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Sample records for human cell models

  1. Rotating cell culture systems for human cell culture: human trophoblast cells as a model.

    PubMed

    Zwezdaryk, Kevin J; Warner, Jessica A; Machado, Heather L; Morris, Cindy A; Höner zu Bentrup, Kerstin

    2012-01-01

    The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines support our understanding of invasion of the uterine wall and remodeling of uterine spiral arteries by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS. The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies. The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS

  2. Generative models: Human embryonic stem cells and multiple modeling relations.

    PubMed

    Fagan, Melinda Bonnie

    2016-04-01

    Model organisms are at once scientific models and concrete living things. It is widely assumed by philosophers of science that (1) model organisms function much like other kinds of models, and (2) that insofar as their scientific role is distinctive, it is in virtue of representing a wide range of biological species and providing a basis for generalizations about those targets. This paper uses the case of human embryonic stem cells (hESC) to challenge both assumptions. I first argue that hESC can be considered model organisms, analogous to classic examples such as Escherichia coli and Drosophila melanogaster. I then discuss four contrasts between the epistemic role of hESC in practice, and the assumptions about model organisms noted above. These contrasts motivate an alternative view of model organisms as a network of systems related constructively and developmentally to one another. I conclude by relating this result to other accounts of model organisms in recent philosophy of science. PMID:27083092

  3. Human pluripotent stem cell models of Fragile X syndrome.

    PubMed

    Bhattacharyya, Anita; Zhao, Xinyu

    2016-06-01

    Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and autism. The causal mutation in FXS is a trinucleotide CGG repeat expansion in the FMR1 gene that leads to human specific epigenetic silencing and loss of Fragile X Mental Retardation Protein (FMRP) expression. Human pluripotent stem cells (PSCs), including human embryonic stem cells (ESCs) and particularly induced PSCs (iPSCs), offer a model system to reveal cellular and molecular events underlying human neuronal development and function in FXS. Human FXS PSCs have been established and have provided insight into the epigenetic silencing of the FMR1 gene as well as aspects of neuronal development. PMID:26640241

  4. Human airway xenograft models of epithelial cell regeneration.

    PubMed

    Puchelle, E; Peault, B

    2000-01-01

    Regeneration and restoration of the airway epithelium after mechanical, viral or bacterial injury have a determinant role in the evolution of numerous respiratory diseases such as chronic bronchitis, asthma and cystic fibrosis. The study in vivo of epithelial regeneration in animal models has shown that airway epithelial cells are able to dedifferentiate, spread, migrate over the denuded basement membrane and progressively redifferentiate to restore a functional respiratory epithelium after several weeks. Recently, human tracheal xenografts have been developed in immunodeficient severe combined immunodeficiency (SCID) and nude mice. In this review we recall that human airway cells implanted in such conditioned host grafts can regenerate a well-differentiated and functional human epithelium; we stress the interest in these humanized mice in assaying candidate progenitor and stem cells of the human airway mucosa. PMID:11667974

  5. Human Pluripotent Stem Cells for Modelling Human Liver Diseases and Cell Therapy

    PubMed Central

    Dianat, Noushin; Steichen, Clara; Vallier, Ludovic; Weber, Anne; Dubart-Kupperschmitt, Anne

    2013-01-01

    The liver is affected by many types of diseases, including metabolic disorders and acute liver failure. Orthotopic liver transplantation (OLT) is currently the only effective treatment for life-threatening liver diseases but transplantation of allogeneic hepatocytes has now become an alternative as it is less invasive than OLT and can be performed repeatedly. However, this approach is hampered by the shortage of organ donors, and the problems related to the isolation of high quality adult hepatocytes, their cryopreservation and their absence of proliferation in culture. Liver is also a key organ to assess the pharmacokinetics and toxicology of xenobiotics and for drug discovery, but appropriate cell culture systems are lacking. All these problems have highlighted the need to explore other sources of cells such as stem cells that could be isolated, expanded to yield sufficiently large populations and then induced to differentiate into functional hepatocytes. The presence of a niche of “facultative” progenitor and stem cells in the normal liver has recently been confirmed but they display no telomerase activity. The recent discovery that human induced pluripotent stem cells can be generated from somatic cells has renewed hopes for regenerative medicine and in vitro disease modelling, as these cells are easily accessible. We review here the present progresses, limits and challenges for the generation of functional hepatocytes from human pluripotent stem cells in view of their potential use in regenerative medicine and drug discovery. PMID:23444872

  6. Development and function of human innate immune cells in a humanized mouse model

    PubMed Central

    Rongvaux, Anthony; Willinger, Tim; Martinek, Jan; Strowig, Till; Gearty, Sofia V.; Teichmann, Lino L.; Saito, Yasuyuki; Marches, Florentina; Halene, Stephanie; Palucka, A. Karolina; Manz, Markus G.; Flavell, Richard A.

    2014-01-01

    Mice repopulated with human hematopoietic cells are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, existing humanized mouse models are unable to support development of human innate immune cells, including myeloid cells and NK cells. Here we describe a mouse strain, called MI(S)TRG, in which human versions of four genes encoding cytokines important for innate immune cell development are knocked in to their respective mouse loci. The human cytokines support the development and function of monocytes/macrophages and natural killer cells derived from human fetal liver or adult CD34+ progenitor cells injected into the mice. Human macrophages infiltrated a human tumor xenograft in MI(S)TRG mice in a manner resembling that observed in tumors obtained from human patients. This humanized mouse model may be used to model the human immune system in scenarios of health and pathology, and may enable evaluation of therapeutic candidates in an in vivo setting relevant to human physiology. PMID:24633240

  7. Humanized Murine Model for HBV and HCV Using Human Induced Pluripotent Stem Cells

    PubMed Central

    Zhou, Xiao-Ling; Sullivan, Gareth J.; Sun, Pingnan; Park, In-Hyun

    2013-01-01

    Infection of hepatitis B virus (HBV) and hepatitis C virus (HCV) results in heterogeneous outcomes from acute asymptomatic infection to chronic infection leading to cirrhosis and hepatocellular carcinoma (HCC). In vitro models using animal hepatocytes, human HCC cell lines, or in vivo transgenic mouse models have contributed invaluably to understanding the pathogenesis of HBV and HCV. A humanized mouse model made by reconstitution of human primary hepatocytes in the liver of the immunodeficient mouse provides a novel experimental opportunity which mimics the in vivo growth of the human hepatocytes. The limited access to primary human hepatocytes necessitated the search for other cellular sources, such as pluripotent stem cells. Human embryonic stem cells (hESCs) have the features of self-renewal and pluripotency and differentiate into cells of all three germ layers, including hepatocytes. Humaninduced pluripotent stem cells (iPSCs) derived from the patient’s or individual’s own cells provide a novel opportunity to generate hepatocyte-like cells with the defined genetic composition. Here, we will review the current perspective of the models used for HBV and HCV study, and introduce the personalized mouse model using human iPSCs. This novel mouse model will facilitate the direct investigation of HBV and HCV in human hepatocytes as well as probing the genetic influence on the susceptibility of hepatocytes to HBV and HCV. PMID:22370780

  8. New paradigms for metabolic modeling of human cells.

    PubMed

    Mardinoglu, Adil; Nielsen, Jens

    2015-08-01

    Abnormalities in cellular functions are associated with the progression of human diseases, often resulting in metabolic reprogramming. GEnome-scale metabolic Models (GEMs) have enabled studying global metabolic reprogramming in connection with disease development in a systematic manner. Here we review recent work on reconstruction of GEMs for human cell/tissue types and cancer, and the use of GEMs for identification of metabolic changes occurring in response to disease development. We further discuss how GEMs can be used for the development of efficient therapeutic strategies. Finally, challenges in integration of cell/tissue models for simulation of whole body functions as well as integration of GEMs with other biological networks for generating complete cell/tissue models are presented. PMID:25559199

  9. Humanized mice as a model to study human hematopoietic stem cell transplantation.

    PubMed

    Tanner, Anne; Taylor, Stephen E; Decottignies, Wittnee; Berges, Bradford K

    2014-01-01

    Hematopoietic stem cell (HSC) transplantation has the potential to treat a variety of human diseases, including genetic deficiencies, immune disorders, and to restore immunity following cancer treatment. However, there are several obstacles that prevent effective HSC transplantation in humans. These include finding a matched donor, having a sufficient number of cells for the transplant, and the potency of the cells in the transplant. Ethical issues prevent effective research in humans that could provide insight into ways to overcome these obstacles. Highly immunodeficient mice can be transplanted with human HSCs and this process is accompanied by HSC homing to the murine bone marrow. This is followed by stem cell expansion, multilineage hematopoiesis, long-term engraftment, and functional human antibody and cellular immune responses. As such, humanized mice serve as a model for human HSC transplantation. A variety of conditions have been analyzed for their impact on HSC transplantation to produce humanized mice, including the type and source of cells used in the transplant, the number of cells transplanted, the expansion of cells with various protocols, and the route of introduction of cells into the mouse. In this review, we summarize what has been learned about HSC transplantation using humanized mice as a recipient model and we comment on how these models may be useful to future preclinical research to determine more effective ways to expand HSCs and to determine their repopulating potential in vivo. PMID:23962058

  10. Cell sources for in vitro human liver cell culture models.

    PubMed

    Zeilinger, Katrin; Freyer, Nora; Damm, Georg; Seehofer, Daniel; Knöspel, Fanny

    2016-09-01

    In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described. PMID:27385595

  11. Differentiation and Genomic Instability in a Human Mammary Cell Model

    NASA Technical Reports Server (NTRS)

    Richmond, R.; Kale, R.; Pettengill, O.; Rose, M. Franklin (Technical Monitor)

    2001-01-01

    Harvest of prophylactic mastectomy specimens from an obligate heterozygote for ataxia-telangiectasia provided autologous fibroblasts as well epithelial cells (HMEC). The routine availability of these autologous cells has provided an opportunity to study cell-cell interactions in coculture and monoculture, and in 3-dimensional cultures grown in the NASA rotating bioreactor. HMEC and stromal fibroblasts grown in 2-dimensional monoculture were both observed to produce extracellular matrix. Similar matrix was encountered in 3-dimensional cultures containing HMEC. Metaphases were analyzed. For stromal fibroblasts, genomic aberrations were found in 18% of metaphase spreads. For HMEC, aberrations were greater such that a majority were found to be abnormal. The level of genomic instability determined for these noncancerous cells in 2-dimensional monoculture should be useful for generating a human cell model that can correlate the effects of differentiation in 3-dimensional coculture on the level of genomic instability.

  12. Culture models of human mammary epithelial cell transformation

    SciTech Connect

    Stampfer, Martha R.; Yaswen, Paul

    2000-11-10

    Human pre-malignant breast diseases, particularly ductal carcinoma in situ (DCIS)3 already display several of the aberrant phenotypes found in primary breast cancers, including chromosomal abnormalities, telomerase activity, inactivation of the p53 gene and overexpression of some oncogenes. Efforts to model early breast carcinogenesis in human cell cultures have largely involved studies in vitro transformation of normal finite lifespan human mammary epithelial cells (HMEC) to immortality and malignancy. We present a model of HMEC immortal transformation consistent with the know in vivo data. This model includes a recently described, presumably epigenetic process, termed conversion, which occurs in cells that have overcome stringent replicative senescence and are thus able to maintain proliferation with critically short telomeres. The conversion process involves reactivation of telomerase activity, and acquisition of good uniform growth in the absence and presence of TFGB. We propose th at overcoming the proliferative constraints set by senescence, and undergoing conversion, represent key rate-limiting steps in human breast carcinogenesis, and occur during early stage breast cancer progression.

  13. Human Induced Pluripotent Stem Cell Models of Inherited Cardiovascular Diseases.

    PubMed

    Jiang, Wenjian; Lan, Feng; Zhang, Hongjia

    2014-10-16

    Cardiovascular cells derived from patient specific induced Pluripotent Stem Cell (iPSC) harbor gene mutations associated with the pathogenesis of inherited cardiac diseases and congenital heart diseases (CHD). Numerous reports have demonstrated the utilization of human induced Pluripotent Stem Cell (hiPSC) to model cardiac diseases as a means of investigating their underlying mechanisms. So far, they have been shown to investigate the molecular mechanisms of many cardiac disorders, such as long-QT syndrome (LQT), catecholaminergic polymorphic ventricular tachycardia (CPVT), dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), LEOPARD syndrome (LS), arrhythmogenic cardiomyopathy (ACM), Friedreich ataxia (FRDA), Barth syndrome (BTHS), hypoplastic left heart syndrome (HLHS), Marfan syndrome (MFS) and other CHD. This article summarizes the growing body of research related to modeling various cardiac diseases using hiPSCs. Moreover, by reviewing the methods used in previous studies, we propose multiple novel applications of hiPSCs to investigate comprehensive cardiovascular disorders and facilitate drug discovery. PMID:25322695

  14. Human normal bronchial epithelial cells: a novel in vitro cell model for toxicity evaluation.

    PubMed

    Feng, Wenqiang; Guo, Juanjuan; Huang, Haiyan; Xia, Bo; Liu, Hongya; Li, Jie; Lin, Shaolin; Li, Tiyuan; Liu, Jianjun; Li, Hui

    2015-01-01

    Human normal cell-based systems are needed for drug discovery and toxicity evaluation. hTERT or viral genes transduced human cells are currently widely used for these studies, while these cells exhibited abnormal differentiation potential or response to biological and chemical signals. In this study, we established human normal bronchial epithelial cells (HNBEC) using a defined primary epithelial cell culture medium without transduction of exogenous genes. This system may involve decreased IL-1 signaling and enhanced Wnt signaling in cells. Our data demonstrated that HNBEC exhibited a normal diploid karyotype. They formed well-defined spheres in matrigel 3D culture while cancer cells (HeLa) formed disorganized aggregates. HNBEC cells possessed a normal cellular response to DNA damage and did not induce tumor formation in vivo by xenograft assays. Importantly, we assessed the potential of these cells in toxicity evaluation of the common occupational toxicants that may affect human respiratory system. Our results demonstrated that HNBEC cells are more sensitive to exposure of 10~20 nm-sized SiO2, Cr(VI) and B(a)P compared to 16HBE cells (a SV40-immortalized human bronchial epithelial cells). This study provides a novel in vitro human cells-based model for toxicity evaluation, may also be facilitating studies in basic cell biology, cancer biology and drug discovery. PMID:25861018

  15. Human Normal Bronchial Epithelial Cells: A Novel In Vitro Cell Model for Toxicity Evaluation

    PubMed Central

    Huang, Haiyan; Xia, Bo; Liu, Hongya; Li, Jie; Lin, Shaolin; Li, Tiyuan; Liu, Jianjun; Li, Hui

    2015-01-01

    Human normal cell-based systems are needed for drug discovery and toxicity evaluation. hTERT or viral genes transduced human cells are currently widely used for these studies, while these cells exhibited abnormal differentiation potential or response to biological and chemical signals. In this study, we established human normal bronchial epithelial cells (HNBEC) using a defined primary epithelial cell culture medium without transduction of exogenous genes. This system may involve decreased IL-1 signaling and enhanced Wnt signaling in cells. Our data demonstrated that HNBEC exhibited a normal diploid karyotype. They formed well-defined spheres in matrigel 3D culture while cancer cells (HeLa) formed disorganized aggregates. HNBEC cells possessed a normal cellular response to DNA damage and did not induce tumor formation in vivo by xenograft assays. Importantly, we assessed the potential of these cells in toxicity evaluation of the common occupational toxicants that may affect human respiratory system. Our results demonstrated that HNBEC cells are more sensitive to exposure of 10~20 nm-sized SiO2, Cr(VI) and B(a)P compared to 16HBE cells (a SV40-immortalized human bronchial epithelial cells). This study provides a novel in vitro human cells-based model for toxicity evaluation, may also be facilitating studies in basic cell biology, cancer biology and drug discovery. PMID:25861018

  16. Being human: The role of pluripotent stem cells in regenerative medicine and humanizing Alzheimer's disease models.

    PubMed

    Sproul, Andrew A

    2015-01-01

    Human pluripotent stem cells (PSCs) have the capacity to revolutionize medicine by allowing the generation of functional cell types such as neurons for cell replacement therapy. However, the more immediate impact of PSCs on treatment of Alzheimer's disease (AD) will be through improved human AD model systems for mechanistic studies and therapeutic screening. This review will first briefly discuss different types of PSCs and genome-editing techniques that can be used to modify PSCs for disease modeling or for personalized medicine. This will be followed by a more in depth analysis of current AD iPSC models and a discussion of the need for more complex multicellular models, including cell types such as microglia. It will finish with a discussion on current clinical trials using PSC-derived cells and the long-term potential of such strategies for treating AD. PMID:26101165

  17. Human vascular tissue models formed from human induced pluripotent stem cell derived endothelial cells

    PubMed Central

    Belair, David G.; Whisler, Jordan A.; Valdez, Jorge; Velazquez, Jeremy; Molenda, James A.; Vickerman, Vernella; Lewis, Rachel; Daigh, Christine; Hansen, Tyler D.; Mann, David A.; Thomson, James A.; Griffith, Linda G.; Kamm, Roger D.; Schwartz, Michael P.; Murphy, William L.

    2015-01-01

    Here we describe a strategy to model blood vessel development using a well-defined iPSC-derived endothelial cell type (iPSC-EC) cultured within engineered platforms that mimic the 3D microenvironment. The iPSC-ECs used here were first characterized by expression of endothelial markers and functional properties that included VEGF responsiveness, TNF-α-induced upregulation of cell adhesion molecules (MCAM/CD146; ICAM1/CD54), thrombin-dependent barrier function, shear stress-induced alignment, and 2D and 3D capillary-like network formation in Matrigel. The iPSC-ECs also formed 3D vascular networks in a variety of engineering contexts, yielded perfusable, interconnected lumen when co-cultured with primary human fibroblasts, and aligned with flow in microfluidics devices. iPSC-EC function during tubule network formation, barrier formation, and sprouting was consistent with that of primary ECs, and the results suggest a VEGF-independent mechanism for sprouting, which is relevant to therapeutic anti-angiogenesis strategies. Our combined results demonstrate the feasibility of using a well-defined, stable source of iPSC-ECs to model blood vessel formation within a variety of contexts using standard in vitro formats. PMID:25190668

  18. Modeling human endothelial cell transformation in vascular neoplasias

    PubMed Central

    Wen, Victoria W.; MacKenzie, Karen L.

    2013-01-01

    Endothelial cell (EC)-derived neoplasias range from benign hemangioma to aggressive metastatic angiosarcoma, which responds poorly to current treatments and has a very high mortality rate. The development of treatments that are more effective for these disorders will be expedited by insight into the processes that promote abnormal proliferation and malignant transformation of human ECs. The study of primary endothelial malignancy has been limited by the rarity of the disease; however, there is potential for carefully characterized EC lines and animal models to play a central role in the discovery, development and testing of molecular targeted therapies for vascular neoplasias. This review describes molecular alterations that have been identified in EC-derived neoplasias, as well as the processes that underpin the immortalization and tumorigenic conversion of ECs. Human EC lines, established through the introduction of defined genetic elements or by culture of primary tumor tissue, are catalogued and discussed in relation to their relevance as models of vascular neoplasia. PMID:24046386

  19. Biological Analysis of Human CML Stem Cells; Xenograft Model of Chronic Phase Human Chronic Myeloid Leukemia.

    PubMed

    Abraham, Sheela A

    2016-01-01

    Xenograft mouse models have been instrumental in expanding our knowledge of hematopoiesis and can provide a functional description of stem cells that possess engrafting potential. Here we describe methodology outlining one way of analyzing human malignant cells that are able to engraft immune compromised mice. Using models such as these will allow researchers to gain valuable insight into the primitive leukemic subtypes that evade current therapy regimes and are critical to understand, in order to eradicate malignancy. PMID:27581148

  20. Assessment of Cell Line Models of Primary Human Cells by Raman Spectral Phenotyping

    PubMed Central

    Swain, Robin J.; Kemp, Sarah J.; Goldstraw, Peter; Tetley, Teresa D.; Stevens, Molly M.

    2010-01-01

    Abstract Researchers have previously questioned the suitability of cell lines as models for primary cells. In this study, we used Raman microspectroscopy to characterize live A549 cells from a unique molecular biochemical perspective to shed light on their suitability as a model for primary human pulmonary alveolar type II (ATII) cells. We also investigated a recently developed transduced type I (TT1) cell line as a model for alveolar type I (ATI) cells. Single-cell Raman spectra provide unique biomolecular fingerprints that can be used to characterize cellular phenotypes. A multivariate statistical analysis of Raman spectra indicated that the spectra of A549 and TT1 cells are characterized by significantly lower phospholipid content compared to ATII and ATI spectra because their cytoplasm contains fewer surfactant lamellar bodies. Furthermore, we found that A549 spectra are statistically more similar to ATI spectra than to ATII spectra. The spectral variation permitted phenotypic classification of cells based on Raman spectral signatures with >99% accuracy. These results suggest that A549 cells are not a good model for ATII cells, but TT1 cells do provide a reasonable model for ATI cells. The findings have far-reaching implications for the assessment of cell lines as suitable primary cellular models in live cultures. PMID:20409492

  1. Modeling Rett Syndrome Using Human Induced Pluripotent Stem Cells.

    PubMed

    Andoh-Noda, Tomoko; Inouye, Michiko O; Miyake, Kunio; Kubota, Takeo; Okano, Hideyuki; Akamatsu, Wado

    2016-01-01

    Rett syndrome (RTT) is one of a group of neurodevelopmental disorders typically characterized by deficits in the X-linked gene MECP2 (methyl-CpG binding protein 2). The MECP2 gene encodes a multifunctional protein involved in transcriptional repression, transcriptional activation, chromatin remodeling, and RNA splicing. Genetic deletion of Mecp2 in mice revealed neuronal disabilities including RTT-like phenotypes and provided an excellent platform for understanding the pathogenesis of RTT. So far, there are no effective pharmacological treatments for RTT because the role of MECP2 in RTT is incompletely understood. Recently, human induced pluripotent stem cell (hiPSC) technologies have improved our knowledge of neurological and neurodevelopmental diseases including RTT because neurons derived from RTT-hiPSCs can be used for disease modeling to understand RTT phenotypes and to perform high throughput pharmaceutical drug screening. In this review, we provide an overview of RTT, including MeCP2 function and mouse models of RTT. In addition, we introduce recent advances in disease modeling of RTT using hiPSC-derived neural cells. PMID:27071793

  2. Neoplastic human embryonic stem cells as a model of radiation resistance of human cancer stem cells

    PubMed Central

    Dingwall, Steve; Lee, Jung Bok; Guezguez, Borhane; Fiebig, Aline; McNicol, Jamie; Boreham, Douglas; Collins, Tony J.; Bhatia, Mick

    2015-01-01

    Studies have implicated that a small sub-population of cells within a tumour, termed cancer stem cells (CSCs), have an enhanced capacity for tumour formation in multiple cancers and may be responsible for recurrence of the disease after treatment, including radiation. Although comparisons have been made between CSCs and bulk-tumour, the more important comparison with respect to therapy is between tumour-sustaining CSC versus normal stem cells that maintain the healthy tissue. However, the absence of normal known counterparts for many CSCs has made it difficult to compare the radiation responses of CSCs with the normal stem cells required for post-radiotherapy tissue regeneration and the maintenance of tissue homeostasis. Here we demonstrate that transformed human embryonic stem cells (t-hESCs), showing features of neoplastic progression produce tumours resistant to radiation relative to their normal counterpart upon injection into immune compromised mice. We reveal that t-hESCs have a reduced capacity for radiation induced cell death via apoptosis and exhibit altered cell cycle arrest relative to hESCs in vitro. t-hESCs have an increased expression of BclXL in comparison to their normal counterparts and re-sensitization of t-hESCs to radiation upon addition of BH3-only mimetic ABT737, suggesting that overexpression of BclXL underpins t-hESC radiation insensitivity. Using this novel discovery platform to investigate radiation resistance in human CSCs, our study indicates that chemotherapy targeting Bcl2-family members may prove to be an adjuvant to radiotherapy capable of targeting CSCs. PMID:26082437

  3. Derivation of Induced Pluripotent Stem Cells for Human Disease Modeling

    PubMed Central

    Narsinh, Kamileh; Narsinh, Kazim H.; Wu, Joseph C.

