dendritic cell differentiation more efficiently. In addition, HIF-MSCs showed a tendency to induce higher attraction of monocytes, which differentiate into suppressor macrophages, and exhibited enhanced resistance to NK cell-mediated lysis, which supports the improved therapeutic capacity of HIF-MSCs. HIF-MSCs also displayed a pro-angiogenic profile characterized by increased expression of CXCL12/SDF1 and CCL5/RANTES and complete loss of CXCL10/IP10 transcription. Immunomodulation and expression of trophic factors by dental MSCs make them perfect candidates for cell therapy. Overexpression of HIF-1 alpha enhances these features and increases their resistance to allogenic NK cell lysis and, hence, their potential in vivo lifespan. Our results further support the use of HIF-1 alpha-expressing dental MSCs for cell therapy in tissue injury and immune disorders.

Generation of three-dimensional retinal organoids expressing rhodopsin and S- and M-cone opsins from mouse stem cells.

PubMed

Ueda, Kaori; Onishi, Akishi; Ito, Shin-Ichiro; Nakamura, Makoto; Takahashi, Masayo

2018-01-22

Three-dimensional retinal organoids can be differentiated from embryonic stem cells/induced pluripotent stem cells (ES/iPS cells) under defined medium conditions. We modified the serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq) culture procedure to obtain retinal organoids expressing more rod photoreceptors and S- and M-cone opsins. Retinal organoids differentiated from mouse Nrl-eGFP iPS cells were cultured in various mediums during photoreceptor development. To promote rod photoreceptor development, organoids were maintained in media containing 9-cis retinoic acids (9cRA). To obtain retinal organoids with M-opsin expression, we cultured in medium with 1% fetal bovine serum (FBS) supplemented with T3, BMP4, and DAPT. Section immunohistochemistry was performed to visualize the expression of photoreceptor markers. In three-dimensional (3D) retinas exposed to 9cRA, rhodopsin was expressed earlier and S-cone opsins were suppressed. We could maintain 3D retinas up to DD 35 in culture media with 1% FBS. The 3D retinas expressed rhodopsin, S- and M-opsins, but most cone photoreceptors expressed either S- or M-opsins. By modifying culture conditions in the SFEBq protocol, we obtained rod-dominated 3D retinas and S- and M-opsin expressing 3D retinas. Copyright © 2017 Elsevier Inc. All rights reserved.

Incorporation of DPP6a and DPP6K variants in ternary Kv4 channel complex reconstitutes properties of A-type K current in rat cerebellar granule cells.

PubMed

Jerng, Henry H; Pfaffinger, Paul J

2012-01-01

Dipeptidyl peptidase-like protein 6 (DPP6) proteins co-assemble with Kv4 channel α-subunits and Kv channel-interacting proteins (KChIPs) to form channel protein complexes underlying neuronal somatodendritic A-type potassium current (I(SA)). DPP6 proteins are expressed as N-terminal variants (DPP6a, DPP6K, DPP6S, DPP6L) that result from alternative mRNA initiation and exhibit overlapping expression patterns. Here, we study the role DPP6 variants play in shaping the functional properties of I(SA) found in cerebellar granule (CG) cells using quantitative RT-PCR and voltage-clamp recordings of whole-cell currents from reconstituted channel complexes and native I(SA) channels. Differential expression of DPP6 variants was detected in rat CG cells, with DPP6K (41 ± 3%)>DPP6a (33 ± 3%)>DPP6S (18 ± 2%)>DPP6L (8 ± 3%). To better understand how DPP6 variants shape native neuronal I(SA), we focused on studying interactions between the two dominant variants, DPP6K and DPP6a. Although previous studies did not identify unique functional effects of DPP6K, we find that the unique N-terminus of DPP6K modulates the effects of KChIP proteins, slowing recovery and producing a negative shift in the steady-state inactivation curve. By contrast, DPP6a uses its distinct N-terminus to directly confer rapid N-type inactivation independently of KChIP3a. When DPP6a and DPP6K are co-expressed in ratios similar to those found in CG cells, their distinct effects compete in modulating channel function. The more rapid inactivation from DPP6a dominates during strong depolarization; however, DPP6K produces a negative shift in the steady-state inactivation curve and introduces a slow phase of recovery from inactivation. A direct comparison to the native CG cell I(SA) shows that these mixed effects are present in the native channels. Our results support the hypothesis that the precise expression and co-assembly of different auxiliary subunit variants are important factors in shaping the I(SA) functional properties in specific neuronal populations.

Use of PCR-Based Methods for Rapid Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis

PubMed Central

Torriani, Sandra; Zapparoli, Giacomo; Dellaglio, Franco

1999-01-01

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412T, which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains. PMID:10508059

Use of PCR-based methods for rapid differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis.

PubMed

Torriani, S; Zapparoli, G; Dellaglio, F

1999-10-01

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412(T), which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains.

Reduced incidence of interstitial pneumonitis after allogeneic hematopoietic stem cell transplantation using a modified technique of total body irradiation.

PubMed

Chiang, Yun; Tsai, Cheng-Hong; Kuo, Sung-Hsin; Liu, Chieh-Yu; Yao, Ming; Li, Chi-Cheng; Huang, Shang-Yi; Ko, Bor-Sheng; Lin, Chien-Ting; Hou, Hsin-An; Chou, Wen-Chien; Liu, Jia-Hau; Lin, Chien-Chin; Wu, Shang-Ju; Hsu, Szu-Chun; Chen, Yao-Chang; Lin, Kai-Hsin; Lin, Dong-Tsamn; Chou, Hsien-Tang; Lu, Meng-Yu; Yang, Yung-Li; Chang, Hsiu-Hao; Liu, Ming-Chih; Liao, Xiu-Wen; Wu, Jian-Kuen; Chou, Sheng-Chieh; Cheng, Chieh-Lung; Chen, Chien-Yuan; Tsay, Woei; Tien, Hwei-Fang; Tang, Jih-Luh; Chen, Yu-Hsuan

2016-11-10

Allogeneic hematopoietic stem cell transplantation is a curative-intent treatment for patients with high-risk hematologic diseases. However, interstitial pneumonitis (IP) and other toxicities remain major concerns after total body irradiation (TBI). We have proposed using linear accelerators with rice-bag compensators for intensity modulation (IM-TBI), as an alternative to the traditional cobalt-60 teletherapy with lung-shielding technique (Co-TBI). Patients who received a TBI-based myeloablative conditioning regimen between 1995 and 2014 were recruited consecutively. Before March 2007, TBI was delivered using Co-TBI (n = 181); afterward, TBI was administered using IM-TBI (n = 126). Forty-four patients developed IP; of these cases, 19 were idiopathic. The IP-related mortality rate was 50% in the total IP cohort and 63% in the idiopathic subgroup. The 1-year cumulative incidences of IP and idiopathic IP were 16.5% and 7.4%, respectively; both rates were significantly higher in the Co-TBI group than in the IM-TBI group. Multivariate analysis revealed that Co-TBI was an independent prognostic factor for both total and idiopathic IP. In the acute myeloid leukemia subgroup, patients with different TBI techniques had similar outcomes for both overall and relapse-free survival. In conclusion, IM-TBI is an easy and effective TBI technique that could substantially reduce the complication rate of IP without compromising treatment efficacy.

Integrative genomics identifies molecular alterations that challenge the linear model of melanoma progression.

PubMed

Rose, Amy E; Poliseno, Laura; Wang, Jinhua; Clark, Michael; Pearlman, Alexander; Wang, Guimin; Vega Y Saenz de Miera, Eleazar C; Medicherla, Ratna; Christos, Paul J; Shapiro, Richard; Pavlick, Anna; Darvishian, Farbod; Zavadil, Jiri; Polsky, David; Hernando, Eva; Ostrer, Harry; Osman, Iman

2011-04-01

Superficial spreading melanoma (SSM) and nodular melanoma (NM) are believed to represent sequential phases of linear progression from radial to vertical growth. Several lines of clinical, pathologic, and epidemiologic evidence suggest, however, that SSM and NM might be the result of independent pathways of tumor development. We utilized an integrative genomic approach that combines single nucleotide polymorphism array (6.0; Affymetrix) with gene expression array (U133A 2.0; Affymetrix) to examine molecular differences between SSM and NM. Pathway analysis of the most differentially expressed genes between SSM and NM (N = 114) revealed significant differences related to metabolic processes. We identified 8 genes (DIS3, FGFR1OP, G3BP2, GALNT7, MTAP, SEC23IP, USO1, and ZNF668) in which NM/SSM-specific copy number alterations correlated with differential gene expression (P < 0.05; Spearman's rank). SSM-specific genomic deletions in G3BP2, MTAP, and SEC23IP were independently verified in two external data sets. Forced overexpression of metabolism-related gene MTAP (methylthioadenosine phosphorylase) in SSM resulted in reduced cell growth. The differential expression of another metabolic-related gene, aldehyde dehydrogenase 7A1 (ALDH7A1), was validated at the protein level by using tissue microarrays of human melanoma. In addition, we show that the decreased ALDH7A1 expression in SSM may be the result of epigenetic modifications. Our data reveal recurrent genomic deletions in SSM not present in NM, which challenge the linear model of melanoma progression. Furthermore, our data suggest a role for altered regulation of metabolism-related genes as a possible cause of the different clinical behavior of SSM and NM.