    2011-01-01

    The successful derivation of human induced pluripotent stem cells (hiPSCs) by de-differentiation of somatic cells offers significant potential to overcome obstacles in the field of cardiovascular disease. hiPSC derivatives offer incredible potential for new disease models and regenerative medicine therapies. However, many questions remain regarding the optimal starting materials and methods to enable safe, efficient derivation of hiPSCs suitable for clinical applications. Initial reprogramming experiments were carried out using lentiviral or retroviral gene delivery methods. More recently, various non-viral methods that avoid permanent and random transgene insertion have emerged as alternatives. These include transient DNA transfection approaches using transposons or minicircle plasmids, protein transduction approaches, and RNA transfection approaches. In addition, several small molecules have been found to significantly augment iPSC derivation efficiency, allowing the use of a fewer number of genes during pluripotency induction. Here, we review these various methods for the derivation of hiPSCs, focusing on their ultimate clinical applicability, with an emphasis on their potential for use as cardiovascular therapies and disease modeling platforms. PMID:21527744

  4. Human hepatocytes derived from pluripotent stem cells: a promising cell model for drug hepatotoxicity screening.

    PubMed

    Gómez-Lechón, María José; Tolosa, Laia

    2016-09-01

    Drug-induced liver injury (DILI) is a frequent cause of failure in both clinical and post-approval stages of drug development, and poses a key challenge to the pharmaceutical industry. Current animal models offer poor prediction of human DILI. Although several human cell-based models have been proposed for the detection of human DILI, human primary hepatocytes remain the gold standard for preclinical toxicological screening. However, their use is hindered by their limited availability, variability and phenotypic instability. In contrast, pluripotent stem cells, which include embryonic and induced pluripotent stem cells (iPSCs), proliferate extensively in vitro and can be differentiated into hepatocytes by the addition of soluble factors. This provides a stable source of hepatocytes for multiple applications, including early preclinical hepatotoxicity screening. In addition, iPSCs also have the potential to establish genotype-specific cells from different individuals, which would increase the predictivity of toxicity assays allowing more successful clinical trials. Therefore, the generation of human hepatocyte-like cells derived from pluripotent stem cells seems to be promising for overcoming limitations of hepatocyte preparations, and it is expected to have a substantial repercussion in preclinical hepatotoxicity risk assessment in early drug development stages. PMID:27325232

  5. Human mast cells decrease SLPI levels in type II – like alveolar cell model, in vitro

    PubMed Central

    Hollander, Camilla; Nyström, Max; Janciauskiene, Sabina; Westin, Ulla

    2003-01-01

    Background Mast cells are known to accumulate at sites of inflammation and upon activation to release their granule content, e.g. histamine, cytokines and proteases. The secretory leukocyte protease inhibitor (SLPI) is produced in the respiratory mucous and plays a role in regulating the activity of the proteases. Result We have used the HMC-1 cell line as a model for human mast cells to investigate their effect on SLPI expression and its levels in cell co-culture experiments, in vitro. In comparison with controls, we found a significant reduction in SLPI levels (by 2.35-fold, p < 0.01) in a SLPI-producing, type II-like alveolar cell line, (A549) when co-cultured with HMC-1 cells, but not in an HMC-1-conditioned medium, for 96 hours. By contrast, increased SLPI mRNA expression (by 1.58-fold, p < 0.05) was found under the same experimental conditions. Immunohistochemical analysis revealed mast cell transmigration in co-culture with SLPI-producing A549 cells for 72 and 96 hours. Conclusion These results indicate that SLPI-producing cells may assist mast cell migration and that the regulation of SLPI release and/or consumption by mast cells requires interaction between these cell types. Therefore, a "local relationship" between mast cells and airway epithelial cells might be an important step in the inflammatory response. PMID:12952550

  6. Modeling adenovirus latency in human lymphocyte cell lines.

    PubMed

    Zhang, Yange; Huang, Wen; Ornelles, David A; Gooding, Linda R

    2010-09-01

    Species C adenovirus establishes a latent infection in lymphocytes of the tonsils and adenoids. To understand how this lytic virus is maintained in these cells, four human lymphocytic cell lines that support the entire virus life cycle were examined. The T-cell line Jurkat ceased proliferation and died shortly after virus infection. BJAB, Ramos (B cells), and KE37 (T cells) continued to divide at nearly normal rates while replicating the virus genome. Viral genome numbers peaked and then declined in BJAB cells below one genome per cell at 130 to 150 days postinfection. Ramos and KE37 cells maintained the virus genome at over 100 copies per cell over a comparable period of time. BJAB cells maintained the viral DNA as a monomeric episome. All three persistently infected cells lost expression of the cell surface coxsackie and adenovirus receptor (CAR) within 24 h postinfection, and CAR expression remained low for at least 340 days postinfection. CAR loss proceeded via a two-stage process. First, an initial loss of cell surface staining for CAR required virus late gene expression and a CAR-binding fiber protein even while CAR protein and mRNA levels remained high. Second, CAR mRNA disappeared at around 30 days postinfection and remained low even after virus DNA was lost from the cells. At late times postinfection (day 180), BJAB cells could not be reinfected with adenovirus, even when CAR was reintroduced to the cells via retroviral transduction, suggesting that the expression of multiple genes had been stably altered in these cells following infection. PMID:20573817

  7. A humanized bone marrow ossicle xenotransplantation model enables improved engraftment of healthy and leukemic human hematopoietic cells.

    PubMed

    Reinisch, Andreas; Thomas, Daniel; Corces, M Ryan; Zhang, Xiaohua; Gratzinger, Dita; Hong, Wan-Jen; Schallmoser, Katharina; Strunk, Dirk; Majeti, Ravindra

    2016-07-01

    Xenotransplantation models represent powerful tools for the investigation of healthy and malignant human hematopoiesis. However, current models do not fully mimic the components of the human bone marrow (BM) microenvironment, and they enable only limited engraftment of samples from some human malignancies. Here we show that a xenotransplantation model bearing subcutaneous humanized ossicles with an accessible BM microenvironment, formed by in situ differentiation of human BM-derived mesenchymal stromal cells, enables the robust engraftment of healthy human hematopoietic stem and progenitor cells, as well as primary acute myeloid leukemia (AML) samples, at levels much greater than those in unmanipulated mice. Direct intraossicle transplantation accelerated engraftment and resulted in the detection of substantially higher leukemia-initiating cell (LIC) frequencies. We also observed robust engraftment of acute promyelocytic leukemia (APL) and myelofibrosis (MF) samples, and identified LICs in these malignancies. This humanized ossicle xenotransplantation approach provides a system for modeling a wide variety of human hematological diseases. PMID:27213817

  8. The Human Glioblastoma Cell Culture Resource: Validated Cell Models Representing All Molecular Subtypes

    PubMed Central

    Xie, Yuan; Bergström, Tobias; Jiang, Yiwen; Johansson, Patrik; Marinescu, Voichita Dana; Lindberg, Nanna; Segerman, Anna; Wicher, Grzegorz; Niklasson, Mia; Baskaran, Sathishkumar; Sreedharan, Smitha; Everlien, Isabelle; Kastemar, Marianne; Hermansson, Annika; Elfineh, Lioudmila; Libard, Sylwia; Holland, Eric Charles; Hesselager, Göran; Alafuzoff, Irina; Westermark, Bengt; Nelander, Sven; Forsberg-Nilsson, Karin; Uhrbom, Lene

    2015-01-01

    Glioblastoma (GBM) is the most frequent and malignant form of primary brain tumor. GBM is essentially incurable and its resistance to therapy is attributed to a subpopulation of cells called glioma stem cells (GSCs). To meet the present shortage of relevant GBM cell (GC) lines we developed a library of annotated and validated cell lines derived from surgical samples of GBM patients, maintained under conditions to preserve GSC characteristics. This collection, which we call the Human Glioblastoma Cell Culture (HGCC) resource, consists of a biobank of 48 GC lines and an associated database containing high-resolution molecular data. We demonstrate that the HGCC lines are tumorigenic, harbor genomic lesions characteristic of GBMs, and represent all four transcriptional subtypes. The HGCC panel provides an open resource for in vitro and in vivo modeling of a large part of GBM diversity useful to both basic and translational GBM research. PMID:26629530

  9. Epithelial Cell Culture from Human Adenoids: A Functional Study Model for Ciliated and Secretory Cells

    PubMed Central

    González, Claudia; Espinosa, Marisol; Sánchez, María Trinidad; Droguett, Karla; Ríos, Mariana; Fonseca, Ximena; Villalón, Manuel

    2013-01-01

    Background. Mucociliary transport (MCT) is a defense mechanism of the airway. To study the underlying mechanisms of MCT, we have both developed an experimental model of cultures, from human adenoid tissue of ciliated and secretory cells, and characterized the response to local chemical signals that control ciliary activity and the secretion of respiratory mucins in vitro. Materials and Methods. In ciliated cell cultures, ciliary beat frequency (CBF) and intracellular Ca2+ levels were measured in response to ATP, UTP, and adenosine. In secretory cultures, mucin synthesis and secretion were identified by using immunodetection. Mucin content was taken from conditioned medium and analyzed in the presence or absence of UTP. Results. Enriched ciliated cell monolayers and secretory cells were obtained. Ciliated cells showed a basal CBF of 10.7 Hz that increased significantly after exposure to ATP, UTP, or adenosine. Mature secretory cells showed active secretion of granules containing different glycoproteins, including MUC5AC. Conclusion. Culture of ciliated and secretory cells grown from adenoid epithelium is a reproducible and feasible experimental model, in which it is possible to observe ciliary and secretory activities, with a potential use as a model to understand mucociliary transport control mechanisms. PMID:23484122

  10. Mucosal-associated invariant T cells from induced pluripotent stem cells: A novel approach for modeling human diseases

    PubMed Central

    Sugimoto, Chie; Fujita, Hiroyoshi; Wakao, Hiroshi

    2016-01-01

    Mice have frequently been used to model human diseases involving immune dysregulation such as autoimmune and inflammatory diseases. These models help elucidate the mechanisms underlying the disease and in the development of novel therapies. However, if mice are deficient in certain cells and/or effectors associated with human diseases, how can their functions be investigated in this species? Mucosal-associated invariant T (MAIT) cells, a novel innate-like T cell family member, are a good example. MAIT cells are abundant in humans but scarce in laboratory mice. MAIT cells harbor an invariant T cell receptor and recognize nonpeptidic antigens vitamin B2 metabolites from bacteria and yeasts. Recent studies have shown that MAIT cells play a pivotal role in human diseases such as bacterial infections and autoimmune and inflammatory diseases. MAIT cells possess granulysin, a human-specific effector molecule, but granulysin and its homologue are absent in mice. Furthermore, MAIT cells show poor proliferation in vitro. To overcome these problems and further our knowledge of MAIT cells, we have established a method to expand MAIT cells via induced pluripotent stem cells (iPSCs). In this review, we describe recent advances in the field of MAIT cell research and our approach for human disease modeling with iPSC-derived MAIT cells. PMID:27114747

  11. Mucosal-associated invariant T cells from induced pluripotent stem cells: A novel approach for modeling human diseases.

    PubMed

    Sugimoto, Chie; Fujita, Hiroyoshi; Wakao, Hiroshi

    2016-04-26

    Mice have frequently been used to model human diseases involving immune dysregulation such as autoimmune and inflammatory diseases. These models help elucidate the mechanisms underlying the disease and in the development of novel therapies. However, if mice are deficient in certain cells and/or effectors associated with human diseases, how can their functions be investigated in this species? Mucosal-associated invariant T (MAIT) cells, a novel innate-like T cell family member, are a good example. MAIT cells are abundant in humans but scarce in laboratory mice. MAIT cells harbor an invariant T cell receptor and recognize nonpeptidic antigens vitamin B2 metabolites from bacteria and yeasts. Recent studies have shown that MAIT cells play a pivotal role in human diseases such as bacterial infections and autoimmune and inflammatory diseases. MAIT cells possess granulysin, a human-specific effector molecule, but granulysin and its homologue are absent in mice. Furthermore, MAIT cells show poor proliferation in vitro. To overcome these problems and further our knowledge of MAIT cells, we have established a method to expand MAIT cells via induced pluripotent stem cells (iPSCs). In this review, we describe recent advances in the field of MAIT cell research and our approach for human disease modeling with iPSC-derived MAIT cells. PMID:27114747

  12. Humanized Mouse Models to Study Cell-Mediated Immune Responses to Liver-Stage Malaria Vaccines.

    PubMed

    Good, Michael F; Hawkes, Michael T; Yanow, Stephanie K

    2015-11-01

    Malaria vaccine development is hampered by the lack of small animal models that recapitulate human immune responses to Plasmodium falciparum. We review the burgeoning literature on humanized mice for P. falciparum infection, including challenges in engraftment of human immune cells, hepatocytes, and erythrocytes. Recent advances in immune-compromised mouse models and stem cell technology have already enabled proof of concept that the entire parasite life cycle can be sustained in a murine model and that adaptive human immune responses to several parasite stages can be measured. Nonetheless, optimization is needed to achieve a reproducible and relevant murine model for malaria vaccine development. This review is focused on the complexities of T cell development in a mouse humanized with both a lymphoid system and hepatocytes. An understanding of this will facilitate the use of humanized mice in the development of liver-stage vaccines. PMID:26458783

  13. Reprogramming of human cancer cells to pluripotency for models of cancer progression

    PubMed Central

    Kim, Jungsun; Zaret, Kenneth S

    2015-01-01

    The ability to study live cells as they progress through the stages of cancer provides the opportunity to discover dynamic networks underlying pathology, markers of early stages, and ways to assess therapeutics. Genetically engineered animal models of cancer, where it is possible to study the consequences of temporal-specific induction of oncogenes or deletion of tumor suppressors, have yielded major insights into cancer progression. Yet differences exist between animal and human cancers, such as in markers of progression and response to therapeutics. Thus, there is a need for human cell models of cancer progression. Most human cell models of cancer are based on tumor cell lines and xenografts of primary tumor cells that resemble the advanced tumor state, from which the cells were derived, and thus do not recapitulate disease progression. Yet a subset of cancer types have been reprogrammed to pluripotency or near-pluripotency by blastocyst injection, by somatic cell nuclear transfer and by induced pluripotent stem cell (iPS) technology. The reprogrammed cancer cells show that pluripotency can transiently dominate over the cancer phenotype. Diverse studies show that reprogrammed cancer cells can, in some cases, exhibit early-stage phenotypes reflective of only partial expression of the cancer genome. In one case, reprogrammed human pancreatic cancer cells have been shown to recapitulate stages of cancer progression, from early to late stages, thus providing a model for studying pancreatic cancer development in human cells where previously such could only be discerned from mouse models. We discuss these findings, the challenges in developing such models and their current limitations, and ways that iPS reprogramming may be enhanced to develop human cell models of cancer progression. PMID:25712212

  14. Cultured human airway epithelial cells (calu-3): a model of human respiratory function, structure, and inflammatory responses.

    PubMed

    Zhu, Yan; Chidekel, Aaron; Shaffer, Thomas H

    2010-01-01

    This article reviews the application of the human airway Calu-3 cell line as a respiratory model for studying the effects of gas concentrations, exposure time, biophysical stress, and biological agents on human airway epithelial cells. Calu-3 cells are grown to confluence at an air-liquid interface on permeable supports. To model human respiratory conditions and treatment modalities, monolayers are placed in an environmental chamber, and exposed to specific levels of oxygen or other therapeutic modalities such as positive pressure and medications to assess the effect of interventions on inflammatory mediators, immunologic proteins, and antibacterial outcomes. Monolayer integrity and permeability and cell histology and viability also measure cellular response to therapeutic interventions. Calu-3 cells exposed to graded oxygen concentrations demonstrate cell dysfunction and inflammation in a dose-dependent manner. Modeling positive airway pressure reveals that pressure may exert a greater injurious effect and cytokine response than oxygen. In experiments with pharmacological agents, Lucinactant is protective of Calu-3 cells compared with Beractant and control, and perfluorocarbons also protect against hyperoxia-induced airway epithelial cell injury. The Calu-3 cell preparation is a sensitive and efficient preclinical model to study human respiratory processes and diseases related to oxygen- and ventilator-induced lung injury. PMID:20948883

  15. Development of human cell models for assessing the carcinogenic potential of chemicals

    SciTech Connect

    Pang Yaqin; Li Wenxue; Ma Rulin; Ji Weidong; Wang Qing; Li Daochuan; Xiao Yongmei; Wei Qing; Lai Yandong; Yang Ping; Chen Liping; Tang Shifu; Lin Yuchun; Zhuang Zhixiong; Zheng Yuxin; Chen Wen

    2008-11-01

    To develop human cell models for assessing the carcinogenic potential of chemicals, we established transgenic human cell lines and tested the sensitivity of known carcinogens using a cell transformation assay. A retroviral vector encoding an oncogenic allele of H-Ras (HBER) or c-Myc (HBEM) was introduced into human bronchial epithelial cells (HBE) immortalized by SV40 large T (LT) antigen, leading to increased cell proliferation but failing to confer a transformed phenotype characterized by anchorage-independent cell growth and tumor formation of immunodeficient mice. When these pre-transformed cells were treated with nickel sulfate (NiSO{sub 4}), we found that it shortened the latency of malignant transformation at least by 19 wk in HBER cells or 16 wk in HBEM cells compared to vector control cells. Similarly, the latency of cell transformation was shorter by 15 wk in HBER cells or 9 wk in HBEM cells when cells were treated with benzo(a)pyrenediol epoxide (BPDE). HBER cells appeared to be more sensitive to TPA, NiSO{sub 4} or BPDE-induced cell transformation compared to human embryonic kidney cells expressing H-Ras (HEKR), implying that cell-type specificity is one of important factors determining the effectiveness of the assay. Using AFB{sub 1} and BaP as the representative pro-carcinogens, we also compared the efficiency of three different metabolic conditions in mediating cell transformation. Low dose chemical induction seems to be a prospective system used for metabolic activation of pro-carcinogens. Our findings provided direct evidence that a genetically modified human cell transformation model can be applied to the assessment of potent carcinogens.

  16. Disease modeling using human induced pluripotent stem cells: Lessons from the liver☆

    PubMed Central

    Gieseck, Richard L.; Colquhoun, Jennifer; Hannan, Nicholas R.F.

    2014-01-01

    Human pluripotent stem cells (hPSCs) have the capacity to differentiate into any of the hundreds of distinct cell types that comprise the human body. This unique characteristic has resulted in considerable interest in the field of regenerative medicine, given the potential for these cells to be used to protect, repair, or replace diseased, injured, and aged cells within the human body. In addition to their potential in therapeutics, hPSCs can be used to study the earliest stages of human development and to provide a platform for both drug screening and disease modeling using human cells. Recently, the description of human induced pluripotent stem cells (hIPSCs) has allowed the field of disease modeling to become far more accessible and physiologically relevant, as pluripotent cells can be generated from patients of any genetic background. Disease models derived from hIPSCs that manifest cellular disease phenotypes have been established to study several monogenic diseases; furthermore, hIPSCs can be used for phenotype-based drug screens to investigate complex diseases for which the underlying genetic mechanism is unknown. As a result, the use of stem cells as research tools has seen an unprecedented growth within the last decade as researchers look for in vitro disease models which closely mimic in vivo responses in humans. Here, we discuss the beginnings of hPSCs, starting with isolation of human embryonic stem cells, moving into the development and optimization of hIPSC technology, and ending with the application of hIPSCs towards disease modeling and drug screening applications, with specific examples highlighting the modeling of inherited metabolic disorders of the liver. This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics. PMID:24943800

  17. Disease modeling using human induced pluripotent stem cells: lessons from the liver.

    PubMed

    Gieseck, Richard L; Colquhoun, Jennifer; Hannan, Nicholas R F

    2015-01-01

    Human pluripotent stem cells (hPSCs) have the capacity to differentiate into any of the hundreds of distinct cell types that comprise the human body. This unique characteristic has resulted in considerable interest in the field of regenerative medicine, given the potential for these cells to be used to protect, repair, or replace diseased, injured, and aged cells within the human body. In addition to their potential in therapeutics, hPSCs can be used to study the earliest stages of human development and to provide a platform for both drug screening and disease modeling using human cells. Recently, the description of human induced pluripotent stem cells (hIPSCs) has allowed the field of disease modeling to become far more accessible and physiologically relevant, as pluripotent cells can be generated from patients of any genetic background. Disease models derived from hIPSCs that manifest cellular disease phenotypes have been established to study several monogenic diseases; furthermore, hIPSCs can be used for phenotype-based drug screens to investigate complex diseases for which the underlying genetic mechanism is unknown. As a result, the use of stem cells as research tools has seen an unprecedented growth within the last decade as researchers look for in vitro disease models which closely mimic in vivo responses in humans. Here, we discuss the beginnings of hPSCs, starting with isolation of human embryonic stem cells, moving into the development and optimization of hIPSC technology, and ending with the application of hIPSCs towards disease modeling and drug screening applications, with specific examples highlighting the modeling of inherited metabolic disorders of the liver. This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics. PMID:24943800

  18. Stem-cell Based Engineered Immunity Against HIV Infection in the Humanized Mouse Model.

    PubMed

    Zhen, Anjie; Rezek, Valerie; Youn, Cindy; Rick, Jonathan; Lam, Brianna; Chang, Nelson; Zack, Jerome; Kamata, Masakazu; Kitchen, Scott

    2016-01-01

    With the rapid development of stem cell-based gene therapies against HIV, there is pressing requirement for an animal model to study the hematopoietic differentiation and immune function of the genetically modified cells. The humanized Bone-marrow/Liver/Thymus (BLT) mouse model allows for full reconstitution of a human immune system in the periphery, which includes T cells, B cells, NK cells and monocytes. The human thymic implant also allows for thymic selection of T cells in autologous thymic tissue. In addition to the study of HIV infection, the model stands as a powerful tool to study differentiation, development and functionality of cells derived from hematopoietic stem cells (HSCs). Here we outline the construction of humanized non-obese diabetic (NOD)-severe combined immunodeficient (SCID)-common gamma chain knockout (cγ(-/-))-Bone-marrow/Liver/Thymus (NSG-BLT) mice with HSCs transduced with CD4 chimeric antigen receptor (CD4CAR) lentivirus vector. We show that the CD4CAR HSCs can successfully differentiate into multiple lineages and have anti-HIV activity. The goal of the study is to demonstrate the use of NSG-BLT mouse model as an in vivo model for engineered immunity against HIV. It is worth noting that, because lentivirus and human tissue is used, experiments and surgeries should be performed in a Class II biosafety cabinet in a Biosafety Level 2 (BSL2) with special precautions (BSL2+) facility. PMID:27404517

  19. Mathematical modeling of gap junction coupling and electrical activity in human β-cells

    NASA Astrophysics Data System (ADS)

    Loppini, Alessandro; Braun, Matthias; Filippi, Simonetta; Gram Pedersen, Morten

    2015-12-01

    Coordinated insulin secretion is controlled by electrical coupling of pancreatic β-cells due to connexin-36 gap junctions. Gap junction coupling not only synchronizes the heterogeneous β-cell population, but can also modify the electrical behavior of the cells. These phenomena have been widely studied with mathematical models based on data from mouse β-cells. However, it is now known that human β-cell electrophysiology shows important differences to its rodent counterpart, and although human pancreatic islets express connexin-36 and show evidence of β-cell coupling, these aspects have been little investigated in human β-cells. Here we investigate theoretically, the gap junction coupling strength required for synchronizing electrical activity in a small cluster of cells simulated with a recent mathematical model of human β-cell electrophysiology. We find a lower limit for the coupling strength of approximately 20 pS (i.e., normalized to cell size, ˜2 pS pF-1) below which spiking electrical activity is asynchronous. To confront this theoretical lower bound with data, we use our model to estimate from an experimental patch clamp recording that the coupling strength is approximately 100-200 pS (10-20 pS pF-1), similar to previous estimates in mouse β-cells. We then investigate the role of gap junction coupling in synchronizing and modifying other forms of electrical activity in human β-cell clusters. We find that electrical coupling can prolong the period of rapid bursting electrical activity, and synchronize metabolically driven slow bursting, in particular when the metabolic oscillators are in phase. Our results show that realistic coupling conductances are sufficient to promote synchrony in small clusters of human β-cells as observed experimentally, and provide motivation for further detailed studies of electrical coupling in human pancreatic islets.