Integrative genomics identifies molecular alterations that challenge the linear model of melanoma progression

PubMed Central

Rose, Amy E.; Poliseno, Laura; Wang, Jinhua; Clark, Michael; Pearlman, Alexander; Wang, Guimin; Vega y Saenz de Miera, Eleazar C.; Medicherla, Ratna; Christos, Paul J.; Shapiro, Richard; Pavlick, Anna; Darvishian, Farbod; Zavadil, Jiri; Polsky, David; Hernando, Eva; Ostrer, Harry; Osman, Iman

2011-01-01

Superficial spreading melanoma (SSM) and nodular melanoma (NM) are believed to represent sequential phases of linear progression from radial to vertical growth. Several lines of clinical, pathological and epidemiologic evidence suggest, however, that SSM and NM might be the result of independent pathways of tumor development. We utilized an integrative genomic approach that combines single nucleotide polymorphism array (SNP 6.0, Affymetrix) with gene expression array (U133A 2.0, Affymetrix) to examine molecular differences between SSM and NM. Pathway analysis of the most differentially expressed genes between SSM and NM (N=114) revealed significant differences related to metabolic processes. We identified 8 genes (DIS3, FGFR1OP, G3BP2, GALNT7, MTAP, SEC23IP, USO1, ZNF668) in which NM/SSM-specific copy number alterations correlated with differential gene expression (P<0.05, Spearman’s rank). SSM-specific genomic deletions in G3BP2, MTAP, and SEC23IP were independently verified in two external data sets. Forced overexpression of metabolism-related gene methylthioadenosine phosphorylase (MTAP) in SSM resulted in reduced cell growth. The differential expression of another metabolic related gene, aldehyde dehydrogenase 7A1 (ALDH7A1), was validated at the protein level using tissue microarrays of human melanoma. In addition, we show that the decreased ALDH7A1 expression in SSM may be the result of epigenetic modifications. Our data reveal recurrent genomic deletions in SSM not present in NM, which challenge the linear model of melanoma progression. Furthermore, our data suggest a role for altered regulation of metabolism-related genes as a possible cause of the different clinical behavior of SSM and NM. PMID:21343389

The prevalence of human papillomavirus infection in Iranian patients with sinonasal inverted papilloma.

PubMed

Jalilvand, Somayeh; Saidi, Masoumeh; Shoja, Zabihollah; Ghavami, Nastaran; Hamkar, Rasool

2016-03-01

Inverted papilloma (IP) is an uncommon disease which arises in the mucosal membrane of the nasal cavity and paranasal sinus. It has been proposed that human papillomavirus (HPV) is the causal agent in the pathogenesis of IP and plays a key role in the progression from benign IP to malignancy. As there are no prior studies that focus on an Iranian population, this study intended to characterize the prevalence of HPV types in benign and malignant forms of IP. In this retrospective study, we included a total of 40 IP patients [37 benign IP and 3 IP/squamous cell carcinoma (SCC)] who were referred to Amiralam Hospital in Tehran from 2004-2006. HPV was detected in 18.9% and 100% of IP and IP/SCC cases, respectively. In all HPV positive cases of IP and IP/SCC cases, HPV6/11 and HPV16/18 were detected, respectively. Therefore, HPV types were different between the IP and IP/SCC patients, and this difference was statistically significant (p = 0.002). This study suggests that HPV6 and 11 may be involved in the development of IP, but HPV16 and 18 likely play an important role in the progression from benign to malignant form of IP. Copyright © 2015. Published by Elsevier Taiwan LLC.

Tumor tropism of intravenously injected human-induced pluripotent stem cell-derived neural stem cells and their gene therapy application in a metastatic breast cancer model.

PubMed

Yang, Jing; Lam, Dang Hoang; Goh, Sally Sallee; Lee, Esther Xingwei; Zhao, Ying; Tay, Felix Chang; Chen, Can; Du, Shouhui; Balasundaram, Ghayathri; Shahbazi, Mohammad; Tham, Chee Kian; Ng, Wai Hoe; Toh, Han Chong; Wang, Shu

2012-05-01

Human pluripotent stem cells can serve as an accessible and reliable source for the generation of functional human cells for medical therapies. In this study, we used a conventional lentiviral transduction method to derive human-induced pluripotent stem (iPS) cells from primary human fibroblasts and then generated neural stem cells (NSCs) from the iPS cells. Using a dual-color whole-body imaging technology, we demonstrated that after tail vein injection, these human NSCs displayed a robust migratory capacity outside the central nervous system in both immunodeficient and immunocompetent mice and homed in on established orthotopic 4T1 mouse mammary tumors. To investigate whether the iPS cell-derived NSCs can be used as a cellular delivery vehicle for cancer gene therapy, the cells were transduced with a baculoviral vector containing the herpes simplex virus thymidine kinase suicide gene and injected through tail vein into 4T1 tumor-bearing mice. The transduced NSCs were effective in inhibiting the growth of the orthotopic 4T1 breast tumor and the metastatic spread of the cancer cells in the presence of ganciclovir, leading to prolonged survival of the tumor-bearing mice. The use of iPS cell-derived NSCs for cancer gene therapy bypasses the sensitive ethical issue surrounding the use of cells derived from human fetal tissues or human embryonic stem cells. This approach may also help to overcome problems associated with allogeneic transplantation of other types of human NSCs. Copyright © 2012 AlphaMed Press.