  20. Amniotic Fluid Stem Cells: A Novel Source for Modeling of Human Genetic Diseases

    PubMed Central

    Antonucci, Ivana; Provenzano, Martina; Rodrigues, Melissa; Pantalone, Andrea; Salini, Vincenzo; Ballerini, Patrizia; Borlongan, Cesar V.; Stuppia, Liborio

    2016-01-01

    In recent years, great interest has been devoted to the use of Induced Pluripotent Stem cells (iPS) for modeling of human genetic diseases, due to the possibility of reprogramming somatic cells of affected patients into pluripotent cells, enabling differentiation into several cell types, and allowing investigations into the molecular mechanisms of the disease. However, the protocol of iPS generation still suffers from technical limitations, showing low efficiency, being expensive and time consuming. Amniotic Fluid Stem cells (AFS) represent a potential alternative novel source of stem cells for modeling of human genetic diseases. In fact, by means of prenatal diagnosis, a number of fetuses affected by chromosomal or Mendelian diseases can be identified, and the amniotic fluid collected for genetic testing can be used, after diagnosis, for the isolation, culture and differentiation of AFS cells. This can provide a useful stem cell model for the investigation of the molecular basis of the diagnosed disease without the necessity of producing iPS, since AFS cells show some features of pluripotency and are able to differentiate in cells derived from all three germ layers “in vitro”. In this article, we describe the potential benefits provided by using AFS cells in the modeling of human genetic diseases. PMID:27110774

  1. Amniotic Fluid Stem Cells: A Novel Source for Modeling of Human Genetic Diseases.

    PubMed

    Antonucci, Ivana; Provenzano, Martina; Rodrigues, Melissa; Pantalone, Andrea; Salini, Vincenzo; Ballerini, Patrizia; Borlongan, Cesar V; Stuppia, Liborio

    2016-01-01

    In recent years, great interest has been devoted to the use of Induced Pluripotent Stem cells (iPS) for modeling of human genetic diseases, due to the possibility of reprogramming somatic cells of affected patients into pluripotent cells, enabling differentiation into several cell types, and allowing investigations into the molecular mechanisms of the disease. However, the protocol of iPS generation still suffers from technical limitations, showing low efficiency, being expensive and time consuming. Amniotic Fluid Stem cells (AFS) represent a potential alternative novel source of stem cells for modeling of human genetic diseases. In fact, by means of prenatal diagnosis, a number of fetuses affected by chromosomal or Mendelian diseases can be identified, and the amniotic fluid collected for genetic testing can be used, after diagnosis, for the isolation, culture and differentiation of AFS cells. This can provide a useful stem cell model for the investigation of the molecular basis of the diagnosed disease without the necessity of producing iPS, since AFS cells show some features of pluripotency and are able to differentiate in cells derived from all three germ layers "in vitro". In this article, we describe the potential benefits provided by using AFS cells in the modeling of human genetic diseases. PMID:27110774

  2. Modeling human risk: Cell & molecular biology in context

    SciTech Connect

    1997-06-01

    It is anticipated that early in the next century manned missions into outer space will occur, with a mission to Mars scheduled between 2015 and 2020. However, before such missions can be undertaken, a realistic estimation of the potential risks to the flight crews is required. One of the uncertainties remaining in this risk estimation is that posed by the effects of exposure to the radiation environment of outer space. Although the composition of this environment is fairly well understood, the biological effects arising from exposure to it are not. The reasons for this are three-fold: (1) A small but highly significant component of the radiation spectrum in outer space consists of highly charged, high energy (HZE) particles which are not routinely experienced on earth, and for which there are insufficient data on biological effects; (2) Most studies on the biological effects of radiation to date have been high-dose, high dose-rate, whereas in space, with the exception of solar particle events, radiation exposures will be low-dose, low dose-rate; (3) Although it has been established that the virtual absence of gravity in space has a profound effect on human physiology, it is not clear whether these effects will act synergistically with those of radiation exposure. A select panel will evaluate the utilizing experiments and models to accurately predict the risks associated with exposure to HZE particles. Topics of research include cellular and tissue response, health effects associated with radiation damage, model animal systems, and critical markers of Radiation response.

  3. Large-scale in silico modeling of metabolic interactions between cell types in the human brain.

    PubMed

    Lewis, Nathan E; Schramm, Gunnar; Bordbar, Aarash; Schellenberger, Jan; Andersen, Michael P; Cheng, Jeffrey K; Patel, Nilam; Yee, Alex; Lewis, Randall A; Eils, Roland; König, Rainer; Palsson, Bernhard Ø

    2010-12-01

    Metabolic interactions between multiple cell types are difficult to model using existing approaches. Here we present a workflow that integrates gene expression data, proteomics data and literature-based manual curation to model human metabolism within and between different types of cells. Transport reactions are used to account for the transfer of metabolites between models of different cell types via the interstitial fluid. We apply the method to create models of brain energy metabolism that recapitulate metabolic interactions between astrocytes and various neuron types relevant to Alzheimer's disease. Analysis of the models identifies genes and pathways that may explain observed experimental phenomena, including the differential effects of the disease on cell types and regions of the brain. Constraint-based modeling can thus contribute to the study and analysis of multicellular metabolic processes in the human tissue microenvironment and provide detailed mechanistic insight into high-throughput data analysis. PMID:21102456

  4. Chemokine receptor CXCR3 agonist prevents human T-cell migration in a humanized model of arthritic inflammation.

    PubMed

    O'Boyle, Graeme; Fox, Christopher R J; Walden, Hannah R; Willet, Joseph D P; Mavin, Emily R; Hine, Dominic W; Palmer, Jeremy M; Barker, Catriona E; Lamb, Christopher A; Ali, Simi; Kirby, John A

    2012-03-20

    The recruitment of T lymphocytes during diseases such as rheumatoid arthritis is regulated by stimulation of the chemokine receptors expressed by these cells. This study was designed to assess the potential of a CXCR3-specific small-molecule agonist to inhibit the migration of activated human T cells toward multiple chemokines. Further experiments defined the molecular mechanism for this anti-inflammatory activity. Analysis in vitro demonstrated agonist induced internalization of both CXCR3 and other chemokine receptors coexpressed by CXCR3(+) T cells. Unlike chemokine receptor-specific antagonists, the CXCR3 agonist inhibited migration of activated T cells toward the chemokine mixture in synovial fluid from patients with active rheumatoid arthritis. A humanized mouse air-pouch model showed that intravenous treatment with the CXCR3 agonist prevented inflammatory migration of activated human T cells toward this synovial fluid. A potential mechanism for this action was defined by demonstration that the CXCR3 agonist induces receptor cross-phosphorylation within CXCR3-CCR5 heterodimers on the surface of activated T cells. This study shows that generalized chemokine receptor desensitization can be induced by specific stimulation of a single chemokine receptor on the surface of activated human T cells. A humanized mouse model was used to demonstrate that this receptor desensitization inhibits the inflammatory response that is normally produced by the chemokines present in synovial fluid from patients with active rheumatoid arthritis. PMID:22392992

  5. Recapitulating amyloid β and tau pathology in human neural cell culture models: clinical implications

    PubMed Central

    Choi, Se Hoon; Kim, Young Hye; D’Avanzo, Carla; Aronson, Jenna; Tanzi, Rudolph E.; Kim, Doo Yeon

    2015-01-01

    Summary The “amyloid β hypothesis” of Alzheimer’s disease (AD) has been the reigning hypothesis explaining pathogenic mechanisms of AD over the last two decades. However, this hypothesis has not been fully validated in animal models, and several major unresolved issues remain. We recently developed a human neural cell culture model of AD based on a three-dimensional (3D) cell culture system. This unique, cellular model recapitulates key events of the AD pathogenic cascade, including β-amyloid plaques and neurofibrillary tangles. Our 3D human neural cell culture model system provides a premise for a new generation of cellular AD models that can serve as a novel platform for studying pathogenic mechanisms and for high-throughput drug screening in a human brain-like environment. PMID:27019672

  6. Modeling the Initiation of Ewing Sarcoma Tumorigenesis in Differentiating Human Embryonic Stem Cells

    PubMed Central

    Gordon, David J.; Motwani, Mona; Pellman, David

    2015-01-01

    Oncogenic transformation in Ewing sarcoma tumors is driven by the fusion oncogene EWS-FLI1. However, despite the well-established role of EWS-FLI1 in tumor initiation, the development of models of Ewing sarcoma in human cells with defined genetic elements has been challenging. Here, we report a novel approach to model the initiation of Ewing sarcoma tumorigenesis that exploits the developmental and pluripotent potential of human embryonic stem cells. The inducible expression of EWS-FLI1 in embryoid bodies, or collections of differentiating stem cells, generates cells with properties of Ewing sarcoma tumors, including characteristics of transformation. These cell lines exhibit anchorage-independent growth, a lack of contact inhibition and a strong Ewing sarcoma gene expression signature. Furthermore, these cells also demonstrate a requirement for the persistent expression of EWS-FLI1 for cell survival and growth, which is a hallmark Ewing sarcoma tumors. PMID:26455317

  7. Validation of Normal Human Bronchial Epithelial Cells as a Model for Influenza A Infections in Human Distal Trachea

    PubMed Central

    Davis, A. Sally; Chertow, Daniel S.; Moyer, Jenna E.; Suzich, Jon; Sandouk, Aline; Dorward, David W.; Logun, Carolea; Shelhamer, James H.

    2015-01-01

    Primary normal human bronchial/tracheal epithelial (NHBE) cells, derived from the distal-most aspect of the trachea at the bifurcation, have been used for a number of studies in respiratory disease research. Differences between the source tissue and the differentiated primary cells may impact infection studies based on this model. Therefore, we examined how well-differentiated NHBE cells compared with their source tissue, the human distal trachea, as well as the ramifications of these differences on influenza A viral pathogenesis research using this model. We employed a histological analysis including morphological measurements, electron microscopy, multi-label immunofluorescence confocal microscopy, lectin histochemistry, and microarray expression analysis to compare differentiated NHBEs to human distal tracheal epithelium. Pseudostratified epithelial height, cell type variety and distribution varied significantly. Electron microscopy confirmed differences in cellular attachment and paracellular junctions. Influenza receptor lectin histochemistry revealed that α2,3 sialic acids were rarely present on the apical aspect of the differentiated NHBE cells, but were present in low numbers in the distal trachea. We bound fluorochrome bioconjugated virus to respiratory tissue and NHBE cells and infected NHBE cells with human influenza A viruses. Both indicated that the pattern of infection progression in these cells correlated with autopsy studies of fatal cases from the 2009 pandemic. PMID:25604814

  8. Derivation of Neural Stem Cells from Human Adult Peripheral CD34+ Cells for an Autologous Model of Neuroinflammation

    PubMed Central

    Wang, Tongguang; Choi, Elliot; Monaco, Maria Chiara G.; Campanac, Emilie; Medynets, Marie; Do, Thao; Rao, Prashant; Johnson, Kory R.; Elkahloun, Abdel G.; Von Geldern, Gloria; Johnson, Tory; Subramaniam, Sriram; Hoffman, Dax; Major, Eugene; Nath, Avindra

    2013-01-01

    Proinflammatory factors from activated T cells inhibit neurogenesis in adult animal brain and cultured human fetal neural stem cells (NSC). However, the role of inhibition of neurogenesis in human neuroinflammatory diseases is still uncertain because of the difficulty in obtaining adult NSC from patients. Recent developments in cell reprogramming suggest that NSC may be derived directly from adult fibroblasts. We generated NSC from adult human peripheral CD34+ cells by transfecting the cells with Sendai virus constructs containing Sox2, Oct3/4, c-Myc and Klf4. The derived NSC could be differentiated to glial cells and action potential firing neurons. Co-culturing NSC with activated autologous T cells or treatment with recombinant granzyme B caused inhibition of neurogenesis as indicated by decreased NSC proliferation and neuronal differentiation. Thus, we have established a unique autologous in vitro model to study the pathophysiology of neuroinflammatory diseases that has potential for usage in personalized medicine. PMID:24303066

  9. 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. PMID:26657644

  10. Characterization of human follicular thyroid cancer cell lines in preclinical mouse models

    PubMed Central

    Reeb, Ashley N; Ziegler, Andrea

    2016-01-01

    Follicular thyroid cancer (FTC) is the second most common type of thyroid cancers. In order to develop more effective personalized therapies, it is necessary to thoroughly evaluate patient-derived cell lines in in vivo preclinical models before using them to test new, targeted therapies. This study evaluates the tumorigenic and metastatic potential of a panel of three human FTC cell lines (WRO, FTC-238, and TT1609-CO2) with defined genetic mutations in two in vivo murine models: an orthotopic thyroid cancer model to study tumor progression and a tail vein injection model to study metastasis. All cell lines developed tumors in the orthotopic model, with take rates of 100%. Notably, WRO-derived tumors grew two to four times faster than tumors arising from the FTC-238 and TT2609-CO2 cell lines. These results mirrored those of a tail vein injection model for lung metastasis: one hundred percent of mice injected with WRO cells in the tail vein exhibited aggressive growth of bilateral lung metastases within 35 days. In contrast, tail vein injection of FTC-238 or TT2609-CO2 cells did not result in lung metastasis. Together, our work demonstrates that these human FTC cell lines display highly varied tumorigenic and metastatic potential in vivo with WRO being the most aggressive cell line in both orthotopic and lung metastasis models. This information will be valuable when selecting cell lines for preclinical drug testing. PMID:26830329

  11. Restorative effect of hair follicular dermal cells on injured human hair follicles in a mouse model.

    PubMed

    Yamao, Mikaru; Inamatsu, Mutsumi; Okada, Taro; Ogawa, Yuko; Ishida, Yuji; Tateno, Chise; Yoshizato, Katsutoshi

    2015-03-01

    No model is available for examining whether in vivo-damaged human hair follicles (hu-HFs) are rescued by transplanting cultured hu-HF dermal cells (dermal papilla and dermal sheath cells). Such a model might be valuable for examining whether in vivo-damaged hu-HFs such as miniaturized hu-HFs in androgenic alopecia are improvable by auto-transplanting hu-HF dermal cells. In this study, we first developed mice with humanized skin composed of hu-keratinocytes and hu-dermal fibroblasts. Then, a 'humanized scalp model mouse' was generated by transplanting hu-scalp HFs into the humanized skin. To demonstrate the usability of the model, the lower halves of the hu-HFs in the model were amputated in situ, and cultured hu-HF dermal cells were injected around the amputated area. The results demonstrated that the transplanted cells contributed to the restoration of the damaged HFs. This model could be used to explore clinically effective technologies for hair restoration therapy by autologous cell transplantation. PMID:25557326

  12. Searching for Classical Brown Fat in Humans: Development of a Novel Human Fetal Brown Stem Cell Model.

    PubMed

    Di Franco, Alessandra; Guasti, Daniele; Squecco, Roberta; Mazzanti, Benedetta; Rossi, Francesca; Idrizaj, Eglantina; Gallego-Escuredo, José M; Villarroya, Francesc; Bani, Daniele; Forti, Gianni; Vannelli, Gabriella Barbara; Luconi, Michaela

    2016-06-01

    The potential therapeutic applications of targeting brown adipose tissue open new clinical avenues in fighting against metabolic pathologies. However, due to the limited extension in adult humans of brown depots, which are dramatically reduced after birth, solid cell models to study human brown adipogenesis and its regulatory factors in pathophysiology are urgently needed. Here, we generated a novel human model of brown adipose stem cells, hfB-ASC, derived for the first time from fetal interscapular brown fat depots. Besides the characterization of their stem and classical brown adipose properties, we demonstrated that these cells retain a specific intrinsic differentiation program to functional brown adipocytes, even spontaneously generating organoid structures with brown features. Moreover, for the first time, we investigated the thermogenic and electrophysiological activity of the in vitro-derived fetal brown adipocytes compared to their undifferentiated precursors hfB-ASC, in basal and norepinephrine-induced conditions. In conclusion, from interscapular brown fat of the human fetus we developed and functionally characterized a novel physiological brown adipose stem cell model early programmed to brown differentiation, which may represent a unique opportunity for further studies on brown adipogenesis processes in humans as well as the most suitable target to study novel therapeutic approaches for stimulating brown activity in metabolic pathologies. Stem Cells 2016;34:1679-1691. PMID:26866833

  13. Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

    PubMed Central

    Vatakis, Dimitrios N.; Bristol, Gregory C.; Kim, Sohn G.; Levin, Bernard; Liu, Wei; Radu, Caius G.; Kitchen, Scott G.; Zack, Jerome A.

    2012-01-01

    Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection. One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8. We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor

  14. Human T cells in silico: Modelling their electrophysiological behaviour in health and disease.

    PubMed

    Ehling, Petra; Meuth, Patrick; Eichinger, Paul; Herrmann, Alexander M; Bittner, Stefan; Pawlowski, Matthias; Pankratz, Susann; Herty, Michael; Budde, Thomas; Meuth, Sven G

    2016-09-01

    Although various types of ion channels are known to have an impact on human T cell effector functions, their exact mechanisms of influence are still poorly understood. The patch clamp technique is a well-established method for the investigation of ion channels in neurons and T cells. However, small cell sizes and limited selectivity of pharmacological blockers restrict the value of this experimental approach. Building a realistic T cell computer model therefore can help to overcome these kinds of limitations as well as reduce the overall experimental effort. The computer model introduced here was fed off ion channel parameters from literature and new experimental data. It is capable of simulating the electrophysiological behaviour of resting and activated human CD4(+) T cells under basal conditions and during extracellular acidification. The latter allows for the very first time to assess the electrophysiological consequences of tissue acidosis accompanying most forms of inflammation. PMID:27288542

  15. Identifying anti-growth factors for human cancer cell lines through genome-scale metabolic modeling

    PubMed Central

    Ghaffari, Pouyan; Mardinoglu, Adil; Asplund, Anna; Shoaie, Saeed; Kampf, Caroline; Uhlen, Mathias; Nielsen, Jens

    2015-01-01

    Human cancer cell lines are used as important model systems to study molecular mechanisms associated with tumor growth, hereunder how genomic and biological heterogeneity found in primary tumors affect cellular phenotypes. We reconstructed Genome scale metabolic models (GEMs) for eleven cell lines based on RNA-Seq data and validated the functionality of these models with data from metabolite profiling. We used cell line-specific GEMs to analyze the differences in the metabolism of cancer cell lines, and to explore the heterogeneous expression of the metabolic subsystems. Furthermore, we predicted 85 antimetabolites that can inhibit growth of, or even kill, any of the cell lines, while at the same time not being toxic for 83 different healthy human cell types. 60 of these antimetabolites were found to inhibit growth in all cell lines. Finally, we experimentally validated one of the predicted antimetabolites using two cell lines with different phenotypic origins, and found that it is effective in inhibiting the growth of these cell lines. Using immunohistochemistry, we also showed high or moderate expression levels of proteins targeted by the validated antimetabolite. Identified anti-growth factors for inhibition of cell growth may provide leads for the development of efficient cancer treatment strategies. PMID:25640694

  16. Identifying anti-growth factors for human cancer cell lines through genome-scale metabolic modeling.

    PubMed

    Ghaffari, Pouyan; Mardinoglu, Adil; Asplund, Anna; Shoaie, Saeed; Kampf, Caroline; Uhlen, Mathias; Nielsen, Jens

    2015-01-01

    Human cancer cell lines are used as important model systems to study molecular mechanisms associated with tumor growth, hereunder how genomic and biological heterogeneity found in primary tumors affect cellular phenotypes. We reconstructed Genome scale metabolic models (GEMs) for eleven cell lines based on RNA-Seq data and validated the functionality of these models with data from metabolite profiling. We used cell line-specific GEMs to analyze the differences in the metabolism of cancer cell lines, and to explore the heterogeneous expression of the metabolic subsystems. Furthermore, we predicted 85 antimetabolites that can inhibit growth of, or even kill, any of the cell lines, while at the same time not being toxic for 83 different healthy human cell types. 60 of these antimetabolites were found to inhibit growth in all cell lines. Finally, we experimentally validated one of the predicted antimetabolites using two cell lines with different phenotypic origins, and found that it is effective in inhibiting the growth of these cell lines. Using immunohistochemistry, we also showed high or moderate expression levels of proteins targeted by the validated antimetabolite. Identified anti-growth factors for inhibition of cell growth may provide leads for the development of efficient cancer treatment strategies. PMID:25640694

  17. Cytotoxicity evaluation of large cyanobacterial strain set using selected human and murine in vitro cell models.

    PubMed

    Hrouzek, Pavel; Kapuścik, Aleksandra; Vacek, Jan; Voráčová, Kateřina; Paichlová, Jindřiška; Kosina, Pavel; Voloshko, Ludmila; Ventura, Stefano; Kopecký, Jiří

    2016-02-01

    The production of cytotoxic molecules interfering with mammalian cells is extensively reported in cyanobacteria. These compounds may have a use in pharmacological applications; however, their potential toxicity needs to be considered. We performed cytotoxicity tests of crude cyanobacterial extracts in six cell models in order to address the frequency of cyanobacterial cytotoxicity to human cells and the level of specificity to a particular cell line. A set of more than 100 cyanobacterial crude extracts isolated from soil habitats (mainly genera Nostoc and Tolypothrix) was tested by MTT test for in vitro toxicity on the hepatic and non-hepatic human cell lines HepG2 and HeLa, and three cell systems of rodent origin: Yac-1, Sp-2 and Balb/c 3T3 fibroblasts. Furthermore, a subset of the extracts was assessed for cytotoxicity against primary cultures of human hepatocytes as a model for evaluating potential hepatotoxicity. Roughly one third of cyanobacterial extracts caused cytotoxic effects (i.e. viability<75%) on human cell lines. Despite the sensitivity differences, high correlation coefficients among the inhibition values were obtained for particular cell systems. This suggests a prevailing general cytotoxic effect of extracts and their constituents. The non-transformed immortalized fibroblasts (Balb/c 3T3) and hepatic cancer line HepG2 exhibited good correlations with primary cultures of human hepatocytes. The presence of cytotoxic fractions in strongly cytotoxic extracts was confirmed by an activity-guided HPLC fractionation, and it was demonstrated that cyanobacterial cytotoxicity is caused by a mixture of components with similar hydrophobic/hydrophilic properties. The data presented here could be used in further research into in vitro testing based on human models for the toxicological monitoring of complex cyanobacterial samples. PMID:26519817

  18. Use of human induced pluripotent stem cell-derived neurons as a model for Cerebral Toxoplasmosis.

    PubMed

    Tanaka, Naomi; Ashour, Danah; Dratz, Edward; Halonen, Sandra

    2016-01-01

    Toxoplasma gondii is a ubiquitous protozoan parasite with approximately one-third of the worlds' population chronically infected. In chronically infected individuals, the parasite resides primarily in cysts within neurons in the central nervous system. The chronic infection in immunocompetent individuals has been considered to be asymptomatic but increasing evidence indicates the chronic infection can lead to neuropsychiatric disorders such as Schizophrenia, prenatal depression and suicidal thoughts. A better understanding of the mechanism(s) by which the parasite exerts effects on human behavior is limited due to lack of suitable human neuronal models. In this paper, we report the use of human neurons derived from normal cord blood CD34+ cells generated via genetic reprogramming, as an in vitro model for the study T. gondii in neurons. This culture method resulted in a relatively pure monolayer of induced human neuronal-like cells that stained positive for neuronal markers, MAP2, NFL, NFH and NeuN. These induced human neuronal-like cells (iHNs) were efficiently infected by the Prugniad strain of the parasite and supported replication of the tachyzoite stage and development of the cyst stage. Infected iHNs could be maintained through 5 days of infection, allowing for formation of large cysts. This induced human neuronal model represents a novel culture method to study both tachyzoite and bradyzoite stages of T. gondii in human neurons. PMID:27083472

  19. Cell therapy for the diseased liver: from stem cell biology to novel models for hepatotropic human pathogens

    PubMed Central

    Brezillon, Nicolas; Kremsdorf, Dina; Weiss, Mary C.