Impact of Environmental Exposures on the Mutagenicity/Carcinogenicity of Heterocyclic Amines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Felton, J S; Knize, M G; Bennett, L M

2003-12-19

Carcinogenic heterocyclic amines are produced from overcooked foods and are highly mutagenic in most short-term test systems. One of the most abundant of these amines, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), induces breast, colon and prostate tumors in rats. Human dietary epidemiology studies suggest a strong correlation between either meat consumption or well-done muscle meat consumption and cancers of the colon, breast, stomach, lung and esophagus. For over 20 years our laboratory has helped define the human exposure to these dietary carcinogens. In this report we describe how various environmental exposures may modulate the risk from exposure to heterocyclic amines, especially PhIP. To assessmore » the impact of foods on PhIP metabolism in humans, we developed an LC/MS/MS method to analyze the four major PhIP urinary metabolites following the consumption of a single portion of grilled chicken. Adding broccoli to the volunteers' diet altered the kinetics of PhIP metabolism. At the cellular level we have found that PhIP itself stimulates a significant estrogenic response in MCF-7 cells, but even more interestingly, co-incubation of the cells with herbal teas appear to enhance the response. Numerous environmental chemicals found in food or the atmosphere can impact the exposure, metabolism, and cell proliferation response of heterocyclic amines.« less

Chloride secretagogues stimulate inositol phosphate formation in shark rectal gland tubules cultured in suspension

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ecay, T.W.; Valentich, J.D.

1991-03-01

Neuroendocrine activation of transepithelial chloride secretion by shark rectal gland cells is associated with increases in cellular cAMP, cGMP, and free calcium concentrations. We report here on the effects of several chloride secretagogues on inositol phosphate formation in cultured rectal gland tubules. Vasoactive intestinal peptide (VIP), atriopeptin (AP), and ionomycin increase the total inositol phosphate levels of cultured tubules, as measured by ion exchange chromatography. Forskolin, a potent chloride secretagogue, has no effect on inositol phosphate formation. The uptake of {sup 3}H-myo-inositol into phospholipids is very slow, preventing the detection of increased levels of inositol trisphosphate. However, significant increases inmore » inositol monophosphate (IP1) and inositol biphosphate (IP2) were measured. The time course of VIP- and AP-stimulated IP1 and IP2 formation is similar to the effects of these agents on the short-circuit current responses of rectal gland monolayer cultures. In addition, aluminum fluoride, an artificial activator of guanine nucleotide-binding proteins, stimulates IP1 and IP2 formation. We conclude that rectal gland cells contain VIP and AP receptors coupled to the activation of phospholipase C. Coupling may be mediated by G-proteins. Receptor-stimulated increases in inositol phospholipid metabolism is one mechanism leading to increased intracellular free calcium concentrations, an important regulatory event in the activation of transepithelial chloride secretion by shark rectal gland epithelial cells.« less

The influence of implantoplasty on the diameter, chemical surface composition, and biocompatibility of titanium implants.

PubMed

Schwarz, Frank; John, Gordon; Becker, Jürgen

2017-09-01

The objective of the study was to assess the influence of implantoplasty (IP) on the diameter, chemical surface composition, and biocompatibility of titanium implants in vitro. Twenty soft tissue-level (TL; machined transmucosal-M and rough endosseous part-SLA) and 20 bone-level (BL; SLA) implants were allocated to IP covering 3 or 6 mm of the structured surface (SLA) area. The samples were subjected to diameter, energy-dispersive X-ray spectroscopy (EDX), and cell viability (ginigval fibroblasts, 6 days) assessments. Median diameter reductions varied between 0.1 (TL 3 mm) and 0.2 mm (TL 6 mm). EDX analysis revealed that IP and M surfaces were characterized by a comparable quantity (Wt%) of elements C, O, Na, Cl, K, and Si, but a significantly different quantity of elements Ti and Al. When compared to SLA surfaces, significant differences were noted for elements C, O, Na, Ti, and Al. At BL implants, the extension of IP (i.e., 3 to 6 mm) was associated with a significant increase in cell viability. IP applied to SLA implants was associated with (i) a minimal diameter reduction, (ii) an undisturbed cell viability, and (iii) a chemical elemental composition comparable to M surfaces. This specific IP procedure appears to be suitable for the management of exposed SLA implant surfaces.

Substance P receptor desensitization requires receptor activation but not phospholipase C

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sugiya, Hiroshi; Putney, J.W. Jr.

1988-08-01

Previous studies have shown that exposure of parotid acinar cells to substance P at 37{degree}C results in activation of phospholipase C, formation of ({sup 3}H)inositol 1,4,5-trisphosphate (IP{sub 3}), and persistent desensitization of the substance P response. In cells treated with antimycin in medium containing glucose, ATP was decreased to {approximately}20% of control values, IP{sub 3} formation was completely inhibited, but desensitization was unaffected. When cells were treated with antimycin in the absence of glucose, cellular ATP was decreased to {approximately}5% of control values, and both IP{sub 3} formation and desensitization were blocked. A series of substance P-related peptides increased themore » formation of ({sup 3}H)IP{sub 3} and induced desensitization of the substance P response with a similar rank order of potencies. The substance P antagonist, (D-Pro{sup 2}, D-Try{sup 7,9})-substance P, inhibited substance P-induced IP{sub 3} formation and desensitization but did not induce desensitization. These results suggest that the desensitization of substance P-induced IP{sub 3} formation requires agonist activation of a P-type substance P receptor, and that one or more cellular ATP-dependent processes are required for this reaction. However, activation of phospholipase C and the generation of inositol phosphates does not seem to be a prerequisite for desensitization.« less

IP3R deficit underlies loss of salivary fluid secretion in Sjögren’s Syndrome

PubMed Central

Teos, Leyla Y.; Zhang, Yu; Cotrim, Ana P.; Swaim, William; Won, Jon H.; Ambrus, Julian; Shen, Long; Bebris, Lolita; Grisius, Margaret; Jang, Shyh-Ing; Yule, David I.; Ambudkar, Indu S.; Alevizos, Ilias

2015-01-01

The autoimmune exocrinopathy, Sjögren’s syndrome (SS), is associated with secretory defects in patients, including individuals with mild lymphocytic infiltration and minimal glandular damage. The mechanism(s) underlying the secretory dysfunction is not known. We have used minor salivary gland biopsies from SS patients and healthy individuals to assess acinar cell function in morphologically intact glandular areas. We report that agonist-regulated intracellular Ca2+ release, critically required for Ca2+ entry and fluid secretion, is defective in acini from SS patients. Importantly, these acini displayed reduction in IP3R2 and IP3R3, but not AQP5 or STIM1. Similar decreases in IP3R and carbachol (CCh)-stimulated [Ca2+]i elevation were detected in acinar cells from lymphotoxin-alpha (LTα) transgenic (TG) mice, a model for (SS). Treatment of salivary glands from healthy individuals with LT α, a cytokine linked to disease progression in SS and IL14α mice, reduced Ca2+ signaling. Together, our findings reveal novel IP3R deficits in acinar cells that underlie secretory dysfunction in SS patients. PMID:26365984

External light activates hair follicle stem cells through eyes via an ipRGC-SCN-sympathetic neural pathway.

PubMed

Fan, Sabrina Mai-Yi; Chang, Yi-Ting; Chen, Chih-Lung; Wang, Wei-Hung; Pan, Ming-Kai; Chen, Wen-Pin; Huang, Wen-Yen; Xu, Zijian; Huang, Hai-En; Chen, Ting; Plikus, Maksim V; Chen, Shih-Kuo; Lin, Sung-Jan

2018-06-29

Changes in external light patterns can alter cell activities in peripheral tissues through slow entrainment of the central clock in suprachiasmatic nucleus (SCN). It remains unclear whether cells in otherwise photo-insensitive tissues can achieve rapid responses to changes in external light. Here we show that light stimulation of animals' eyes results in rapid activation of hair follicle stem cells with prominent hair regeneration. Mechanistically, light signals are interpreted by M1-type intrinsically photosensitive retinal ganglion cells (ipRGCs), which signal to the SCN via melanopsin. Subsequently, efferent sympathetic nerves are immediately activated. Increased norepinephrine release in skin promotes hedgehog signaling to activate hair follicle stem cells. Thus, external light can directly regulate tissue stem cells via an ipRGC-SCN autonomic nervous system circuit. Since activation of sympathetic nerves is not limited to skin, this circuit can also facilitate rapid adaptive responses to external light in other homeostatic tissues.