    2008-01-01

    It has long been known that hepatocytes possess the potential to replicate through many cell generations because regeneration can be achieved in rodents after serial two-thirds hepatectomy. It has taken considerable time and effort to harness this potential, with liver regeneration models involving hepatocyte transplantation developing over the past 15 years. This review will describe the experiments that have established the models and methodology for liver repopulation, and the use of cells other than adult hepatocytes in liver repopulation, including hepatic cell lines and hematopoietic, cord blood, hepatic and embryonic stem cells. Emphasis will be placed on the characteristics of the models and how they can influence the outcome of the experiments. Finally, an account of the development of murine models that are competent to accept human hepatocytes is provided. In these models, liver deficiencies are induced in immunodeficient mice, where healthy human cells have a selective advantage. These mice with humanized livers provide a powerful new experimental tool for the study of human hepatotropic pathogens. PMID:19048074

  20. Human mesenchymal stem cells towards non-alcoholic steatohepatitis in an immunodeficient mouse model

    SciTech Connect

    Winkler, Sandra; Borkham-Kamphorst, Erawan; Stock, Peggy; Brückner, Sandra; Dollinger, Matthias; Weiskirchen, Ralf; Christ, Bruno

    2014-08-15

    Non-alcoholic steatohepatitis (NASH) is a frequent clinical picture characterised by hepatic inflammation, lipid accumulation and fibrosis. When untreated, NASH bears a high risk of developing liver cirrhosis and consecutive hepatocellular carcinoma requiring liver transplantation in its end-stage. However, donor organ scarcity has prompted the search for alternatives, of which hepatocyte or stem cell-derived hepatocyte transplantation are regarded auspicious options of treatment. Mesenchymal stem cells (MSC) are able to differentiate into hepatocyte-like cells and thus may represent an alternative cell source to primary hepatocytes. In addition these cells feature anti-inflammatory and pro-regenerative characteristics, which might favour liver recovery from NASH. The aim of this study was to investigate the potential benefit of hepatocyte-like cells derived from human bone marrow MSC in a mouse model of diet-induced NASH. Seven days post-transplant, human hepatocyte-like cells were found in the mouse liver parenchyma. Triglyceride depositions were lowered in the liver but restored to normal in the blood. Hepatic inflammation was attenuated as verified by decreased expression of the acute phase protein serum amyloid A, inflammation-associated markers (e.g. lipocalin 2), as well as the pro-inflammatory cytokine TNFα. Moreover, the proliferation of host hepatocytes that indicate the regenerative capacity in livers receiving cell transplants was enhanced. Transplantation of MSC-derived human hepatocyte-like cells corrects NASH in mice by restoring triglyceride depositions, reducing inflammation and augmenting the regenerative capacity of the liver. - Highlights: • First time to show NASH in an immune-deficient mouse model. • Human MSC attenuate NASH and improve lipid homeostasis. • MSC act anti-fibrotic and augment liver regeneration by stimulation of proliferation. • Pre-clinical assessment of human MSC for stem cell-based therapy of NASH.

  1. Cell senescence abrogates the therapeutic potential of human mesenchymal stem cells in the lethal endotoxemia model.

    PubMed

    Sepúlveda, Juan Carlos; Tomé, María; Fernández, María Eugenia; Delgado, Mario; Campisi, Judith; Bernad, Antonio; González, Manuel A

    2014-07-01

    Mesenchymal stem cells (MSCs) possess unique paracrine and immunosuppressive properties, which make them useful candidates for cellular therapy. Here, we address how cellular senescence influences the therapeutic potential of human MSCs (hMSCs). Senescence was induced in bone marrow-derived hMSC cultures with gamma irradiation. Control and senescent cells were tested for their immunoregulatory activity in vitro and in vivo, and an extensive molecular characterization of the phenotypic changes induced by senescence was performed. We also compared the gene expression profiles of senescent hMSCs with a collection of hMSCs used in an ongoing clinical study of Graft Versus Host disease (GVHD). Our results show that senescence induces extensive phenotypic changes in hMSCs and abrogates their protective activity in a murine model of LPS-induced lethal endotoxemia. Although senescent hMSCs retain an ability to regulate the inflammatory response on macrophages in vitro, and, in part retain their capacity to significantly inhibit lymphocyte proliferation, they have a severely impaired migratory capacity in response to proinflammatory signals, which is associated with an inhibition of the AP-1 pathway. Additionally, expression analysis identified PLEC, C8orf48, TRPC4, and ZNF14, as differentially regulated genes in senescent hMSCs that were similarly regulated in those hMSCs which failed to produce a therapeutic effect in a GVHD trial. All the observed phenotypic alterations were confirmed in replicative-senescent hMSCs. In conclusion, this study highlights important changes in the immunomodulatory phenotype of senescent hMSCs and provides candidate gene signatures which may be useful to evaluate the therapeutic potential of hMSCs used in future clinical studies. PMID:24496748

  2. Cortical Differentiation of Human Pluripotent Cells for In Vitro Modeling of Alzheimer's Disease.

    PubMed

    Saurat, Nathalie G; Livesey, Frederick J; Moore, Steven

    2016-01-01

    Stem cell models of Alzheimer's disease provide an opportunity to study the mechanisms underlying disease pathology at a resolution that is not possible in animal models. Furthermore, the ability to reprogram patient somatic cells to a pluripotent state ensures that the disease can be investigated in the correct genetic context. Here, we describe the directed differentiation of human pluripotent cells to cortical progenitors by recapitulating key developmental signaling events in vitro. Over a timeframe that mirrors human development, these progenitors give rise to functional lower and upper layer neurons. We also describe biochemical and imaging based methods to analyse key APP and Tau phenotypes in neurons generated from pluripotent stem cells from individuals with either monogenic familial Alzheimer's disease or Down's syndrome. PMID:26235073

  3. In vitro modeling of the interaction between human epithelial cells and lymphocytes upon influenza infection.

    PubMed

    Ilyushina, Natalia A; Wright, Peter F

    2016-09-01

    Influenza viruses are a continuous threat to humans because of their ability to cross species barriers and adapt to new hosts. Data from murine studies, along with limited human data, suggest that CD8(+) cytotoxic T lymphocytes (CTL) that recognize conserved epitopes of structural influenza proteins are the main mediators of influenza virus clearance. Additionally, the fact that many CTLs recognize epitopes shared between different influenza strains offers the potential for broad cross-strain immunity. However, the mechanisms of cellular immunity against influenza viruses are poorly defined in humans, where the CTL response has been hard to measure and interpret. We developed a novel CTL assay that utilizes fully differentiated nasal human epithelial cells taken from volunteers as permissive targets for autologous peripheral blood-derived influenza virus-specific cytotoxic T lymphocytes. This in vitro system of human lymphocyte-epithelial cell co-cultures can be considered as the closest approximation to events in vivo and can be employed for studying the interactions between the pathogen and human host. Modeling of the natural interaction process between the primary cell type that supports the productive replication of influenza and immune cells may allow us to put in perspective CTLs as a correlate of immunity to influenza in humans. PMID:27102577

  4. Concise review: the relevance of human stem cell-derived organoid models for epithelial translational medicine.

    PubMed

    Hynds, Robert E; Giangreco, Adam

    2013-03-01

    Epithelial organ remodeling is a major contributing factor to worldwide death and disease, costing healthcare systems billions of dollars every year. Despite this, most fundamental epithelial organ research fails to produce new therapies and mortality rates for epithelial organ diseases remain unacceptably high. In large part, this failure in translating basic epithelial research into clinical therapy is due to a lack of relevance in existing preclinical models. To correct this, new models are required that improve preclinical target identification, pharmacological lead validation, and compound optimization. In this review, we discuss the relevance of human stem cell-derived, three-dimensional organoid models for addressing each of these challenges. We highlight the advantages of stem cell-derived organoid models over existing culture systems, discuss recent advances in epithelial tissue-specific organoids, and present a paradigm for using organoid models in human translational medicine. PMID:23203919

  5. Characterization of Human Neural Progenitor Cell Models for Developmental Neurotoxicity Screening

    EPA Science Inventory

    Current testing methods for developmental neurotoxicity (DNT) make evaluation of the effects of large numbers of chemicals impractical and prohibitively expensive. As such, we are evaluating two different human neural progenitor cell (hNPC) models for their utility in screens for...

  6. Cell-Free Transmission of Human Adenovirus by Passive Mass Transfer in Cell Culture Simulated in a Computer Model

    PubMed Central

    Yakimovich, Artur; Gumpert, Heidi; Burckhardt, Christoph J.; Lütschg, Verena A.; Jurgeit, Andreas; Sbalzarini, Ivo F.

    2012-01-01

    Viruses spread between cells, tissues, and organisms by cell-free and cell-cell transmissions. Both mechanisms enhance disease development, but it is difficult to distinguish between them. Here, we analyzed the transmission mode of human adenovirus (HAdV) in monolayers of epithelial cells by wet laboratory experimentation and a computer simulation. Using live-cell fluorescence microscopy and replication-competent HAdV2 expressing green fluorescent protein, we found that the spread of infection invariably occurred after cell lysis. It was affected by convection and blocked by neutralizing antibodies but was independent of second-round infections. If cells were overlaid with agarose, convection was blocked and round plaques developed around lytic infected cells. Infected cells that did not lyse did not give rise to plaques, highlighting the importance of cell-free transmission. Key parameters for cell-free virus transmission were the time from infection to lysis, the dose of free viruses determining infection probability, and the diffusion of single HAdV particles in aqueous medium. With these parameters, we developed an in silico model using multiscale hybrid dynamics, cellular automata, and particle strength exchange. This so-called white box model is based on experimentally determined parameters and reproduces viral infection spreading as a function of the local concentration of free viruses. These analyses imply that the extent of lytic infections can be determined by either direct plaque assays or can be predicted by calculations of virus diffusion constants and modeling. PMID:22787215

  7. Modeling human development and disease in pluripotent stem cell-derived gastric organoids

    PubMed Central

    McCracken, Kyle W.; Catá, Emily M.; Crawford, Calyn M.; Sinagoga, Katie L.; Schumacher, Michael; Rockich, Briana E.; Tsai, Yu-Hwai; Mayhew, Christopher N.; Spence, Jason R.; Zavros, Yana; Wells, James M.

    2014-01-01

    Gastric diseases, including peptic ulcer disease and gastric cancer, affect 10% of the world’s population and are largely due to chronic H. pylori infection1–3. Species differences in embryonic development and architecture of the adult stomach make animal models suboptimal for studying human stomach organogenesis and pathogenesis4, and there is no experimental model of normal human gastric mucosa. Here we report the de novo generation of three-dimensional human gastric tissue in vitro through the directed differentiation of human pluripotent stem cells (hPSCs). We identified that temporal manipulation of the FGF, WNT, BMP, retinoic acid and EGF signaling pathways and three-dimensional growth are sufficient to generate human gastric organoids (hGOs). Developing hGOs progressed through molecular and morphogenetic stages that were nearly identical to the developing antrum of the mouse stomach. Organoids formed primitive gastric gland- and pit-like domains, proliferative zones containing LGR5-expressing cells, surface and antral mucous cells, and a diversity of gastric endocrine cells. We used hGO cultures to identify novel signaling mechanisms that regulate early endoderm patterning and gastric endocrine cell differentiation upstream of the transcription factor NEUROG3. Using hGOs to model pathogenesis of human disease, we found that H. pylori infection resulted in rapid association of the virulence factor CagA with the c-Met receptor, activation of signaling and induction of epithelial proliferation. Together, these studies describe a novel and robust in vitro system for elucidating the mechanisms underlying human stomach development and disease. PMID:25363776

  8. Three-dimensional cultures modeling premalignant progression of human breast epithelial cells: role of cysteine cathepsins.

    PubMed

    Mullins, Stefanie R; Sameni, Mansoureth; Blum, Galia; Bogyo, Matthew; Sloane, Bonnie F; Moin, Kamiar

    2012-12-01

    The expression of the cysteine protease cathepsin B is increased in early stages of human breast cancer.To assess the potential role of cathepsin B in premalignant progression of breast epithelial cells, we employed a 3D reconstituted basement membrane overlay culture model of MCF10A human breast epithelial cells and isogenic variants that replicate the in vivo phenotypes of hyper plasia(MCF10AneoT) and atypical hyperplasia (MCF10AT1). MCF10A cells developed into polarized acinar structures with central lumens. In contrast, MCF10AneoT and MCF10AT1 cells form larger structures in which the lumens are filled with cells. CA074Me, a cell-permeable inhibitor selective for the cysteine cathepsins B and L,reduced proliferation and increased apoptosis of MCF10A, MCF10AneoT and MCF10AT1 cells in 3D culture. We detected active cysteine cathepsins in the isogenic MCF10 variants in 3D culture with GB111, a cell-permeable activity based probe, and established differential inhibition of cathepsin B in our 3D cultures. We conclude that cathepsin B promotes proliferation and premalignant progression of breast epithelial cells. These findings are consistent with studies by others showing that deletion of cathepsin B in the transgenic MMTV-PyMT mice, a murine model that is predisposed to development of mammary cancer, reduces malignant progression. PMID:23667900

  9. Animals Models of Human T Cell Leukemia Virus Type I Leukemogenesis.

    PubMed

    Niewiesk, Stefan

    2016-03-31

    Infection with human T cell leukemia virus type I (HTLV-I) causes adult T cell leukemia (ATL) in a minority of infected individuals after long periods of viral persistence. The various stages of HTLV-I infection and leukemia development are studied by using several different animal models: (1) the rabbit (and mouse) model of persistent HTLV-I infection, (2) transgenic mice to model tumorigenesis by HTLV-I specific protein expression, (3) ATL cell transfers into immune-deficient mice, and (4) infection of humanized mice with HTLV-I. After infection, virus replicates without clinical disease in rabbits and to a lesser extent in mice. Transgenic expression of both the transactivator protein (Tax) and the HTLV-I bZIP factor (HBZ) protein have provided insight into factors important in leukemia/lymphoma development. To investigate factors relating to tumor spread and tissue invasion, a number of immune-deficient mice based on the severe combined immunodeficiency (SCID) or non-obese diabetic/SCID background have been used. Inoculation of adult T cell leukemia cell (lines) leads to lymphoma with osteolytic bone lesions and to a lesser degree to leukemia development. These mice have been used extensively for the testing of anticancer drugs and virotherapy. A recent development is the use of so-called humanized mice, which, upon transfer of CD34(+)human umbilical cord stem cells, generate human lymphocytes. Infection with HTLV-I leads to leukemia/lymphoma development, thus providing an opportunity to investigate disease development with the aid of molecularly cloned viruses. However, further improvements of this mouse model, particularly in respect to the development of adaptive immune responses, are necessary. PMID:27034390

  10. Targeting Tumor Vasculature Endothelial Cells and Tumor Cells for Immunotherapy of Human Melanoma in a Mouse Xenograft Model

    NASA Astrophysics Data System (ADS)

    Hu, Zhiwei; Sun, Ying; Garen, Alan

    1999-07-01

    An immunotherapy treatment for cancer that targets both the tumor vasculature and tumor cells has shown promising results in a severe combined immunodeficient mouse xenograft model of human melanoma. The treatment involves systemic delivery of an immunoconjugate molecule composed of a tumor-targeting domain conjugated to the Fc effector domain of human IgG1. The effector domain induces a cytolytic immune response against the targeted cells by natural killer cells and complement. Two types of targeting domains were used. One targeting domain is a human single-chain Fv molecule that binds to a chondroitin sulfate proteoglycan expressed on the surface of most human melanoma cells. Another targeting domain is factor VII (fVII), a zymogen that binds with high specificity and affinity to the transmembrane receptor tissue factor (TF) to initiate the blood coagulation cascade. TF is expressed by endothelial cells lining the tumor vasculature but not the normal vasculature, and also by many types of tumor cells including melanoma. Because the binding of a fVII immunoconjugate to TF might cause disseminated intravascular coagulation, the active site of fVII was mutated to inhibit coagulation without affecting the affinity for TF. The immunoconjugates were encoded as secreted molecules in a replication-defective adenovirus vector, which was injected into the tail vein of severe combined immunodeficient mice. The results demonstrate that a mutated fVII immunoconjugate, administered separately or together with a single-chain Fv immunoconjugate that binds to the tumor cells, can inhibit the growth or cause regression of an established human tumor xenograft. This procedure could be effective in treating a broad spectrum of human solid tumors that express TF on vascular endothelial cells and tumor cells.

  11. Human induced pluripotent stem cells for monogenic disease modelling and therapy

    PubMed Central

    Spitalieri, Paola; Talarico, Valentina Rosa; Murdocca, Michela; Novelli, Giuseppe; Sangiuolo, Federica

    2016-01-01

    Recent and advanced protocols are now available to derive human induced pluripotent stem cells (hiPSCs) from patients affected by genetic diseases. No curative treatments are available for many of these diseases; thus, hiPSCs represent a major impact on patient’ health. hiPSCs represent a valid model for the in vitro study of monogenic diseases, together with a better comprehension of the pathogenic mechanisms of the pathology, for both cell and gene therapy protocol applications. Moreover, these pluripotent cells represent a good opportunity to test innovative pharmacological treatments focused on evaluating the efficacy and toxicity of novel drugs. Today, innovative gene therapy protocols, especially gene editing-based, are being developed, allowing the use of these cells not only as in vitro disease models but also as an unlimited source of cells useful for tissue regeneration and regenerative medicine, eluding ethical and immune rejection problems. In this review, we will provide an up-to-date of modelling monogenic disease by using hiPSCs and the ultimate applications of these in vitro models for cell therapy. We consider and summarize some peculiar aspects such as the type of parental cells used for reprogramming, the methods currently used to induce the transcription of the reprogramming factors, and the type of iPSC-derived differentiated cells, relating them to the genetic basis of diseases and to their inheritance model. PMID:27114745

  12. Human induced pluripotent stem cells for monogenic disease modelling and therapy.

    PubMed

    Spitalieri, Paola; Talarico, Valentina Rosa; Murdocca, Michela; Novelli, Giuseppe; Sangiuolo, Federica

    2016-04-26

    Recent and advanced protocols are now available to derive human induced pluripotent stem cells (hiPSCs) from patients affected by genetic diseases. No curative treatments are available for many of these diseases; thus, hiPSCs represent a major impact on patient' health. hiPSCs represent a valid model for the in vitro study of monogenic diseases, together with a better comprehension of the pathogenic mechanisms of the pathology, for both cell and gene therapy protocol applications. Moreover, these pluripotent cells represent a good opportunity to test innovative pharmacological treatments focused on evaluating the efficacy and toxicity of novel drugs. Today, innovative gene therapy protocols, especially gene editing-based, are being developed, allowing the use of these cells not only as in vitro disease models but also as an unlimited source of cells useful for tissue regeneration and regenerative medicine, eluding ethical and immune rejection problems. In this review, we will provide an up-to-date of modelling monogenic disease by using hiPSCs and the ultimate applications of these in vitro models for cell therapy. We consider and summarize some peculiar aspects such as the type of parental cells used for reprogramming, the methods currently used to induce the transcription of the reprogramming factors, and the type of iPSC-derived differentiated cells, relating them to the genetic basis of diseases and to their inheritance model. PMID:27114745

  13. Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Afford New Opportunities in Inherited Cardiovascular Disease Modeling

    PubMed Central

    Bayzigitov, Daniel R.; Medvedev, Sergey P.; Dementyeva, Elena V.; Bayramova, Sevda A.; Pokushalov, Evgeny A.; Karaskov, Alexander M.; Zakian, Suren M.

    2016-01-01

    Fundamental studies of molecular and cellular mechanisms of cardiovascular disease pathogenesis are required to create more effective and safer methods of their therapy. The studies can be carried out only when model systems that fully recapitulate pathological phenotype seen in patients are used. Application of laboratory animals for cardiovascular disease modeling is limited because of physiological differences with humans. Since discovery of induced pluripotency generating induced pluripotent stem cells has become a breakthrough technology in human disease modeling. In this review, we discuss a progress that has been made in modeling inherited arrhythmias and cardiomyopathies, studying molecular mechanisms of the diseases, and searching for and testing drug compounds using patient-specific induced pluripotent stem cell-derived cardiomyocytes. PMID:27110425

  14. Modeling Cardiovascular Diseases with Patient-Specific Human Pluripotent Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Burridge, Paul W.; Diecke, Sebastian; Matsa, Elena; Sharma, Arun; Wu, Haodi; Wu, Joseph C.