Cigarette Smoke–Induced CXCR3 Receptor Up-Regulation Mediates Endothelial Apoptosis

PubMed Central

Green, Linden A.; Petrusca, Daniela; Rajashekhar, Gangaraju; Gianaris, Tom; Schweitzer, Kelly S.; Wang, Liang; Justice, Matthew J.; Petrache, Irina

2012-01-01

Endothelial monocyte–activating polypeptide II (EMAP II) and interferon-inducible protein (IP)–10 are proinflammatory mediators, which in addition to their chemokine activities, selectively induce apoptosis in endothelial cells and are up-regulated in the lungs of cigarette smoke–exposed humans. Previously, we showed that EMAP II is an essential mediator of cigarette smoke–induced lung emphysema in mice linking endothelial cell apoptosis with inflammation. Here we addressed the role of the CXCR3 receptor in EMAP II–induced and IP-10–induced apoptosis in endothelial cells and its regulation by cigarette smoke. We found that both neutralizing antibodies and small inhibitory RNA to CXCR3 abrogated EMAP II–induced and IP-10–induced endothelial caspase-3 activation and DNA fragmentation. CXCR3 receptor surface expression in human lung microvascular endothelial cells and in lung tissue endothelium was up-regulated by exposure to cigarette smoke. In tissue culture conditions, EMAP II–induced and IP-10–induced apoptosis was enhanced by preincubation with cigarette smoke extract. Interestingly, serum starvation also induced CXCR3 up-regulation and enhanced EMAP II–induced endothelial apoptosis. Signal transduction via p38 mitogen-activated protein kinase activation was essential for CXCR3-induced cell death, but not for CXCR3 receptor up-regulation by cigarette smoke. In turn, protein nitration was required for CXCR3 receptor up-regulation by cigarette smoke and consequently for subsequent CXCR3-induced cell death. In conclusion, the concerted up-regulation of proinflammatory EMAP II, IP-10, and CXCR3 by cigarette smoke could sustain a cascade of cell death that may promote the alveolar tissue loss noted in human emphysema. PMID:22936405

Discrimination Between Human Leukocyte Antigen Class I-Bound and Co-Purified HIV-Derived Peptides in Immunopeptidomics Workflows

PubMed Central

Partridge, Thomas; Nicastri, Annalisa; Kliszczak, Anna E.; Yindom, Louis-Marie; Kessler, Benedikt M.; Ternette, Nicola; Borrow, Persephone

2018-01-01

Elucidation of novel peptides presented by human leukocyte antigen (HLA) class I alleles by immunopeptidomics constitutes a powerful approach that can inform the rational design of CD8+ T cell inducing vaccines to control infection with pathogens such as human immunodeficiency virus type 1 (HIV-1) or to combat tumors. Recent advances in the sensitivity of liquid chromatography tandem mass spectrometry instrumentation have facilitated the discovery of thousands of natural HLA-restricted peptides in a single measurement. However, the extent of contamination of class I-bound peptides identified using HLA immunoprecipitation (IP)-based immunopeptidomics approaches with peptides from other sources has not previously been evaluated in depth. Here, we investigated the specificity of the IP-based immunopeptidomics methodology using HLA class I- or II-deficient cell lines and membrane protein-specific antibody IPs. We demonstrate that the 721.221 B lymphoblastoid cell line, widely regarded to be HLA class Ia-deficient, actually expresses and presents peptides on HLA-C*01:02. Using this cell line and the C8166 (HLA class I- and II-expressing) cell line, we show that some HLA class II-bound peptides were co-purified non-specifically during HLA class I and membrane protein IPs. Furthermore, IPs of “irrelevant” membrane proteins from HIV-1-infected HLA class I- and/or II-expressing cells revealed that unusually long HIV-1-derived peptides previously reported by us and other immunopeptidomics studies as potentially novel CD8+ T cell epitopes were non-specifically co-isolated, and so constitute a source of contamination in HLA class I IPs. For example, a 16-mer (FLGKIWPSYKGRPGNF), which was detected in all samples studied represents the full p1 segment of the abundant intracellular or virion-associated proteolytically-processed HIV-1 Gag protein. This result is of importance, as these long co-purified HIV-1 Gag peptides may not elicit CD8+ T cell responses when incorporated into candidate vaccines. These results have wider implications for HLA epitope discovery from abundant or membrane-associated antigens by immunopeptidomics in the context of infectious diseases, cancer, and autoimmunity. PMID:29780384

Muscle fiber type specific induction of slow myosin heavy chain 2 gene expression by electrical stimulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crew, Jennifer R.; Falzari, Kanakeshwari; DiMario, Joseph X., E-mail: joseph.dimario@rosalindfranklin.edu