    2016-01-01

    The generation of cardiomyocytes from human induced pluripotent stem cells (hiPSCs) provides a source of cells that accurately recapitulate the human cardiac pathophysiology. The application of these cells allows for modeling of cardiovascular diseases, providing a novel understanding of human disease mechanisms and assessment of therapies. Here, we describe a stepwise protocol developed in our laboratory for the generation of hiPSCs from patients with a specific disease phenotype, long-term hiPSC culture and cryopreservation, differentiation of hiPSCs to cardiomyocytes, and assessment of disease phenotypes. Our protocol combines a number of innovative tools that include a codon-optimized mini intronic plasmid (CoMiP), chemically defined culture conditions to achieve high efficiencies of reprogramming and differentiation, and calcium imaging for assessment of cardiomyocyte phenotypes. Thus, this protocol provides a complete guide to use a patient cohort on a testable cardiomyocyte platform for pharmacological drug assessment. PMID:25690476

  15. Human Umbilical Cord Mesenchymal Stem Cells Therapy in Cyclophosphamide-Induced Premature Ovarian Failure Rat Model

    PubMed Central

    Song, Dan; Zhong, Yun; Qian, Chunfeng; Zou, Qinyan; Ou, Jian; Shi, Yichao; Gao, Liang; Wang, Gaigai; Liu, Zhenxing; Li, Haibo; Ding, Hailei; Wu, Huihua; Wang, Fuxin; Wang, Jing

    2016-01-01

    Premature ovarian failure (POF) is one of the most common causes of infertility in women. In our present study, we established cyclophosphamide- (CTX-) induced POF rat model and elucidated its effect on ovarian function. We detected the serum estrogen, follicle stimulating hormone, and anti-Müllerian hormone of mice models by ELISA and evaluated their folliculogenesis by histopathology examination. Our study revealed that CTX administration could severely disturb hormone secretion and influence folliculogenesis in rat. This study also detected ovarian cells apoptosis by deoxy-UTP-digoxigenin nick end labeling (TUNEL) and demonstrated marked ovarian cells apoptosis in rat models following CTX administration. In order to explore the potential of human umbilical cord mesenchymal stem cells (UCMSCs) in POF treatment, the above indexes were used to evaluate ovarian function. We found that human UCMSCs transplantation recovered disturbed hormone secretion and folliculogenesis in POF rat, in addition to reduced ovarian cell apoptosis. We also tracked transplanted UCMSCs in ovaries by fluorescence in situ hybridization (FISH). The results manifested that the transplanted human UCMSCs could reside in ovarian tissues and could survive for a comparatively long time without obvious proliferation. Our present study provides new insights into the great clinical potential of human UCMSCs in POF treatment. PMID:27047962

  16. Follicular Helper CD4+ T Cells in Human Neuroautoimmune Diseases and Their Animal Models

    PubMed Central

    Fan, Xueli; Lin, Chenhong; Han, Jinming; Jiang, Xinmei; Zhu, Jie; Jin, Tao

    2015-01-01

    Follicular helper CD4+ T (TFH) cells play a fundamental role in humoral immunity deriving from their ability to provide help for germinal center (GC) formation, B cell differentiation into plasma cells and memory cells, and antibody production in secondary lymphoid tissues. TFH cells can be identified by a combination of markers, including the chemokine receptor CXCR5, costimulatory molecules ICOS and PD-1, transcription repressor Bcl-6, and cytokine IL-21. It is difficult and impossible to get access to secondary lymphoid tissues in humans, so studies are usually performed with human peripheral blood samples as circulating counterparts of tissue TFH cells. A balance of TFH cell generation and function is critical for protective antibody response, whereas overactivation of TFH cells or overexpression of TFH-associated molecules may result in autoimmune diseases. Emerging data have shown that TFH cells and TFH-associated molecules may be involved in the pathogenesis of neuroautoimmune diseases including multiple sclerosis (MS), neuromyelitis optica (NMO)/neuromyelitis optica spectrum disorders (NMOSD), and myasthenia gravis (MG). This review summarizes the features of TFH cells, including their development, function, and roles as well as TFH-associated molecules in neuroautoimmune diseases and their animal models. PMID:26300592

  17. Human purified CD8+ T cells: Ex vivo expansion model to generate a maximum yield of functional cytotoxic cells.

    PubMed

    Al-Shanti, Nasser; Aldahoudi, Ziyad

    2007-01-01

    CD8+ T cells are a critical component of the cellular immune response. They play an important role in the control of viral infection and eliminating cells with malignant potential. However, attempts to generate and expand human CD8+ T cells in vitro for an adoptive immunotherapy have been conducted with limitation of the very low frequency of CD8+ T cells in blood. Therefore, several expansion protocols have been developed to obtain large and efficient numbers of human CD8+ T cells for use in adoptive immunotherapies. In this study various common culture conditions using different cytokines IL-2, IL-4, IL-7, IL-10, IL-12 and IL-15 and autologous feeders and sera were investigated to expand human purified CD8+ T cells. The importance and the influence of these factors on the growth and phenotype of CD8+ T cell were assessed by serially sampling cultures using flow cytometry. We demonstrated that combination of IL-2 (50 U/ml) and autologous feeders induced maximal CD8+ T cell proliferation (40-50 folds) compared to other cytokines. Immunophenotypic analysis of cultured cells showed that expanded CD8+ T cells were activated and differentiated. Furthermore our expansion model also demonstrated that expanded CD8+ T cells are functionally cytotoxic active by killing Allogeneic LCLs cells. In conclusion, we have developed a reliable, simple method that uses minimal cell numbers to generate a high yield of functional cytotoxic CD8+ T cells, which can be used for the development of cellular immunotherapies. PMID:17190652

  18. Human pluripotent stem cells as a model of trophoblast differentiation in both normal development and disease.

    PubMed

    Horii, Mariko; Li, Yingchun; Wakeland, Anna K; Pizzo, Donald P; Nelson, Katharine K; Sabatini, Karen; Laurent, Louise Chang; Liu, Ying; Parast, Mana M

    2016-07-01

    Trophoblast is the primary epithelial cell type in the placenta, a transient organ required for proper fetal growth and development. Different trophoblast subtypes are responsible for gas/nutrient exchange (syncytiotrophoblasts, STBs) and invasion and maternal vascular remodeling (extravillous trophoblasts, EVTs). Studies of early human placental development are severely hampered by the lack of a representative trophoblast stem cell (TSC) model with the capacity for self-renewal and the ability to differentiate into both STBs and EVTs. Primary cytotrophoblasts (CTBs) isolated from early-gestation (6-8 wk) human placentas are bipotential, a phenotype that is lost with increasing gestational age. We have identified a CDX2(+)/p63(+) CTB subpopulation in the early postimplantation human placenta that is significantly reduced later in gestation. We describe a reproducible protocol, using defined medium containing bone morphogenetic protein 4 by which human pluripotent stem cells (hPSCs) can be differentiated into CDX2(+)/p63(+) CTB stem-like cells. These cells can be replated and further differentiated into STB- and EVT-like cells, based on marker expression, hormone secretion, and invasive ability. As in primary CTBs, differentiation of hPSC-derived CTBs in low oxygen leads to reduced human chorionic gonadotropin secretion and STB-associated gene expression, instead promoting differentiation into HLA-G(+) EVTs in an hypoxia-inducible, factor-dependent manner. To validate further the utility of hPSC-derived CTBs, we demonstrated that differentiation of trisomy 21 (T21) hPSCs recapitulates the delayed CTB maturation and blunted STB differentiation seen in T21 placentae. Collectively, our data suggest that hPSCs are a valuable model of human placental development, enabling us to recapitulate processes that result in both normal and diseased pregnancies. PMID:27325764

  19. Technical challenges in using human induced pluripotent stem cells to model disease

    PubMed Central

    Saha, Krishanu; Jaenisch, Rudolf

    2009-01-01

    SUMMARY Reprogramming of human somatic cells uses readily accessible tissue, such as skin or blood, to generate embryonic-like induced pluripotent stem cells (iPSCs). This procedure has been applied to somatic cells from patients who are classified into a disease group, thus creating ‘disease-specific’ iPSCs. Here we examine the challenges and assumptions in creating a disease model from a single cell of the patient. Both the kinetics of disease onset and progression as well as the spatial localization of disease in the patient’s body are challenges to model construction. New tools in genetic modification, reprogramming, biomaterials, and animal models can be used to address these challenges. PMID:19951687

  20. A human breast cell model of pre-invasive to invasive transition

    SciTech Connect

    Bissell, Mina J; Rizki, Aylin; Weaver, Valerie M.; Lee, Sun-Young; Rozenberg, Gabriela I.; Chin, Koei; Myers, Connie A.; Bascom, Jamie L.; Mott, Joni D.; Semeiks, Jeremy R.; Grate, Leslie R.; Mian, I. Saira; Borowsky, Alexander D.; Jensen, Roy A.; Idowu, Michael O.; Chen, Fanqing; Chen, David J.; Petersen, Ole W.; Gray, Joe W.; Bissell, Mina J.

    2008-03-10

    A crucial step in human breast cancer progression is the acquisition of invasiveness. There is a distinct lack of human cell culture models to study the transition from pre-invasive to invasive phenotype as it may occur 'spontaneously' in vivo. To delineate molecular alterations important for this transition, we isolated human breast epithelial cell lines that showed partial loss of tissue polarity in three-dimensional reconstituted-basement membrane cultures. These cells remained non-invasive; however, unlike their non-malignant counterparts, they exhibited a high propensity to acquire invasiveness through basement membrane in culture. The genomic aberrations and gene expression profiles of the cells in this model showed a high degree of similarity to primary breast tumor profiles. The xenograft tumors formed by the cell lines in three different microenvironments in nude mice displayed metaplastic phenotypes, including squamous and basal characteristics, with invasive cells exhibiting features of higher grade tumors. To find functionally significant changes in transition from pre-invasive to invasive phenotype, we performed attribute profile clustering analysis on the list of genes differentially expressed between pre-invasive and invasive cells. We found integral membrane proteins, transcription factors, kinases, transport molecules, and chemokines to be highly represented. In addition, expression of matrix metalloproteinases MMP-9,-13,-15,-17 was up regulated in the invasive cells. Using siRNA based approaches, we found these MMPs to be required for the invasive phenotype. This model provides a new tool for dissection of mechanisms by which pre-invasive breast cells could acquire invasiveness in a metaplastic context.

  1. Minireview: Genome Editing of Human Pluripotent Stem Cells for Modeling Metabolic Disease.

    PubMed

    Yu, Haojie; Cowan, Chad A

    2016-06-01

    The pathophysiology of metabolic diseases such as coronary artery disease, diabetes, and obesity is complex and multifactorial. Developing new strategies to prevent or treat these diseases requires in vitro models with which researchers can extensively study the molecular mechanisms that lead to disease. Human pluripotent stem cells and their differentiated derivatives have the potential to provide an unlimited source of disease-relevant cell types and, when combined with recent advances in genome editing, make the goal of generating functional metabolic disease models, for the first time, consistently attainable. However, this approach still has certain limitations including lack of robust differentiation methods and potential off-target effects. This review describes the current progress in human pluripotent stem cell-based metabolic disease research using genome-editing technology. PMID:27075706

  2. Human alveolar epithelial cells expressing tight junctions to model the air-blood barrier.

    PubMed

    Kuehn, Anna; Kletting, Stephanie; de Souza Carvalho-Wodarz, Cristiane; Repnik, Urska; Griffiths, Gareth; Fischer, Ulrike; Meese, Eckart; Huwer, Hanno; Wirth, Dagmar; May, Tobias; Schneider-Daum, Nicole; Lehr, Claus-Michael

    2016-01-01

    This paper describes a new human alveolar epithelial cell line (hAELVi - human Alveolar Epithelial Lentivirus immortalized) with type I-like characteristics and functional tight junctions, suitable to model the air-blood barrier of the peripheral lung. Primary human alveolar epithelial cells were immortalized by a novel regimen, grown as monolayers on permeable filter supports and characterized morphologically, biochemically and biophysically. hAELVi cells maintain the capacity to form tight intercellular junctions, with high trans-epithelial electrical resistance (> 1000 Ω*cm²). The cells could be kept in culture over several days, up to passage 75, under liquid-liquid as well as air-liquid conditions. Ultrastructural analysis and real time PCR revealed type I-like cell properties, such as the presence of caveolae, expression of caveolin-1, and absence of surfactant protein C. Accounting for the barrier properties, inter-digitations sealed with tight junctions and desmosomes were also observed. Low permeability of the hydrophilic marker sodium fluorescein confirmed the suitability of hAELVi cells for in vitro transport studies across the alveolar epithelium. These results suggest that hAELVi cells reflect the essential features of the air-blood barrier, as needed for an alternative to animal testing to study absorption and toxicity of inhaled drugs, chemicals and nanomaterials. PMID:26985677

  3. Comparison of human adipose-derived stem cells and bone marrow-derived stem cells in a myocardial infarction model.

    PubMed

    Rasmussen, Jeppe Grøndahl; Frøbert, Ole; Holst-Hansen, Claus; Kastrup, Jens; Baandrup, Ulrik; Zachar, Vladimir; Fink, Trine; Simonsen, Ulf

    2014-02-01

    Treatment of myocardial infarction (MI) with bone marrow-derived mesenchymal stem cells and recently also adipose-derived stem cells has shown promising results. In contrast to clinical trials and their use of autologous bone marrow-derived cells from the ischemic patient, the animal MI models are often using young donors and young, often immune-compromised, recipient animals. Our objective was to compare bone marrow-derived mesenchymal stem cells with adipose-derived stem cells from an elderly ischemic patient in the treatment of MI using a fully grown non-immune-compromised rat model. Mesenchymal stem cells were isolated from adipose tissue and bone marrow and compared with respect to surface markers and proliferative capability. To compare the regenerative potential of the two stem cell populations, male Sprague-Dawley rats were randomized to receive intramyocardial injections of adipose-derived stem cells, bone marrow-derived mesenchymal stem cells, or phosphate-buffered saline 1 week following induction of MI. After 4 weeks, left ventricular ejection fraction (LVEF) was improved in the adipose-derived stem cell group, and scar wall thickness was greater compared with the saline group. Adipose-derived as well as bone marrow-derived mesenchymal stem cells prevented left ventricular end diastolic dilation. Neither of the cell groups displayed increased angiogenesis in the myocardium compared with the saline group. Adipose-derived stem cells from a human ischemic patient preserved cardiac function following MI, whereas this could not be demonstrated for bone marrow-derived mesenchymal stem cells, with only adipose-derived stem cells leading to an improvement in LVEF. Neither of the stem cell types induced myocardial angiogenesis, raising the question whether donor age and health have an effect on the efficacy of stem cells used in the treatment of MI. PMID:23211469

  4. Visualizing tropoelastin in a long-term human elastic fibre cell culture model

    PubMed Central

    Halm, M.; Schenke-Layland, K.; Jaspers, S.; Wenck, H.; Fischer, F.

    2016-01-01

    Elastin is an essential protein found in a variety of tissues where resilience and flexibility are needed, such as the skin and the heart. When aiming to engineer suitable implants, elastic fibres are needed to allow adequate tissue renewal. However, the visualization of human elastogenesis remains in the dark. To date, the visualization of human tropoelastin (TE) production in a human cell context and its fibre assembly under live cell conditions has not been achieved. Here, we present a long-term cell culture model of human dermal fibroblasts expressing fluorescence-labelled human TE. We employed a lentiviral system to stably overexpress Citrine-labelled TE to build a fluorescent fibre network. Using immunofluorescence, we confirmed the functionality of the Citrine-tagged TE. Furthermore, we visualized the fibre assembly over the course of several days using confocal microscopy. Applying super resolution microscopy, we were able to investigate the inner structure of the elastin–fibrillin-1 fibre network. Future investigations will allow the tracking of TE produced under various conditions. In tissue engineering applications the fluorescent fibre network can be visualized under various conditions or it serves as a tool for investigating fibre degradation processes in disease-in-a-dish-models. PMID:26842906

  5. Insensitivity of Human Prolactin Receptors to Nonhuman Prolactins: Relevance for Experimental Modeling of Prolactin Receptor-Expressing Human Cells

    PubMed Central

    Utama, Fransiscus E.; Tran, Thai H.; Ryder, Amy; LeBaron, Matthew J.; Parlow, Albert F.; Rui, Hallgeir

    2009-01-01

    Prolactin (PRL) receptors are expressed in a broad range of human cell types and in a majority of human breast and prostate cancers. Experimentally, normal and malignant human cells are typically cultured in vitro in media containing bovine PRL (bPRL) from fetal bovine serum or as xenotransplants in vivo in the presence of murine PRL (mPRL). The biological efficacy of bPRL toward hPRL receptors (hPRLR) is controversial, and hPRLR are insensitive to mPRL, but the mechanism is not known. To clarify limitations of current in vitro and in vivo experimental model systems for studies of hPRLR-expressing cells, we tested human and relevant subprimate prolactins in multiple hPRLR bioassays. bPRL and ovine PRL were 10-fold less potent hPRLR agonists than hPRL, although maximal responses at high ligand concentrations (efficacies) equaled that of hPRL. mPRL and rat PRL had greater than 50-fold lower potencies toward hPRLR than hPRL and had 50% reduced efficacies. In fact, mPRL and rat PRL were less effective hPRLR agonists than murine GH. Unexpectedly, mPRL was an effective competitive inhibitor of hPRL binding to hPRLR with an inhibitory constant of 1.3 nm and showed partial antagonist activity, suggesting reduced site-2 binding. Collectively, low bioactivities of bPRL and mPRL toward hPRLR suggest that existing laboratory cancer cell lines grown in 10% bovine serum-supplemented media or in mice are selected for growth under lactogen-depleted conditions. The biology and drug responsiveness of existing human cell lines may therefore not be representative of clinical cancers that are sensitive to circulating PRL. PMID:19022890

  6. Nephron organoids derived from human pluripotent stem cells model kidney development and injury.

    PubMed

    Morizane, Ryuji; Lam, Albert Q; Freedman, Benjamin S; Kishi, Seiji; Valerius, M Todd; Bonventre, Joseph V

    2015-11-01

    Kidney cells and tissues derived from human pluripotent stem cells (hPSCs) may enable organ regeneration, disease modeling and drug screening. We report an efficient, chemically defined protocol for differentiating hPSCs into multipotent nephron progenitor cells (NPCs) that can form nephron-like structures. By recapitulating metanephric kidney development in vitro, we generate SIX2+ SALL1+ WT1+ PAX2+ NPCs with 90% efficiency within 9 days of differentiation. The NPCs possess the developmental potential of their in vivo counterparts and form PAX8+ LHX1+ renal vesicles that self-organize into nephron structures. In both two- and three-dimensional culture, NPCs form kidney organoids containing epithelial nephron-like structures expressing markers of podocytes, proximal tubules, loops of Henle and distal tubules in an organized, continuous arrangement that resembles the nephron in vivo. We also show that this organoid culture system can be used to study mechanisms of human kidney development and toxicity. PMID:26458176

  7. The development of a functionally relevant cell culture model of progressive human breast cancer.

    PubMed

    Weaver, V M; Howlett, A R; Langton-Webster, B; Petersen, O W; Bissell, M J

    1995-06-01

    Normal mammary homeostasis, and by implication tumorigenesis, are dependent upon the dynamic interplay between epithelial cells, stromal components and the extracellular matrix. To study the evolution of human breast cancer, a functionally relevant cell culture model is required which recognizes the complexity of the mammary gland's microenvironment. The development of an appropriate breast epithelial cancer cell model will be dependent on the ability to recreate the 'normal' and 'neoplastic' tissue microenvironment in culture. Towards this goal, a 3-dimensional extracellular matrix (ECM) assay, employing a reconstituted basement membrane, has been developed which allows for the rapid and accurate discrimination of normal and neoplastic cells when cultured. To investigate stromal/epithelial cell interactions, we have developed a tumor environment assay which essentially mirrors the tumor microenvironment histologically. The use of a novel, near diploid, human breast epithelial cell line, HMT-3522, which has transformed spontaneously with passage in culture, together with these 3-dimensional culture assays is expected to provide meaningful markers of initiation and progression. PMID:7495986

  8. Subretinal transplantation of putative retinal pigment epithelial cells derived from human embryonic stem cells in rat retinal degeneration model

    PubMed Central

    Park, Un Chul; Cho, Myung Soo; Park, Jung Hyun; Kim, Sang Jin; Ku, Seung-Yup; Choi, Young Min; Moon, Shin Yong

    2011-01-01

    Objective To differentiate the human embryonic stem cells (hESCs) into the retinal pigment epithelium (RPE) in the defined culture condition and determine its therapeutic potential for the treatment of retinal degenerative diseases. Methods The embryoid bodies were formed from hESCs and attached on the matrigel coated culture dishes. The neural structures consisting neural precursors were selected and expanded to form rosette structures. The mechanically isolated neural rosettes were differentiated into pigmented cells in the media comprised of N2 and B27. Expression profiles of markers related to RPE development were analyzed by reverse transcription-polymerase chain reaction and immunostaining. Dissociated putative RPE cells (105 cells/5 µL) were transplanted into the subretinal space of rat retinal degeneration model induced by intravenous sodium iodate injection. Animals were sacrificed at 1, 2, and 4 weeks after transplantation, and immnohistochemistry study was performed to verify the survival of the transplanted cells. Results The putative RPE cells derived from hESC showed characteristics of the human RPE cells morphologically and expressed molecular markers and associated with RPE fate. Grafted RPE cells were found to survive in the subretinal space up to 4 weeks after transplantation, and the expression of RPE markers was confirmed with immunohistochemistry. Conclusion Transplanted RPE cells derived from hESC in the defined culture condition successfully survived and migrated within subretinal space of rat retinal degeneration model. These results support the feasibility of the hESC derived RPE cells for cell-based therapies for retinal degenerative disease. PMID:22384445

  9. Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation

    PubMed Central

    Sampaziotis, Fotios; Bertero, Alessandro; Saeb-Parsy, Kourosh; Soares, Filipa A. C.; Schrumpf, Elisabeth; Melum, Espen; Karlsen, Tom H.; Bradley, J. Andrew; Gelson, William TH; Davies, Susan; Baker, Alastair; Kaser, Arthur; Alexander, Graeme J.