Vertebrate skeletal muscle fiber types are defined by a broad array of differentially expressed contractile and metabolic protein genes. The mechanisms that establish and maintain these different fiber types vary throughout development and with changing functional demand. Chicken skeletal muscle fibers can be generally categorized as fast and fast/slow based on expression of the slow myosin heavy chain 2 (MyHC2) gene in fast/slow muscle fibers. To investigate the cellular and molecular mechanisms that control fiber type formation in secondary or fetal muscle fibers, myoblasts from the fast pectoralis major (PM) and fast/slow medial adductor (MA) muscles were isolated, allowed tomore » differentiate in vitro, and electrically stimulated. MA muscle fibers were induced to express the slow MyHC2 gene by electrical stimulation, whereas PM muscle fibers did not express the slow MyHC2 gene under identical stimulation conditions. However, PM muscle fibers did express the slow MyHC2 gene when electrical stimulation was combined with inhibition of inositol triphosphate receptor (IP3R) activity. Electrical stimulation was sufficient to increase nuclear localization of expressed nuclear-factor-of-activated-T-cells (NFAT), NFAT-mediated transcription, and slow MyHC2 promoter activity in MA muscle fibers. In contrast, both electrical stimulation and inhibitors of IP3R activity were required for these effects in PM muscle fibers. Electrical stimulation also increased levels of peroxisome-proliferator-activated receptor-{gamma} co-activator-1 (PGC-1{alpha}) protein in PM and MA muscle fibers. These results indicate that MA muscle fibers can be induced by electrical stimulation to express the slow MyHC2 gene and that fast PM muscle fibers are refractory to stimulation-induced slow MyHC2 gene expression due to fast PM muscle fiber specific cellular mechanisms involving IP3R activity.« less

Genome-Wide Profiling of Histone Modifications (H3K9me2 and H4K12ac) and Gene Expression in Rust (Uromyces appendiculatus) Inoculated Common Bean (Phaseolus vulgaris L.).

PubMed

Ayyappan, Vasudevan; Kalavacharla, Venu; Thimmapuram, Jyothi; Bhide, Ketaki P; Sripathi, Venkateswara R; Smolinski, Tomasz G; Manoharan, Muthusamy; Thurston, Yaqoob; Todd, Antonette; Kingham, Bruce

2015-01-01

Histone modifications such as methylation and acetylation play a significant role in controlling gene expression in unstressed and stressed plants. Genome-wide analysis of such stress-responsive modifications and genes in non-model crops is limited. We report the genome-wide profiling of histone methylation (H3K9me2) and acetylation (H4K12ac) in common bean (Phaseolus vulgaris L.) under rust (Uromyces appendiculatus) stress using two high-throughput approaches, chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq). ChIP-Seq analysis revealed 1,235 and 556 histone methylation and acetylation responsive genes from common bean leaves treated with the rust pathogen at 0, 12 and 84 hour-after-inoculation (hai), while RNA-Seq analysis identified 145 and 1,763 genes differentially expressed between mock-inoculated and inoculated plants. The combined ChIP-Seq and RNA-Seq analyses identified some key defense responsive genes (calmodulin, cytochrome p450, chitinase, DNA Pol II, and LRR) and transcription factors (WRKY, bZIP, MYB, HSFB3, GRAS, NAC, and NMRA) in bean-rust interaction. Differential methylation and acetylation affected a large proportion of stress-responsive genes including resistant (R) proteins, detoxifying enzymes, and genes involved in ion flux and cell death. The genes identified were functionally classified using Gene Ontology (GO) and EuKaryotic Orthologous Groups (KOGs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified a putative pathway with ten key genes involved in plant-pathogen interactions. This first report of an integrated analysis of histone modifications and gene expression involved in the bean-rust interaction as reported here provides a comprehensive resource for other epigenomic regulation studies in non-model species under stress.

Genome-Wide Profiling of Histone Modifications (H3K9me2 and H4K12ac) and Gene Expression in Rust (Uromyces appendiculatus) Inoculated Common Bean (Phaseolus vulgaris L.)

PubMed Central

Thimmapuram, Jyothi; Bhide, Ketaki P.; Sripathi, Venkateswara R.; Smolinski, Tomasz G.; Manoharan, Muthusamy; Thurston, Yaqoob; Todd, Antonette; Kingham, Bruce

2015-01-01

Histone modifications such as methylation and acetylation play a significant role in controlling gene expression in unstressed and stressed plants. Genome-wide analysis of such stress-responsive modifications and genes in non-model crops is limited. We report the genome-wide profiling of histone methylation (H3K9me2) and acetylation (H4K12ac) in common bean (Phaseolus vulgaris L.) under rust (Uromyces appendiculatus) stress using two high-throughput approaches, chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq). ChIP-Seq analysis revealed 1,235 and 556 histone methylation and acetylation responsive genes from common bean leaves treated with the rust pathogen at 0, 12 and 84 hour-after-inoculation (hai), while RNA-Seq analysis identified 145 and 1,763 genes differentially expressed between mock-inoculated and inoculated plants. The combined ChIP-Seq and RNA-Seq analyses identified some key defense responsive genes (calmodulin, cytochrome p450, chitinase, DNA Pol II, and LRR) and transcription factors (WRKY, bZIP, MYB, HSFB3, GRAS, NAC, and NMRA) in bean-rust interaction. Differential methylation and acetylation affected a large proportion of stress-responsive genes including resistant (R) proteins, detoxifying enzymes, and genes involved in ion flux and cell death. The genes identified were functionally classified using Gene Ontology (GO) and EuKaryotic Orthologous Groups (KOGs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified a putative pathway with ten key genes involved in plant-pathogen interactions. This first report of an integrated analysis of histone modifications and gene expression involved in the bean-rust interaction as reported here provides a comprehensive resource for other epigenomic regulation studies in non-model species under stress. PMID:26167691