    2016-01-01

    The study of biliary disease has been constrained by a lack of primary human cholangiocytes. Here we present an efficient, serum-free protocol for directed differentiation of human induced pluripotent stem cells into cholangiocyte-like cells (CLCs). CLCs show functional characteristics of cholangiocytes, including bile acids transfer, alkaline phosphatase activity, gamma-glutamyl-transpeptidase activity and physiological responses to secretin, somatostatin and VEGF. We use CLCs to model in vitro key features of Alagille syndrome, polycystic liver disease and cystic fibrosis (CF)-associated cholangiopathy. Furthermore, we use CLCs generated from healthy individuals and patients with polycystic liver disease to reproduce the effects of the drugs verapamil and octreotide, and we show that the experimental CF drug VX809 rescues the disease phenotype of CF cholangiopathy in vitro. Our differentiation protocol will facilitate the study of biological mechanisms controlling biliary development as well as disease modeling and drug screening. PMID:26167629

  10. Cholangiocytes derived from human induced pluripotent stem cells for disease modeling and drug validation.

    PubMed

    Sampaziotis, Fotios; Cardoso de Brito, Miguel; Madrigal, Pedro; Bertero, Alessandro; Saeb-Parsy, Kourosh; Soares, Filipa A C; Schrumpf, Elisabeth; Melum, Espen; Karlsen, Tom H; Bradley, J Andrew; Gelson, William T H; Davies, Susan; Baker, Alastair; Kaser, Arthur; Alexander, Graeme J; Hannan, Nicholas R F; Vallier, Ludovic

    2015-08-01

    The study of biliary disease has been constrained by a lack of primary human cholangiocytes. Here we present an efficient, serum-free protocol for directed differentiation of human induced pluripotent stem cells into cholangiocyte-like cells (CLCs). CLCs show functional characteristics of cholangiocytes, including bile acids transfer, alkaline phosphatase activity, γ-glutamyl-transpeptidase activity and physiological responses to secretin, somatostatin and vascular endothelial growth factor. We use CLCs to model in vitro key features of Alagille syndrome, polycystic liver disease and cystic fibrosis (CF)-associated cholangiopathy. Furthermore, we use CLCs generated from healthy individuals and patients with polycystic liver disease to reproduce the effects of the drugs verapamil and octreotide, and we show that the experimental CF drug VX809 rescues the disease phenotype of CF cholangiopathy in vitro. Our differentiation protocol will facilitate the study of biological mechanisms controlling biliary development, as well as disease modeling and drug screening. PMID:26167629

  11. Standardized 3D Bioprinting of Soft Tissue Models with Human Primary Cells.

    PubMed

    Rimann, Markus; Bono, Epifania; Annaheim, Helene; Bleisch, Matthias; Graf-Hausner, Ursula

    2016-08-01

    Cells grown in 3D are more physiologically relevant than cells cultured in 2D. To use 3D models in substance testing and regenerative medicine, reproducibility and standardization are important. Bioprinting offers not only automated standardizable processes but also the production of complex tissue-like structures in an additive manner. We developed an all-in-one bioprinting solution to produce soft tissue models. The holistic approach included (1) a bioprinter in a sterile environment, (2) a light-induced bioink polymerization unit, (3) a user-friendly software, (4) the capability to print in standard labware for high-throughput screening, (5) cell-compatible inkjet-based printheads, (6) a cell-compatible ready-to-use BioInk, and (7) standard operating procedures. In a proof-of-concept study, skin as a reference soft tissue model was printed. To produce dermal equivalents, primary human dermal fibroblasts were printed in alternating layers with BioInk and cultured for up to 7 weeks. During long-term cultures, the models were remodeled and fully populated with viable and spreaded fibroblasts. Primary human dermal keratinocytes were seeded on top of dermal equivalents, and epidermis-like structures were formed as verified with hematoxylin and eosin staining and immunostaining. However, a fully stratified epidermis was not achieved. Nevertheless, this is one of the first reports of an integrative bioprinting strategy for industrial routine application. PMID:25609254

  12. Behavioral outcome measures used for human neural stem cell transplantation in rat stroke models

    PubMed Central

    Jensen, Matthew B.; Han, Dong Y.; Sawaf, Abdullah Al; Krishnaney-Davison, Rajeev

    2011-01-01

    Stroke is a leading cause of death and disability, leading to the development of various stroke models to test new treatments, most commonly in the rat. Human stroke trials focus on disability, related primarily to neurological deficits. To better model the clinical application of these treatments, many behavioral tests have been developed using the rat stroke model. We performed a systematic review of all the behavioral outcome measures used in published studies of human neural stem cell transplantation in rat stroke models. The reviewed tests include motor, sensory, cognitive, activity, and combination tests. For each test, we give a brief description, trace the origin of the test, and discuss test performance in the reviewed studies. We conclude that while many behavioral tests are available for this purpose, there does not appear to be consensus on an optimal testing strategy. PMID:22053257

  13. Hep G2 cell line as a human model for sulphate conjugation of drugs.

    PubMed

    Shwed, J A; Walle, U K; Walle, T

    1992-08-01

    1. The objective of this study was to examine the usefulness of the hepatoma cell line Hep G2 as a model for human sulphoconjugation of drugs, in particular stereoselective conjugation. 2. Using the substrates p-nitrophenol and dopamine, we found sulphation activities consistent with the presence of both the phenol (P) and the monoamine (M) form of the human phenolsulphotransferases in these cells. 3. The Kmapp was 3.0 microM for the sulphation of p-nitrophenol. This activity was inhibited selectively by 2,6-dichloro-4-nitrophenol, IC50 6 microM. The Kmapp was 39 microM for the sulphation of dopamine. This activity was selectively inhibited by elevated temperature. 4. The chiral adrenergic drugs (+/-)-terbutaline and (+/-)-4-hydroxypropranolol were both sulphated stereoselectively with Kmapp and Vmaxapp values for each enantiomer virtually identical to previous observations with human liver cytosol. 5. In a direct comparison, the estimated activity of the P form of phenolsulphotransferase in the Hep G2 cell line was 30% of that in human liver, whereas, surprisingly, the activity of the M form of phenolsulphotransferase was 4.5 times higher in the Hep G2 cells than in the liver. PMID:1329363

  14. Examining and elucidation of human weight cycle model adopting e-cell simulation system

    PubMed Central

    Rajesh, Durairaj; Muthukumar, Subramanian; Siva, Durairaj; Saibaba, Ganesan; Dhanasekaran, Dharumadhurai; Archunan, Govindaraju

    2015-01-01

    Cellular rhythms regulate various physiological functions in circadian oscillatory mechanisms. Weight cycling or ‘yo-yo’ dieting is an evitable process in human, because of subsequent loss and regain of body weight due to irregular diet. Human weight cycle (HWC) is the major factor for causing global epidemic diseases in human beings. Understanding the HWC process would provide potent additional knowledge to prevent obesity. However till date, there is no study dealing with examine the HWC model using virtual cell simulation based on system biological approach. Therefore, the present study was designed to develop a computational HWC model, which was simulated using E-cell system v3.0. The developed model has the cyclic feedback reactions of three significant variables (the consecutive cycles of weight loss in continuous food intake (Q) and regain of body weight (P) at highest threshold point of cognitive restraint (R)) which are obtained by mathematical modelling. The dynamic plot results supported that the PQR variables depicted sustained oscillation with reversible modification due to protein diet. By contrast, the virtual model simulation would provide extensive information on HWC, which might provide knowledge to develop HWC linked with obesity pathway. The presents study concludes that optimization of body weight is essential to prevent the obesity based diseases. PMID:26339149

  15. Examining and elucidation of human weight cycle model adopting e-cell simulation system.

    PubMed

    Rajesh, Durairaj; Muthukumar, Subramanian; Siva, Durairaj; Saibaba, Ganesan; Dhanasekaran, Dharumadhurai; Archunan, Govindaraju

    2015-01-01

    Cellular rhythms regulate various physiological functions in circadian oscillatory mechanisms. Weight cycling or 'yo-yo' dieting is an evitable process in human, because of subsequent loss and regain of body weight due to irregular diet. Human weight cycle (HWC) is the major factor for causing global epidemic diseases in human beings. Understanding the HWC process would provide potent additional knowledge to prevent obesity. However till date, there is no study dealing with examine the HWC model using virtual cell simulation based on system biological approach. Therefore, the present study was designed to develop a computational HWC model, which was simulated using E-cell system v3.0. The developed model has the cyclic feedback reactions of three significant variables (the consecutive cycles of weight loss in continuous food intake (Q) and regain of body weight (P) at highest threshold point of cognitive restraint (R)) which are obtained by mathematical modelling. The dynamic plot results supported that the PQR variables depicted sustained oscillation with reversible modification due to protein diet. By contrast, the virtual model simulation would provide extensive information on HWC, which might provide knowledge to develop HWC linked with obesity pathway. The presents study concludes that optimization of body weight is essential to prevent the obesity based diseases. PMID:26339149

  16. A 3D human neural cell culture system for modeling Alzheimer's disease.

    PubMed

    Kim, Young Hye; Choi, Se Hoon; D'Avanzo, Carla; Hebisch, Matthias; Sliwinski, Christopher; Bylykbashi, Enjana; Washicosky, Kevin J; Klee, Justin B; Brüstle, Oliver; Tanzi, Rudolph E; Kim, Doo Yeon

    2015-07-01

    Stem cell technologies have facilitated the development of human cellular disease models that can be used to study pathogenesis and test therapeutic candidates. These models hold promise for complex neurological diseases such as Alzheimer's disease (AD), because existing animal models have been unable to fully recapitulate all aspects of pathology. We recently reported the characterization of a novel 3D culture system that exhibits key events in AD pathogenesis, including extracellular aggregation of amyloid-β (Aβ) and accumulation of hyperphosphorylated tau. Here we provide instructions for the generation and analysis of 3D human neural cell cultures, including the production of genetically modified human neural progenitor cells (hNPCs) with familial AD mutations, the differentiation of the hNPCs in a 3D matrix and the analysis of AD pathogenesis. The 3D culture generation takes 1-2 d. The aggregation of Aβ is observed after 6 weeks of differentiation, followed by robust tau pathology after 10-14 weeks. PMID:26068894

  17. A 3D human neural cell culture system for modeling Alzheimer’s disease

    PubMed Central

    Kim, Young Hye; Choi, Se Hoon; D’Avanzo, Carla; Hebisch, Matthias; Sliwinski, Christopher; Bylykbashi, Enjana; Washicosky, Kevin J.; Klee, Justin B.; Brüstle, Oliver; Tanzi, Rudolph E.; Kim, Doo Yeon

    2015-01-01

    Stem cell technologies have facilitated the development of human cellular disease models that can be used to study pathogenesis and test therapeutic candidates. These models hold promise for complex neurological diseases such as Alzheimer’s disease (AD) because existing animal models have been unable to fully recapitulate all aspects of pathology. We recently reported the characterization of a novel three-dimensional (3D) culture system that exhibits key events in AD pathogenesis, including extracellular aggregation of β-amyloid and accumulation of hyperphosphorylated tau. Here we provide instructions for the generation and analysis of 3D human neural cell cultures, including the production of genetically modified human neural progenitor cells (hNPCs) with familial AD mutations, the differentiation of the hNPCs in a 3D matrix, and the analysis of AD pathogenesis. The 3D culture generation takes 1–2 days. The aggregation of β-amyloid is observed after 6-weeks of differentiation followed by robust tau pathology after 10–14 weeks. PMID:26068894

  18. Human periodontal ligament cell sheets can regenerate periodontal ligament tissue in an athymic rat model.

    PubMed

    Hasegawa, Masateru; Yamato, Masayuki; Kikuchi, Akihiko; Okano, Teruo; Ishikawa, Isao

    2005-01-01

    Conventional periodontal regeneration methods remain insufficient to attain complete and reliable clinical regeneration of periodontal tissues. We have developed a new method of cell transplantation using cell sheet engineering and have applied it to this problem. The purpose of this study was to investigate the characteristics of human periodontal ligament (HPDL) cell sheets retrieved from culture on unique temperature-responsive culture dishes, and to examine whether these cell sheets can regenerate periodontal tissues. The HPDL cell sheets were examined histologically and biochemically, and also were transplanted into a mesial dehiscence model in athymic rats. HPDL cells were harvested from culture dishes as a contiguous cell sheet with abundant extracellular matrix and retained intact integrins that are susceptible to trypsin-EDTA treatment. In the animal study, periodontal ligament-like tissues that include an acellular cementum-like layer and fibrils anchoring into this layer were identified in all the athymic rats transplanted with HPDL cell sheets. This fibril anchoring highly resembles native periodontal ligament fibers; such regeneration was not observed in nontransplanted controls. These results suggest that this technique, based on the concept of cell sheet engineering, can be useful for periodontal tissue regeneration. PMID:15869425

  19. Paracellular and passive transcellular permeability in immortalized human corneal epithelial cell culture model.

    PubMed

    Toropainen, Elisa; Ranta, Veli-Pekka; Vellonen, Kati-Sisko; Palmgrén, Joni; Talvitie, Anu; Laavola, Mirka; Suhonen, Pekka; Hämäläinen, Kaisa Mari; Auriola, Seppo; Urtti, Arto

    2003-09-01

    A cell culture model of human corneal epithelium (HCE-model) was recently introduced [Invest. Ophthalmol. Vis. Sci. 42 (2001) 2942] as a tool for ocular drug permeation studies. In this study, passive permeability and esterase activity of the HCE-model were characterised. Immortalised human corneal epithelial cells were grown on collagen coated filters under air-lift. The sensitivity of transcellular permeability to lipophilicity was tested in studies using nine beta-blockers. The size selectivity of the paracellular route was investigated using 16 polyethylene glycol oligomers (PEG). An effusion-like approach was used to estimate porosity and pore sizes of the paracellular space in HCE membrane. Permeability and degradation of fluorescein diacetate to fluorescein in HCE-cells was used to probe the esterase activity of the HCE-model. Drug concentrations were analyzed using HPLC (beta-blockers), LC-MS (PEGs), and fluorometry (fluorescein). Permeabilities were compared to those in the excised rabbit cornea. Penetration of beta-blockers increased with lipophilicity according to a sigmoidal relationship. This was almost similar to the profile in excised cornea. No apical to basolateral directionality was seen in the permeation of beta-blockers. Paracellular permeability of the HCE-model was generally slightly higher than that of the excised rabbit cornea. The HCE-model has larger paracellular pores, but lower pore density than the excised cornea, but the overall paracellular space was fairly similar in both models. The HCE-model shows significant esterase activity (i.e. fluorescein diacetate was converted to free fluorescein). These data on permeability of 27 compounds demonstrate that the barrier of the HCE-model closely resembles that of the excised rabbit cornea. Therefore, the HCE-model is a promising alternative corneal substitute for ocular drug delivery studies. PMID:13678798

  20. Current status of drug screening and disease modelling in human pluripotent stem cells

    PubMed Central

    Rajamohan, Divya; Matsa, Elena; Kalra, Spandan; Crutchley, James; Patel, Asha; George, Vinoj; Denning, Chris

    2013-01-01

    The emphasis in human pluripotent stem cell (hPSC) technologies has shifted from cell therapy to in vitro disease modelling and drug screening. This review examines why this shift has occurred, and how current technological limitations might be overcome to fully realise the potential of hPSCs. Details are provided for all disease-specific human induced pluripotent stem cell lines spanning a dozen dysfunctional organ systems. Phenotype and pharmacology have been examined in only 17 of 63 lines, primarily those that model neurological and cardiac conditions. Drug screening is most advanced in hPSC-cardiomyocytes. Responses for almost 60 agents include examples of how careful tests in hPSC-cardiomyocytes have improved on existing in vitro assays, and how these cells have been integrated into high throughput imaging and electrophysiology industrial platforms. Such successes will provide an incentive to overcome bottlenecks in hPSC technology such as improving cell maturity and industrial scalability whilst reducing cost. PMID:22886688

  1. The BIANCA model/code of radiation-induced cell death: application to human cells exposed to different radiation types.

    PubMed

    Ballarini, Francesca; Altieri, Saverio; Bortolussi, Silva; Carante, Mario; Giroletti, Elio; Protti, Nicoletta

    2014-08-01

    This paper presents a biophysical model of radiation-induced cell death, implemented as a Monte Carlo code called BIophysical ANalysis of Cell death and chromosome Aberrations (BIANCA), based on the assumption that some chromosome aberrations (dicentrics, rings, and large deletions, called ‘‘lethal aberrations’’) lead to clonogenic inactivation. In turn, chromosome aberrations are assumed to derive from clustered, and thus severe, DNA lesions (called ‘‘cluster lesions,’’ or CL) interacting at the micrometer scale; the CL yield and the threshold distance governing CL interaction are the only model parameters. After a pilot study on V79 hamster cells exposed to protons and carbon ions, in the present work the model was extended and applied to AG1522 human cells exposed to photons, He ions, and heavier ions including carbon and neon. The agreement with experimental survival data taken from the literature supported the assumptions. In particular, the inactivation of AG1522 cells was explained by lethal aberrations not only for X-rays, as already reported by others, but also for the aforementioned radiation types. Furthermore, the results are consistent with the hypothesis that the critical initial lesions leading to cell death are DNA cluster lesions having yields in the order of *2 CL Gy-1 cell-1 at low LET and*20 CL Gy-1 cell-1 at high LET, and that the processing of these lesions is modulated by proximity effects at the micrometer scale related to interphase chromatin organization. The model was then applied to calculate the fraction of inactivated cells, as well as the yields of lethal aberrations and cluster lesions, as a function of LET; the results showed a maximum around 130 keV/lm, and such maximum was much higher for cluster lesions and lethal aberrations than for cell inactivation. PMID:24659413

  2. Use of human stem cells in Huntington disease modeling and translational research.

    PubMed

    Golas, Monika M; Sander, Bjoern

    2016-04-01

    Huntington disease (HD) is a devastating neurological disorder caused by an extended CAG repeat in exon 1 of the gene that encodes the huntingtin (HTT) protein. HD pathology involves a loss of striatal medium spiny neurons (MSNs) and progressive neurodegeneration affects the striatum and other brain regions. Because HTT is involved in multiple cellular processes, the molecular mechanisms of HD pathogenesis should be investigated on multiple levels. On the cellular level, in vitro stem cell models, such as induced pluripotent stem cells (iPSCs) derived from HD patients and HD embryonic stem cells (ESCs), have yielded progress. Approaches to differentiate functional MSNs from ESCs, iPSCs, and neural stem/progenitor cells (NSCs/NPCs) have been established, enabling MSN differentiation to be studied and disease phenotypes to be recapitulated. Isolation of target stem cells and precursor cells may also provide a resource for grafting. In animal models, transplantation of striatal precursors differentiated in vitro to the striatum has been reported to improve disease phenotype. Initial clinical trials examining intrastriatal transplantation of fetal neural tissue suggest a more favorable clinical course in a subset of HD patients, though shortcomings persist. Here, we review recent advances in the development of cellular HD models and approaches aimed at cell regeneration with human stem cells. We also describe how genome editing tools could be used to correct the HTT mutation in patient-specific stem cells. Finally, we discuss the potential and the remaining challenges of stem cell-based approaches in HD research and therapy development. PMID:26826449

  3. Chemical form of selenium affects its uptake, transport and glutathione peroxidase activity in the human intestinal Caco-2 cell model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Determining the effect of selenium (Se) chemical form on uptake and transport in human intestinal cells is critical to assess Se bioavailability. In the present study, we measured the uptake and transport of various Se compounds in the human intestinal Caco-2 cell model. We found that two sources...

  4. In Vitro Modeling of Alcohol-Induced Liver Injury Using Human-Induced Pluripotent Stem Cells.

    PubMed

    Tian, Lipeng; Prasad, Neha; Jang, Yoon-Young

    2016-01-01

    Alcohol consumption has long been associated with a majority of liver diseases and has been found to influence both fetal and adult liver functions. In spite of being one of the major causes of morbidity and mortality in the world, currently, there are no effective strategies that can prevent or treat alcoholic liver disease (ALD), due to a lack of human-relevant research models. Recent success in generation of functionally active mature hepatocyte-like cells from human-induced pluripotent cells (iPSCs) enables us to better understand the effects of alcohol on liver functions. Here, we describe the method and effect of alcohol exposure on multistage hepatic cell types derived from human iPSCs, in an attempt to recapitulate the early stages of liver tissue injury associated with ALD. We exposed different stages of iPSC-induced hepatic cells to ethanol at a pathophysiological concentration. In addition to stage-specific molecular markers, we measured several key cellular parameters of hepatocyte injury, including apoptosis, proliferation, and lipid accumulation. PMID:25520290

  5. Increased complexity in carcinomas: Analyzing and modeling the interaction of human cancer cells with their microenvironment.