Dopamine Signaling Leads to Loss of Polycomb Repression and Aberrant Gene Activation in Experimental Parkinsonism

PubMed Central

Lerdrup, Mads; Gomes, Ana-Luisa; Kryh, Hanna; Spigolon, Giada; Caboche, Jocelyne; Fisone, Gilberto; Hansen, Klaus

2014-01-01

Polycomb group (PcG) proteins bind to and repress genes in embryonic stem cells through lineage commitment to the terminal differentiated state. PcG repressed genes are commonly characterized by the presence of the epigenetic histone mark H3K27me3, catalyzed by the Polycomb repressive complex 2. Here, we present in vivo evidence for a previously unrecognized plasticity of PcG-repressed genes in terminally differentiated brain neurons of parkisonian mice. We show that acute administration of the dopamine precursor, L-DOPA, induces a remarkable increase in H3K27me3S28 phosphorylation. The induction of the H3K27me3S28p histone mark specifically occurs in medium spiny neurons expressing dopamine D1 receptors and is dependent on Msk1 kinase activity and DARPP-32-mediated inhibition of protein phosphatase-1. Chromatin immunoprecipitation (ChIP) experiments showed that increased H3K27me3S28p was accompanied by reduced PcG binding to regulatory regions of genes. An analysis of the genome wide distribution of L-DOPA-induced H3K27me3S28 phosphorylation by ChIP sequencing (ChIP-seq) in combination with expression analysis by RNA-sequencing (RNA-seq) showed that the induction of H3K27me3S28p correlated with increased expression of a subset of PcG repressed genes. We found that induction of H3K27me3S28p persisted during chronic L-DOPA administration to parkisonian mice and correlated with aberrant gene expression. We propose that dopaminergic transmission can activate PcG repressed genes in the adult brain and thereby contribute to long-term maladaptive responses including the motor complications, or dyskinesia, caused by prolonged administration of L-DOPA in Parkinson's disease. PMID:25254549

Amniotic membrane extract differentially regulates human peripheral blood T cell subsets, monocyte subpopulations and myeloid dendritic cells.

PubMed

Laranjeira, Paula; Duque, Marta; Vojtek, Martin; Inácio, Maria J; Silva, Isabel; Mamede, Ana C; Laranjo, Mafalda; Pedreiro, Susana; Carvalho, Maria J; Moura, Paulo; Abrantes, Ana M; Maia, Cláudio J; Domingues, Pedro; Domingues, Rosário; Martinho, António; Botelho, Maria F; Trindade, Hélder; Paiva, Artur

2018-03-26

The discovery of the immunoregulatory potential of human amniotic membrane (hAM) propelled several studies focusing on its application for the treatment of immunological disorders. However, there is little information regarding the effects of hAM on distinct activation and differentiation stages of immune cells. Here, we aim to investigate the effect of human amniotic membrane extract (hAME) on the pattern of cytokine production by T cells, monocytes and myeloid dendritic cells (mDCs). For this purpose, peripheral blood mononuclear cells (PBMCs) from eight healthy individuals were stimulated in vitro in the presence or absence of hAME. Mitogen-induced proliferation of PBMCs and cytokine production among the distinct T cell functional compartments, monocyte subpopulations and mDCs were evaluated. hAME displayed an anti-proliferative effect and decreased the frequency of T cells producing tumor necrosis factor (TNF)α, interferon (IFN)γ and interleukin (IL)-2, for all T cell functional compartments. The frequency of IL-17 and IL-9-producing T cells was also reduced. The inhibition of mRNA expression of granzyme B, perforin and NKG2D by CD8 + T cells and γδ T cells and the augment of FoxP3 and IL-10 in CD4 + T cells and IL-10 in regulatory T cells were also observed. Furthermore, hAME inhibited IFNγ-induced protein (IP)-10 expression by classical and non-classical monocytes, without hampering the production of TNFα and IL-6 by monocytes and mDCs. These results suggest that hAME exerts an anti-inflammatory effect on T cells, still at a different extent for distinct T cell functional compartments.