    PubMed

    Stadler, Mira; Walter, Stefanie; Walzl, Angelika; Kramer, Nina; Unger, Christine; Scherzer, Martin; Unterleuthner, Daniela; Hengstschläger, Markus; Krupitza, Georg; Dolznig, Helmut

    2015-12-01

    Solid cancers are not simple accumulations of malignant tumor cells but rather represent complex organ-like structures. Despite a more chaotic general appearance as compared to the highly organized setup of healthy tissues, cancers still show highly differentiated structures and a close interaction with and dependency on the interwoven connective tissue. This complexity within cancers is not known in detail at the molecular level so far. The first part of this article will shortly describe the technology and strategies to quantify and dissect the heterogeneity in human solid cancers. Moreover, there is urgent need to better understand human cancer biology since the development of novel anti-cancer drugs is far from being efficient, predominantly due to the scarcity of predictive preclinical models. Hence, in vivo and in vitro models were developed, which better recapitulate the complexity of human cancers, by their intrinsic three-dimensional nature and the cellular heterogeneity and allow functional intervention for hypothesis testing. Therefore, in the second part 3D in vitro cancer models are presented that analyze and depict the heterogeneity in human cancers. Advantages and drawbacks of each model are highlighted and their suitability to preclinical drug testing is discussed. PMID:26320002

  6. Cultured human chromaffin cells grafted in spinal subarachnoid space relieves allodynia in a pain rat model

    PubMed Central

    Jeon, Younghoon; Baek, Woon Yi; Chung, Seung Hyun; Shin, Nari; Kim, Hye Rim

    2011-01-01

    Background Implantation of xenogenic chromaffin cells into the spinal subarachnoid space can produce analgesia in neuropathic pain models. However, transplantation of xenogeneic chromaffin cell has a potential risk of viral or bacterial infections from animals to humans including encephalopathy due to prion transmission. The aim of this study was to investigate the possibility of developing a homogeneic source of therapeutic chromaffin cells. Methods Anti-allodynic effects of human chromaffin cells (HCCs) were evaluated in a neuropathic pain model in rats induced by chronic constriction injury of the sciatic nerve. HCCs encapsulated with alginate-poly-L-lysine-alginate were intrathecally implanted into rats (n = 10), while empty capsules were intrathecally implanted as a control (n = 8). Levels of norepinephrine from encapsulated HCCs before and after nicotinic stimulation were measured. We then perfomed a behavior test (cold allodynia) with acetone. In addition, to assess the potential contribution to pain reduction of opioid peptides released from the HCCs, all animals were injected with naloxone. Results The concentration of norepinephrine after nicotine stimulation was significantly increased compared to basal levels. Intrathecal implantation of encapsulated HCCs, significantly reduced cold allodynia as compared to rats receiving empty capsules (P < 0.05). Fifteen minutes after the injection of naloxone, cold allodynia significantly decreased in rats with HCCs (P < 0.05), while the degree of cold allodynia in control animals was unaltered. Conclusions From these results, it appears that HCCs have a possibility as an analgesic source for transplants delivering pain-reducing neuroactive substances. PMID:21716909

  7. Analysis of enzymopathies in the human red blood cells by constraint-based stoichiometric modeling approaches.

    PubMed

    Durmuş Tekir, Saliha; Cakir, Tunahan; Ulgen, Kutlu O

    2006-10-01

    The human red blood cell (RBC) metabolism is investigated by calculating steady state fluxes using constraint-based stoichiometric modeling approaches. For the normal RBC metabolism, flux balance analysis (FBA) is performed via optimization of various alternative objective functions, and the maximization of production of ATP and NADPH is found to be the primary objective of the RBC metabolism. FBA and two novel approaches, minimization of metabolic adjustment (MOMA) and regulatory on-off minimization (ROOM), which can describe the behavior of the metabolic networks in case of enzymopathies, are applied to observe the relative changes in the flux distribution of the deficient network. The deficiencies in several enzymes in RBC metabolism are investigated and the flux distributions are compared with the non-deficient FBA distribution to elucidate the metabolic changes in response to enzymopathies. It is found that the metabolism is mostly affected by the glucose-6-phosphate dehydrogenase (G6PDH) and phosphoglycerate kinase (PGK) enzymopathies, whereas the effects of the deficiency in DPGM on the metabolism are negligible. These stoichiometric modeling results are found to be in accordance with the experimental findings in the literature related to metabolic behavior of the human red blood cells, showing that human RBC metabolism can be modeled stoichiometrically. PMID:16987707

  8. Genomic Editing Tools to Model Human Diseases with Isogenic Pluripotent Stem Cells

    PubMed Central

    Kim, Huen Suk; Bernitz, Jeffrey M.; Lee, Dung-Fang

    2014-01-01

    Patient-specific induced pluripotent stem cells (iPSCs) are considered a versatile resource in the field of biomedicine. As iPSCs are generated on an individual basis, iPSCs may be the optimal cellular material to use for disease modeling, drug discovery, and the development of patient-specific cellular therapies. Recently, to gain an in-depth understanding of human pathologies, patient-specific iPSCs have been used to model human diseases with some iPSC-derived cells recapitulating pathological phenotypes in vitro. However, complex multigenic diseases generally have not resulted in concise conclusions regarding the underlying mechanisms of disease, in large part due to genetic variations between disease-state and control iPSCs. To circumvent this, the use of genomic editing tools to generate perfect isogenic controls is gaining momentum. To date, DNA binding domain-based zinc finger nucleases and transcription activator-like effector nucleases have been utilized to create genetically defined conditions in patient-specific iPSCs, with some examples leading to the successful identification of novel mechanisms of disease. As the feasibility and utility of genomic editing tools in iPSCs improve, along with the introduction of the clustered regularly interspaced short palindromic repeat system, understanding the features and limitations of genomic editing tools and their applications to iPSC technology is critical to expending the field of human disease modeling. PMID:25075441

  9. Vectorial bicarbonate transport by Capan-1 cells: a model for human pancreatic ductal secretion.

    PubMed

    Szucs, Akos; Demeter, Irma; Burghardt, Beáta; Ovári, Gabriella; Case, R Maynard; Steward, Martin C; Varga, Gábor

    2006-01-01

    Human pancreatic ducts secrete a bicarbonate-rich fluid but our knowledge of the secretory process is based mainly on studies of animal models. Our aim was to determine whether the HCO(3)(-) transport mechanisms in a human ductal cell line are similar to those previously identified in guinea-pig pancreatic ducts. Intracellular pH was measured by microfluorometry in Capan-1 cell monolayers grown on permeable filters and loaded with BCECF. Epithelial polarization was assessed by immunolocalization of occludin. Expression of mRNA for key electrolyte transporters and receptors was evaluated by RT-PCR. Capan-1 cells grown on permeable supports formed confluent, polarized monolayers with well developed tight junctions. The recovery of pH(i) from an acid load, induced by a short NH(4)(+) pulse, was mediated by Na(+)-dependent transporters located exclusively at the basolateral membrane. One was independent of HCO(3)(-) and blocked by EIPA (probably NHE1) while the other was HCO(3)(-)-dependent and blocked by H(2)DIDS (probably pNBC1). Changes in pH(i) following blockade of basolateral HCO(3)(-) accumulation confirmed that the cells achieve vectorial HCO(3)(-) secretion. Dose-dependent increases in HCO(3)(-) secretion were observed in response to stimulation of both secretin and VPAC receptors. ATP and UTP applied to the apical membrane stimulated HCO(3)(-) secretion but were inhibitory when applied to the basolateral membrane. HCO(3)(-) secretion in guinea-pig ducts and Capan-1 cell monolayers share many common features, suggesting that the latter is an excellent model for studies of human pancreatic HCO(3)(-) secretion. PMID:17167230

  10. Enzyme replacement therapy of a novel humanized mouse model of globoid cell leukodystrophy.

    PubMed

    Matthes, Frank; Andersson, Claes; Stein, Axel; Eistrup, Carl; Fogh, Jens; Gieselmann, Volkmar; Wenger, David A; Matzner, Ulrich

    2015-09-01

    An inherited deficiency of β-galactosylceramidase (GALC) causes the lysosomal storage disease globoid cell leukodystrophy (GLD). The disease is characterized by the accumulation of the cytotoxic metabolite psychosine (galactosylsphingosine), causing rapid degeneration of myelinating cells. Most patients suffer from the infantile form of GLD with onset of disease between 3 and 6 months after birth and death by 2 years of age. The most widely used animal model of GLD, the twitcher mouse, presents with an even more rapid course of disease and death around 40 days of age. We have generated a novel "humanized" mouse model of GLD by inserting a human GALC cDNA containing an adult-onset patient mutation into the murine GALC gene. Humanized GALC mice exhibit pathological hallmarks of GLD including psychosine accumulation, neuroinflammation, CNS infiltration of macrophages, astrogliosis and demyelination. Residual GALC activities in mouse tissues are low and the mice display a median lifespan of 46 days. Due to the expression of the human transgene, the mice do not develop an immune response against rhGALC, rendering the animal model suitable for therapies based on human enzyme. Intravenously injected rhGALC was able to surmount the blood-brain barrier and was targeted to lysosomes of brain macrophages, astrocytes and neurons. High-dose enzyme replacement therapy started at postnatal day 21 reduced the elevated psychosine levels in the peripheral and central nervous system by 14-16%, but did not ameliorate neuroinflammation, demyelination and lifespan. These results may indicate that treatment must be started earlier before pathology occurs. PMID:25956830

  11. Interaction between human placental microvascular endothelial cells and a model of human trophoblasts: effects on growth cycle and angiogenic profile.

    PubMed

    Troja, Weston; Kil, Kicheol; Klanke, Charles; Jones, Helen N

    2014-01-01

    Abstract Intrauterine growth restriction (IUGR) is a leading cause of perinatal complications, and is commonly associated with reduced placental vasculature. Recent studies demonstrated over-expression of IGF-1 in IUGR animal models maintains placental vasculature. However, the cellular environment of the placental chorionic villous is unknown. The close proximity of trophoblasts and microvascular endothelial cells in vivo alludes to autocrine/paracrine regulation following Ad-HuIGF-1 treatment. We investigated the co-culturing of BeWo Choriocarcinoma and Human Placental Microvascular Endothelial Cells (HPMVECs) on the endothelial angiogenic profile and the effect Ad-HuIGF-1 treatment of one cell has on the other. HPMVECs were isolated from human term placentas and cultured in EGM-2 at 37°C with 5% CO2. BeWo cells were maintained in Ham's F12 nutrient mix with 10% FBS and 1% pen/strep. Co-cultured HPMVECS+BeWo cells were incubated in serum-free control media, Ad-HuIGF-1, or Ad-LacZ at MOI 0 and MOI 100:1 for 48 h. Non-treated cells and mono-cultured cells were compared to co-cultured cells. Angiogenic gene expression and proliferative and apoptotic protein expression were analysed by RT-qPCR and immunocytochemistry, respectively. Statistical analyses was performed using student's t-test with P < 0.05 considered significant. Direct Ad-HuIGF-1 treatment increased HPMVEC proliferation (n = 4) and reduced apoptosis (n = 3). Co-culturing HPMVECs+BeWo cells significantly altered RNA expression of the angiogenic profile compared to mono-cultured HPMVECs (n = 8). Direct Ad-HuIGF-1 treatment significantly increased Ang-1 (n = 4) in BeWo cells. Ad-HuIGF-1 treatment of HPMVECs did not alter the RNA expression of angiogenic factors. Trophoblastic factors may play a key role in placental vascular development and IGF-1 may have an important role in HPMVEC growth. PMID:24760505

  12. Complete suppression of in vivo growth of human leukemia cells by specific immunotoxins: nude mouse models

    SciTech Connect

    Hara, H.; Seon, B.K.

    1987-05-01

    In this study, immunotoxins containing monoclonal anti-human T-cell leukemia antibodies are shown to be capable of completely suppressing the tumor growth of human T-cell leukemia cells in vivo without any overt undersirable toxicity. These immunotoxins were prepared by conjugating ricin A chain (RA) with our monoclonal antibodies, SN1 and SN2, directed specifically to the human T-cell leukemia cell surface antigens TALLA and GP37, respectively. The authors have shown that these monoclonal antibodies are highly specific for human T-cell leukemia cells and do not react with various normal cells including normal T and B cells, thymocytes, and bone marrow cells. Ascitic and solid human T-cell leukemia cell tumors were generated in nude mice. The ascitic tumor was generated by transplanting Ichikawa cells (a human T-cell leukemia cell) i.p. into nude mice, whereas the solid tumor was generated by transplanting s.c. MOLT-4 cells (a human T-cell leukemia cell line) and x-irradiated human fibrosarcoma cells into x-irradiated nude mice. To investigate the efficacy of specific immunotoxins in suppression the in vivo growth of the ascitic tumor, they divided 40 nude mice that were injected with Ichikawa cells into four groups. None of the mice in group 4 that were treated with SN1-RA and SN2-RA showed any signs of a tumor or undesirable toxic effects for the 20 weeks that they were followed after the transplantation. Treatment with SN1-RA plus SN2-RA completely suppressed solid tumor growth in 4 of 10 nude mice carrying solid tumors and partially suppressed the tumor growth in the remaining 6 nude mice. These results strongly suggest that SN1-RA and SN2-RA may be useful for clinical treatment.

  13. Immunotoxicity and genotoxicity testing of PLGA-PEO nanoparticles in human blood cell model.

    PubMed

    Tulinska, Jana; Kazimirova, Alena; Kuricova, Miroslava; Barancokova, Magdalena; Liskova, Aurelia; Neubauerova, Eva; Drlickova, Martina; Ciampor, Fedor; Vavra, Ivo; Bilanicova, Dagmar; Pojana, Giulio; Staruchova, Marta; Horvathova, Mira; Jahnova, Eva; Volkovova, Katarina; Bartusova, Maria; Cagalinec, Michal; Dusinska, Maria

    2015-05-01

    A human blood cell model for immunotoxicity and genotoxicity testing was used to measure the response to polylactic-co-glycolic acid (PLGA-PEO) nanoparticle (NP) (0.12, 3, 15 and 75 μg/cm(2) exposure in fresh peripheral whole blood cultures/isolated peripheral blood mononuclear cell cultures from human volunteers (n = 9-13). PLGA-PEO NPs were not toxic up to dose 3 μg/cm(2); dose of 75 μg/cm(2) displays significant decrease in [(3)H]-thymidine incorporation into DNA of proliferating cells after 4 h (70% of control) and 48 h (84%) exposure to NPs. In non-cytotoxic concentrations, in vitro assessment of the immunotoxic effects displayed moderate but significant suppression of proliferative activity of T-lymphocytes and T-dependent B-cell response in cultures stimulated with PWM > CON A, and no changes in PHA cultures. Decrease in proliferative function was the most significant in T-cells stimulated with CD3 antigen (up to 84%). Cytotoxicity of natural killer cells was suppressed moderately (92%) but significantly in middle-dosed cultures (4 h exposure). On the other hand, in low PLGA-PEO NPs dosed cultures, significant stimulation of phagocytic activity of granulocytes (119%) > monocytes (117%) and respiratory burst of phagocytes (122%) was recorded. Genotoxicity assessment revealed no increase in the number of micronucleated binucleated cells and no induction of SBs or oxidised DNA bases in PLGA-PEO-treated cells. To conclude on immuno- and genotoxicity of PLGA-PEO NPs, more experiments with various particle size, charge and composition need to be done. PMID:23859252

  14. Differential susceptibility of primary cultured human skin cells to hypericin PDT in an in vitro model.

    PubMed

    Popovic, A; Wiggins, T; Davids, L M

    2015-08-01

    Skin cancer is the most common cancer worldwide, and its incidence rate in South Africa is increasing. Photodynamic therapy (PDT) has been shown to be an effective treatment modality, through topical administration, for treatment of non-melanoma skin cancers. Our group investigates hypericin-induced PDT (HYP-PDT) for the treatment of both non-melanoma and melanoma skin cancers. However, a prerequisite for effective cancer treatments is efficient and selective targeting of the tumoral cells with minimal collateral damage to the surrounding normal cells, as it is well established that cancer therapies have bystander effects on normal cells in the body, often causing undesirable side effects. The aim of this study was to investigate the cellular and molecular effects of HYP-PDT on normal primary human keratinocytes (Kc), melanocytes (Mc) and fibroblasts (Fb) in an in vitro tissue culture model which represented both the epidermal and dermal cellular compartments of human skin. Cell viability analysis revealed a differential cytotoxic response to a range of HYP-PDT doses in all the human skin cell types, showing that Fb (LD50=1.75μM) were the most susceptible to HYP-PDT, followed by Mc (LD50=3.5μM) and Kc (LD50>4μM HYP-PDT) These results correlated with the morphological analysis which displayed distinct morphological changes in Fb and Mc, 24h post treatment with non-lethal (1μM) and lethal (3μM) doses of HYP-PDT, but the highest HYP-PDT doses had no effect on Kc morphology. Fluorescent microscopy displayed cytoplasmic localization of HYP in all the 3 skin cell types and additionally, HYP was excluded from the nuclei in all the cell types. Intracellular ROS levels measured in Fb at 3μM HYP-PDT, displayed a significant 3.8 fold (p<0.05) increase in ROS, but no significant difference in ROS levels occurred in Mc or Kc. Furthermore, 64% (p<0.005) early apoptotic Fb and 20% (p<0.05) early apoptotic Mc were evident; using fluorescence activated cell sorting (FACS), 24

  15. Gastrospheres of human gastric mucosa cells: an in vitro model of stromal and epithelial stem cell niche reconstruction.

    PubMed

    Santos, Carlos A N; Andrade, Leonardo R; Costa, Márcia H M; Souza, Heitor S P; Granjeiro, José M; Takiya, Christina M; Borojevic, Radovan; Nasciutti, Luiz E

    2016-08-01

    The molecular characterization of mechanisms involved in the gastrointestinal tract disorders needs an in vitro 3D culture model able to mimic the in vivo gastric microenvironment. Herein, we propose a 3D coculture system where gastric epithelial and stromal cells are grown together building spherical and solid structures using the NASA bioreactor - cell culture system (RCCS), a bioreactor. Epithelial and stromal cells from human antral gastric mucosa were isolated from endoscopic gastric biopsies. Thereafter, these cells were mechanically and enzymatically dispersed by treatment with dispase and collagenase, respectively. Using specific culture procedures, these cells formed 3D structures by using a RCCS, named "gastrospheres". Briefly, gastrospheres were obtained by initial seeding of 2.5x10⁴ cells/well in 96 well culture plates. At 24 h after their formation, they were transferred into RCCS, and maintained for 7, 14, 21, and 28 days. The gastrospheres were morphologically characterized by immunocytochemisty to evaluate extracellular matrix (ECM), and by electron microscopy. These analysis of gastrospheres revealed that the epithelial cells were cytokeratin (CK) and lectin reactive and were arranged in the outer layer; stromal cells presented long cytoplasmic processes and were localized inside the gastrosphere. They were vimentin (VIM) and α-smooth muscle actin (α-SMA) positive and expressed ECM components such as laminin (LN), fibronectin (FN), and type IV collagen (CIV). Electron microscopy revealed groups of cohesive gastric cells surrounded by complex stromal structures, with multiple microvilli, and tight cellular junctions interspersed with extracellular matrix fibrils and fibers. The presence of some nestin-positive cells was observed in the inner region of the gastrospheres, suggesting an intermediary localization between epithelial and stromal cells. Altogether, our data suggest that in vitro gastrospheres recapitulate the in vivo gastric niche

  16. Human neural precursor cells promote neurologic recovery in a viral model of multiple sclerosis.

    PubMed

    Chen, Lu; Coleman, Ronald; Leang, Ronika; Tran, Ha; Kopf, Alexandra; Walsh, Craig M; Sears-Kraxberger, Ilse; Steward, Oswald; Macklin, Wendy B; Loring, Jeanne F; Lane, Thomas E

    2014-06-01

    Using a viral model of the demyelinating disease multiple sclerosis (MS), we show that intraspinal transplantation of human embryonic stem cell-derived neural precursor cells (hNPCs) results in sustained clinical recovery, although hNPCs were not detectable beyond day 8 posttransplantation. Improved motor skills were associated with a reduction in neuroinflammation, decreased demyelination, and enhanced remyelination. Evidence indicates that the reduced neuroinflammation is correlated with an increased number of CD4(+)CD25(+)FOXP3(+) regulatory T cells (Tregs) within the spinal cords. Coculture of hNPCs with activated T cells resulted in reduced T cell proliferation and increased Treg numbers. The hNPCs acted, in part, through secretion of TGF-β1 and TGF-β2. These findings indicate that the transient presence of hNPCs transplanted in an animal model of MS has powerful immunomodulatory effects and mediates recovery. Further investigation of the restorative effects of hNPC transplantation may aid in the development of clinically relevant MS treatments. PMID:24936469

  17. Human amniotic fluid stem cell injection therapy for urethral sphincter regeneration in an animal model

    PubMed Central

    2012-01-01

    Background Stem cell injection therapies have been proposed to overcome the limited efficacy and adverse reactions of bulking agents. However, most have significant limitations, including painful procurement, requirement for anesthesia, donor site infection and a frequently low cell yield. Recently, human amniotic fluid stem cells (hAFSCs) have been proposed as an ideal cell therapy source. In this study, we investigated whether periurethral injection of hAFSCs can restore urethral sphincter competency in a mouse model. Methods Amniotic fluids were collected and harvested cells were analyzed for stem cell characteristics and in vitro myogenic differentiation potency. Mice underwent bilateral pudendal nerve transection to generate a stress urinary incontinence (SUI) model and received either periurethral injection of hAFSCs, periurethral injection of Plasma-Lyte (control group), or underwent a sham (normal control group). For in vivo cell tracking, cells were labeled with silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate (MNPs@SiO2 (RITC)) and were injected into the urethral sphincter region (n = 9). Signals were detected by optical imaging. Leak point pressure and closing pressure were recorded serially after injection. Tumorigenicity of hAFSCs was evaluated by implanting hAFSCs into the subcapsular space of the kidney, followed two weeks later by retrieval and histologic analysis. Results Flow activated cell sorting showed that hAFSCs expressed mesenchymal stem cell (MSC) markers, but no hematopoietic stem cell markers. Induction of myogenic differentiation in the hAFSCs resulted in expression of PAX7 and MYOD at Day 3, and DYSTROPHIN at Day 7. The nanoparticle-labeled hAFSCs could be tracked in vivo with optical imaging for up to 10 days after injection. Four weeks after injection, the mean LPP and CP were significantly increased in the hAFSC-injected group compared with the control group. Nerve regeneration and neuromuscular junction

  18. Human umbilical cord mesenchymal stem cells alleviate nasal mucosa radiation damage in a guinea pig model.

    PubMed

    Duan, Hong-Gang; Ji, Fang; Zheng, Chun-Quan; Wang, Chun-Hua; Li, Jing

    2015-02-01

    Nasal complications after radiotherapy severely affect the quality of life of nasopharyngeal carcinoma patients, and there is a compelling need to find novel therapies for nasal epithelial cell radiation damage. Therefore, we investigated the therapeutic effect of human umbilical cord mesenchymal stem cells (hUC-MSCs) in guinea pig model of nasal mucosa radiation damage and explored its therapeutic mechanism. Cultured hUC-MSCs were injected intravenously immediately after radiation in the nasal mucosa-radiation-damage guinea pig model. Migration of hUC-MSCs into the nasal mucosa and the potential for differentiation into nasal epithelial cells were evaluated by immunofluorescence. The therapeutic effects of hUC-MSCs were evaluated by mucus clearance time (MCT), degree of nasal mucosa edema, and the nasal mucosa cilia form and coverage ratio. Results indicate that the hUC-MSCs migrated to the nasal mucosa lamina propria and did not differentiate into nasal epithelial cells in this model. The MCT and degree of mucosal edema were improved at 1 week and 1 month after radiation, respectively, but no difference was found at 3 months and 6 months after radiation. The nasal mucosa cilia form and coverage ratio was not improved 6 months after radiation. Thus, hUC-MSCs can migrate to the nasal mucosa lamina propria and improve MCT and mucosa edema within a short time period, but these cells are unable to differentiate into nasal epithelial cells and improve nasal epithelial regeneration in the nasal mucosa radiation damage guinea pig model. PMID:25209829

  19. Characterization of the replication timing program of 6 human model cell lines.

    PubMed

    Hadjadj, Djihad; Denecker, Thomas; Maric, Chrystelle; Fauchereau, Fabien; Baldacci, Giuseppe; Cadoret, Jean-Charles

    2016-09-01

    During the S-phase, the DNA replication process is finely orchestrated and regulated by two programs: the spatial program that determines where replication will start in the genome (Cadoret et al. (2008 Oct 14), Cayrou et al. (2011 Sep), Picard et al. (2014 May 1) [1], [2], [3]), and the temporal program that determines when during the S phase different parts of the genome are replicated and when origins are activated. The temporal program is so well conserved for each cell type from independent individuals [4] that it is possible to identify a cell type from an unknown sample just by determining its replication timing program. Moreover, replicative domains are strongly correlated with the partition of the genome into topological domains (determined by the Hi-C method, Lieberman-Aiden et al. (2009 Oct 9), Pope et al. (2014 Nov 20) [5], [6]). On the one hand, replicative areas are well defined and participate in shaping the spatial organization of the genome for a given cell type. On the other hand, studies on the timing program during cell differentiation showed a certain plasticity of this program according to the stage of cell differentiation Hiratani et al. (2008 Oct 7, 2010 Feb) [7], [8]. Domains where a replication timing change was observed went through a nuclear re-localization. Thus the temporal program of replication can be considered as an epigenetic mark Hiratani and Gilbert (2009 Feb 16) [9]. We present the genomic data of replication timing in 6 human model cell lines: U2OS (GSM2111308), RKO (GSM2111309), HEK 293T (GSM2111310), HeLa (GSM2111311), MRC5-SV (GSM2111312) and K562 (GSM2111313). A short comparative analysis was performed that allowed us to define regions common to the 6 cell lines. These replication timing data can be taken into account when performing studies that use these model cell lines. PMID:27508120

  20. Selective destruction of mouse islet beta cells by human T lymphocytes in a newly-established humanized type 1 diabetic model

    SciTech Connect

    Zhao, Yong; Guo, Chengshan; Hwang, David; Lin, Brian; Dingeldein, Michael; Mihailescu, Dan; Sam, Susan; Sidhwani, Seema; Zhang, Yongkang; Jain, Sumit; Skidgel, Randal A.; Prabhakar, Bellur S.; Mazzone, Theodore; Holterman, Mark J.

    2010-09-03

    Research highlights: {yields} Establish a human immune-mediated type 1 diabetic model in NOD-scid IL2r{gamma}{sup null} mice. {yields} Using the irradiated diabetic NOD mouse spleen mononuclear cells as trigger. {yields} The islet {beta} cells were selectively destroyed by infiltrated human T cells. {yields} The model can facilitate translational research to find a cure for type 1 diabetes. -- Abstract: Type 1 diabetes (T1D) is caused by a T cell-mediated autoimmune response that leads to the loss of insulin-producing {beta} cells. The optimal preclinical testing of promising therapies would be aided by a humanized immune-mediated T1D model. We develop this model in NOD-scid IL2r{gamma}{sup null} mice. The selective destruction of pancreatic islet {beta} cells was mediated by human T lymphocytes after an initial trigger was supplied by the injection of irradiated spleen mononuclear cells (SMC) from diabetic nonobese diabetic (NOD) mice. This resulted in severe insulitis, a marked loss of total {beta}-cell mass, and other related phenotypes of T1D. The migration of human T cells to pancreatic islets was controlled by the {beta} cell-produced highly conserved chemokine stromal cell-derived factor 1 (SDF-1) and its receptor C-X-C chemokine receptor (CXCR) 4, as demonstrated by in vivo blocking experiments using antibody to CXCR4. The specificity of humanized T cell-mediated immune responses against islet {beta} cells was generated by the local inflammatory microenvironment in pancreatic islets including human CD4{sup +} T cell infiltration and clonal expansion, and the mouse islet {beta}-cell-derived CD1d-mediated human iNKT activation. The selective destruction of mouse islet {beta} cells by a human T cell-mediated immune response in this humanized T1D model can mimic those observed in T1D patients. This model can provide a valuable tool for translational research into T1D.

  1. Modeling Electrophysiological Coupling and Fusion between Human Mesenchymal Stem Cells and Cardiomyocytes

    PubMed Central

    Mayourian, Joshua; Savizky, Ruben M.; Sobie, Eric A.; Costa, Kevin D.

    2016-01-01

    Human mesenchymal stem cell (hMSC) delivery has demonstrated promise in preclinical and clinical trials for myocardial infarction therapy; however, broad acceptance is hindered by limited understanding of hMSC-human cardiomyocyte (hCM) interactions. To better understand the electrophysiological consequences of direct heterocellular connections between hMSCs and hCMs, three original mathematical models were developed, representing an experimentally verified triad of hMSC families with distinct functional ion channel currents. The arrhythmogenic risk of such direct electrical interactions in the setting of healthy adult myocardium was predicted by coupling and fusing these hMSC models to the published ten Tusscher midcardial hCM model. Substantial variations in action potential waveform—such as decreased action potential duration (APD) and plateau height—were found when hCMs were coupled to the two hMSC models expressing functional delayed rectifier-like human ether à-go-go K+ channel 1 (hEAG1); the effects were exacerbated for fused hMSC-hCM hybrid cells. The third family of hMSCs (Type C), absent of hEAG1 activity, led to smaller single-cell action potential alterations during coupling and fusion, translating to longer tissue-level mean action potential wavelength. In a simulated 2-D monolayer of cardiac tissue, re-entry vulnerability with low (5%) hMSC insertion was approximately eight-fold lower with Type C hMSCs compared to hEAG1-functional hMSCs. A 20% decrease in APD dispersion by Type C hMSCs compared to hEAG1-active hMSCs supports the claim of reduced arrhythmogenic potential of this cell type with low hMSC insertion. However, at moderate (15%) and high (25%) hMSC insertion, the vulnerable window increased independent of hMSC type. In summary, this study provides novel electrophysiological models of hMSCs, predicts possible arrhythmogenic effects of hMSCs when directly coupled to healthy hCMs, and proposes that isolating a subset of hMSCs absent of h

  2. Absence of WASp Enhances Hematopoietic and Megakaryocytic Differentiation in a Human Embryonic Stem Cell Model.

    PubMed

    Toscano, Miguel G; Muñoz, Pilar; Sánchez-Gilabert, Almudena; Cobo, Marién; Benabdellah, Karim; Anderson, Per; Ramos-Mejía, Verónica; Real, Pedro J; Neth, Olaf; Molinos-Quintana, Agueda; Gregory, Philip D; Holmes, Michael C; Martin, Francisco

    2016-02-01

    The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency caused by mutations in the WAS gene and characterized by severe thrombocytopenia. Although the role of WASp in terminally differentiated lymphocytes and myeloid cells is well characterized, its role in early hematopoietic differentiation and in platelets (Plts) biology is poorly understood. In the present manuscript, we have used zinc finger nucleases targeted to the WAS locus for the development of two isogenic WAS knockout (WASKO) human embryonic stem cell lines (hESCs). Upon hematopoietic differentiation, hESCs-WASKO generated increased ratios of CD34(+)CD45(+) progenitors with altered responses to stem cell factor compared to hESCs-WT. When differentiated toward the megakaryocytic linage, hESCs-WASKO produced increased numbers of CD34(+)CD41(+) progenitors, megakaryocytes (MKs), and Plts. hESCs-WASKO-derived MKs and Plts showed altered phenotype as well as defective responses to agonist, mimicking WAS patients MKs and Plts defects. Interestingly, the defects were more evident in WASp-deficient MKs than in WASp-deficient Plts. Importantly, ectopic WAS expression using lentiviral vectors restored normal Plts development and MKs responses. These data validate the AND-1_WASKO cell lines as a human cellular model for basic research and for preclinical studies for WAS. PMID:26502776

  3. Biodistribution of Infused Human Umbilical Cord Blood Cells in Alzheimer's Disease-Like Murine Model.

    PubMed

    Ehrhart, Jared; Darlington, Donna; Kuzmin-Nichols, Nicole; Sanberg, Cyndy D; Sawmiller, Darrell R; Sanberg, Paul R; Tan, Jun

    2016-01-01

    Human umbilical cord blood cells (HUCBCs), a prolific source of non-embryonic or adult stem cells, have emerged as effective and relatively safe immunomodulators and neuroprotectors, reducing behavioral impairment in animal models of Alzheimer's disease (AD), Parkinson's disease, amyotrophic lateral sclerosis, traumatic brain injury, spinal cord injury, and stroke. In this report, we followed the bioavailability of HUCBCs in AD-like transgenic PSAPP mice and nontransgenic Sprague-Dawley rats. HUCBCs were injected into tail veins of mice or rats at a single dose of 1 × 10(6) or 2.2 × 10(6) cells, respectively, prior to harvesting of tissues at 24 h, 7 days, and 30 days after injection. For determination of HUCBC distribution, tissues from both species were subjected to total DNA isolation and polymerase chain reaction (PCR) amplification of the gene for human glycerol-3-phosphate dehydrogenase. Our results show a relatively similar biodistribution and retention of HUCBCs in both mouse and rat organs. HUCBCs were broadly detected both in the brain and several peripheral organs, including the liver, kidney, and bone marrow, of both species, starting within 7 days and continuing up to 30 days posttransplantation. No HUCBCs were recovered in the peripheral circulation, even at 24 h posttransplantation. Therefore, HUCBCs reach several tissues including the brain following a single intravenous treatment, suggesting that this route can be a viable method of administration of these cells for the treatment of neurodegenerative diseases. PMID:26414627

  4. Absence of WASp Enhances Hematopoietic and Megakaryocytic Differentiation in a Human Embryonic Stem Cell Model

    PubMed Central

    Toscano, Miguel G; Muñoz, Pilar; Sánchez-Gilabert, Almudena; Cobo, Marién; Benabdellah, Karim; Anderson, Per; Ramos-Mejía, Verónica; Real, Pedro J; Neth, Olaf; Molinos-Quintana, Agueda; Gregory, Philip D; Holmes, Michael C; Martin, Francisco

    2016-01-01

    The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency caused by mutations in the WAS gene and characterized by severe thrombocytopenia. Although the role of WASp in terminally differentiated lymphocytes and myeloid cells is well characterized, its role in early hematopoietic differentiation and in platelets (Plts) biology is poorly understood. In the present manuscript, we have used zinc finger nucleases targeted to the WAS locus for the development of two isogenic WAS knockout (WASKO) human embryonic stem cell lines (hESCs). Upon hematopoietic differentiation, hESCs-WASKO generated increased ratios of CD34+CD45+ progenitors with altered responses to stem cell factor compared to hESCs-WT. When differentiated toward the megakaryocytic linage, hESCs-WASKO produced increased numbers of CD34+CD41+ progenitors, megakaryocytes (MKs), and Plts. hESCs-WASKO-derived MKs and Plts showed altered phenotype as well as defective responses to agonist, mimicking WAS patients MKs and Plts defects. Interestingly, the defects were more evident in WASp-deficient MKs than in WASp-deficient Plts. Importantly, ectopic WAS expression using lentiviral vectors restored normal Plts development and MKs responses. These data validate the AND-1_WASKO cell lines as a human cellular model for basic research and for preclinical studies for WAS. PMID:26502776

  5. Derivation of Epithelial Cells from Human Embryonic Stem Cells as an In Vitro Model of Vocal Mucosa.

    PubMed

    Lungova, Vlasta; Leydon, Ciara; Thibeault, Susan

    2016-01-01

    Vocal fold epithelial cells are very difficult to study as the vocal fold epithelial cell lines do not exist and they cannot be removed from the healthy larynx without engendering a significant and unacceptable risk to vocal fold function. Here, we describe the procedure to create an engineered vocal fold tissue construct consisting of the scaffold composed of the collagen 1 gel seeded with human fibroblasts and simple epithelial progenitors seeded on the scaffold and cultivated at air-liquid interface for 19-21 days to derive the stratified squamous epithelium. This model of vocal fold mucosa is very similar in morphology, gene expression, and phenotypic characteristics to native vocal fold epithelial cells and the underlying lamina propria and, therefore, offers a promising approach to studying vocal fold biology and biomechanics in health and disease. PMID:25403465

  6. Cytoplasmic-nuclear trafficking of G1/S cell cycle molecules and adult human β-cell replication: a revised model of human β-cell G1/S control.

    PubMed

    Fiaschi-Taesch, Nathalie M; Kleinberger, Jeffrey W; Salim, Fatimah G; Troxell, Ronnie; Wills, Rachel; Tanwir, Mansoor; Casinelli, Gabriella; Cox, Amy E; Takane, Karen K; Srinivas, Harish; Scott, Donald K; Stewart, Andrew F

    2013-07-01

    Harnessing control of human β-cell proliferation has proven frustratingly difficult. Most G1/S control molecules, generally presumed to be nuclear proteins in the human β-cell, are in fact constrained to the cytoplasm. Here, we asked whether G1/S molecules might traffic into and out of the cytoplasmic compartment in association with activation of cell cycle progression. Cdk6 and cyclin D3 were used to drive human β-cell proliferation and promptly translocated into the nucleus in association with proliferation. In contrast, the cell cycle inhibitors p15, p18, and p19 did not alter their location, remaining cytoplasmic. Conversely, p16, p21, and p27 increased their nuclear frequency. In contrast once again, p57 decreased its nuclear frequency. Whereas proliferating β-cells contained nuclear cyclin D3 and cdk6, proliferation generally did not occur in β-cells that contained nuclear cell cycle inhibitors, except p21. Dynamic cytoplasmic-nuclear trafficking of cdk6 was confirmed using green fluorescent protein-tagged cdk6 and live cell imaging. Thus, we provide novel working models describing the control of cell cycle progression in the human β-cell. In addition to known obstacles to β-cell proliferation, cytoplasmic-to-nuclear trafficking of G1/S molecules may represent an obstacle as well as a therapeutic opportunity for human β-cell expansion. PMID:23493571

  7. Effectivity of pazopanib treatment in orthotopic models of human testicular germ cell tumors

    PubMed Central

    2013-01-01

    Background Cisplatin (CDDP) resistance in testicular germ cell tumors (GCTs) is still a clinical challenge, and one associated with poor prognosis. The purpose of this work was to test pazopanib, an anti-tumoral and anti-angiogenic multikinase inhibitor, and its combination with lapatinib (an anti-ErbB inhibitor) in mouse orthotopic models of human testicular GCTs. Methods We used two different models of human testicular GCTs orthotopically grown in nude mice; a CDDP-sensitive choriocarcinoma (TGT38) and a new orthotopic model generated from a metastatic GCT refractory to first-line CDDP chemotherapy (TGT44). Nude mice implanted with these orthotopic tumors were treated with the inhibitors and the effect on tumoral growth and angiogenesis was evaluated. Results TGT44 refractory tumor had an immunohistochemical profile similar to the original metastasis, with characteristics of yolk sac tumor. TGT44 did not respond when treated with cisplatin. In contrast, pazopanib had an anti-angiogenic effect and anti-tumor efficacy in this model. Pazopanib in combination with lapatinib in TGT38, an orthotopic model of choriocarcinoma had an additive effect blocking tumor growth. Conclusions We present pazopanib as a possible agent for the alternative treatment of CDDP-sensitive and CDDP-refractory GCT patients, alone or in combination with anti-ErbB therapies. PMID:23937707

  8. Modeling human protein aggregation cardiomyopathy using murine induced pluripotent stem cells.

    PubMed

    Limphong, Pattraranee; Zhang, Huali; Christians, Elisabeth; Liu, Qiang; Riedel, Michael; Ivey, Kathryn; Cheng, Paul; Mitzelfelt, Katie; Taylor, Graydon; Winge, Dennis; Srivastava, Deepak; Benjamin, Ivor

    2013-03-01

    Several mutations in αB-crystallin (CryAB), a heat shock protein with chaperone-like activities, are causally linked to skeletal and cardiac myopathies in humans. To better understand the underlying pathogenic mechanisms, we had previously generated transgenic (TG) mice expressing R120GCryAB, which recapitulated distinguishing features of the myopathic disorder (e.g., protein aggregates, hypertrophic cardiomyopathy). To determine whether induced pluripotent stem cell (iPSC)-derived cardiomyocytes, a new experimental approach for human disease modeling, would be relevant to aggregation-prone disorders, we decided to exploit the existing transgenic mouse model to derive iPSCs from tail tip fibroblasts. Several iPSC lines were generated from TG and non-TG mice and validated for pluripotency. TG iPSC-derived cardiomyocytes contained perinuclear aggregates positive for CryAB staining, whereas CryAB protein accumulated in both detergent-soluble and insoluble fractions. iPSC-derived cardiomyocytes identified by cardiac troponin T staining were significantly larger when expressing R120GCryAB at a high level in comparison with TG low expressor or non-TG cells. Expression of fetal genes such as atrial natriuretic factor, B-type natriuretic peptide, and α-skeletal α-actin, assessed by quantitative reverse transcription-polymerase chain reaction, were increased in TG cardiomyocytes compared with non-TG, indicating the activation of the hypertrophic genetic program in vitro. Our study demonstrates for the first time that differentiation of R120G iPSCs into cardiomyocytes causes protein aggregation and cellular hypertrophy, recapitulating in vitro key pathognomonic hallmarks found in both animal models and patients. Our findings pave the way for further studies exploiting this cell model system for mechanistic and therapeutic investigations. PMID:23430692

  9. Development of a combined model of tissue kinetics and radiation response of human bronchiolar epithelium with single cell resolution

    NASA Astrophysics Data System (ADS)

    Ostrovskaya, Natela Grigoryevna

    2005-07-01

    Lack of accurate data for epidemiological studies of low dose radiation effects necessitates development of dosimetric models allowing prediction of cancer risks for different organs. The objective of this work is to develop a model of the radiation response of human bronchiolar tissue with single cell resolution. The computer model describes epithelial tissue as an ensemble of individual cells, with the geometry of a human bronchiole and the properties of different cell types are taken into account. The model simulates the tissue kinetics and radiation exposure in four dimensions: three spatial dimensions and a temporal dimension. The bronchiole is modeled as a regular hollow cylinder with the epithelial cells of three different types (basal, secretory, and ciliated) lining its interior. For the purposes of assessment of radiation damage to the cells only the nuclei of the cells have been modeled. Subroutines describing cellular kinetics have been developed to simulate cell turnover in a normal epithelial tissue. Monte Carlo subroutines have been developed to simulate exposure to alpha particles; the GEANT4 toolkit has been used to simulate exposure to low LET radiation. Each hit cell is provided with a record of energy deposition, and this record is passed to the progeny if the cell survives. The model output provides data on the number of basal progenitor cells in different phases of a cell life-cycle and secretory to ciliated cell ratio after several generations of cell proliferation. The model calculates labeling and mitotic indices and estimates the average cell turnover time for the bronchiolar tissue. Microdosimetric calculations are performed for cells traversed by ionizing particles. The model will be used to assess the accumulation of damage in cells due to protracted low level radiation exposure. The model output may provide directions for the future experimental design.

  10. Combined Effects of Lipophilic Phycotoxins (Okadaic Acid, Azapsiracid-1 and Yessotoxin) on Human Intestinal Cells Models

    PubMed Central

    Ferron, Pierre-Jean; Dumazeau, Kevin; Beaulieu, Jean-François; Le Hégarat, Ludovic; Fessard, Valérie

    2016-01-01

    Phycotoxins are monitored in seafood because they can cause food poisonings in humans. Phycotoxins do not only occur singly but also as mixtures in shellfish. The aim of this study was to evaluate the in vitro toxic interactions of binary combinations of three lipophilic phycotoxins commonly found in Europe (okadaic acid (OA), yessotoxin (YTX) and azaspiracid-1 (AZA-1)) using the neutral red uptake assay on two human intestinal cell models, Caco-2 and the human intestinal epithelial crypt-like cells (HIEC). Based on the cytotoxicity of individual toxins, we studied the interactions between toxins in binary mixtures using the combination index-isobologram equation, a method widely used in pharmacology to study drug interactions. This method quantitatively classifies interactions between toxins in mixtures as synergistic, additive or antagonistic. AZA-1/OA, and YTX/OA mixtures showed increasing antagonism with increasing toxin concentrations. In contrast, the AZA-1/YTX mixture showed increasing synergism with increasing concentrations, especially for mixtures with high YTX concentrations. These results highlight the hazard potency of AZA-1/YTX mixtures with regard to seafood intoxication. PMID:26907345

  11. In vivo imaging of human adipose-derived stem cells in Alzheimer's disease animal model

    NASA Astrophysics Data System (ADS)

    Ha, Sungji; Ahn, Sangzin; Kim, Saeromi; Joo, Yuyoung; Chong, Young Hae; Suh, Yoo-Hun; Chang, Keun-A.

    2014-05-01

    Stem cell therapy is a promising tool for the treatment of diverse conditions, including neurodegenerative diseases such as Alzheimer's disease (AD). To understand transplanted stem cell biology, in vivo imaging is necessary. Nanomaterial has great potential for in vivo imaging and several noninvasive methods are used, such as magnetic resonance imaging, positron emission tomography, fluorescence imaging (FI) and near-infrared FI. However, each method has limitations for in vivo imaging. To overcome these limitations, multimodal nanoprobes have been developed. In the present study, we intravenously injected human adipose-derived stem cells (hASCs) that were labeled with a multimodal nanoparticle, LEO-LIVE™-Magnoxide 675 or 797 (BITERIALS, Seoul, Korea), into Tg2576 mice, an AD mouse model. After sequential in vivo tracking using Maestro Imaging System, we found fluorescence signals up to 10 days after injection. We also found strong signals in the brains extracted from hASC-transplanted Tg2576 mice up to 12 days after injection. With these results, we suggest that in vivo imaging with this multimodal nanoparticle may provide a useful tool for stem cell tracking and understanding stem cell biology in other neurodegenerative diseases.

  12. A novel in vitro model system for smooth muscle differentiation from human embryonic stem cell-derived mesenchymal cells

    PubMed Central

    Guo, Xia; Stice, Steven L.; Boyd, Nolan L.

    2013-01-01

    The objective of this study was to develop a novel in vitro model for smooth muscle cell (SMC) differentiation from human embryonic stem cell-derived mesenchymal cells (hES-MCs). We found that hES-MCs were differentiated to SMCs by transforming growth factor-β (TGF-β) in a dose- and time-dependent manner as demonstrated by the expression of SMC-specific genes smooth muscle α-actin, calponin, and smooth muscle myosin heavy chain. Under normal growth conditions, however, the differentiation capacity of hES-MCs was very limited. hES-MC-derived SMCs had an elongated and spindle-shaped morphology and contracted in response to the induction of carbachol and KCl. KCl-induced calcium transient was also evident in these cells. Compared with the parental cells, TGF-β-treated hES-MCs sustained the endothelial tube formation for a longer time due to the sustained SMC phenotype. Mechanistically, TGF-β-induced differentiation was both Smad- and serum response factor/myocardin dependent. TGF-β regulated myocardin expression via multiple signaling pathways including Smad2/3, p38 MAPK, and PI3K. Importantly, we found that a low level of myocardin was present in mesoderm prior to SMC lineage determination, and a high level of myocardin was not induced until the differentiation process was initiated. Taken together, our study characterized a novel SMC differentiation model that can be used for studying human SMC differentiation from mesoderm during vascular development. PMID:23220114

  13. A retinoic acid-enhanced, multicellular human blood-brain barrier model derived from stem cell sources

    NASA Astrophysics Data System (ADS)

    Lippmann, Ethan S.; Al-Ahmad, Abraham; Azarin, Samira M.; Palecek, Sean P.; Shusta, Eric V.

    2014-02-01

    Blood-brain barrier (BBB) models are often used to investigate BBB function and screen brain-penetrating therapeutics, but it has been difficult to construct a human model that possesses an optimal BBB phenotype and is readily scalable. To address this challenge, we developed a human in vitro BBB model comprising brain microvascular endothelial cells (BMECs), pericytes, astrocytes and neurons derived from renewable cell sources. First, retinoic acid (RA) was used to substantially enhance BBB phenotypes in human pluripotent stem cell (hPSC)-derived BMECs, particularly through adherens junction, tight junction, and multidrug resistance protein regulation. RA-treated hPSC-derived BMECs were subsequently co-cultured with primary human brain pericytes and human astrocytes and neurons derived from human neural progenitor cells (NPCs) to yield a fully human BBB model that possessed significant tightness as measured by transendothelial electrical resistance (~5,000 Ωxcm2). Overall, this scalable human BBB model may enable a wide range of neuroscience studies.

  14. Modeling Doxorubicin-Induced Cardiotoxicity in Human Pluripotent Stem Cell Derived-Cardiomyocytes

    PubMed Central

    Maillet, Agnes; Tan, Kim; Chai, Xiaoran; Sadananda, Singh N.; Mehta, Ashish; Ooi, Jolene; Hayden, Michael R.; Pouladi, Mahmoud A.; Ghosh, Sujoy; Shim, Winston; Brunham, Liam R.

    2016-01-01

    Doxorubicin is a highly efficacious anti-cancer drug but causes cardiotoxicity in many patients. The mechanisms of doxorubicin-induced cardiotoxicity (DIC) remain incompletely understood. We investigated the characteristics and molecular mechanisms of DIC in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). We found that doxorubicin causes dose-dependent increases in apoptotic and necrotic cell death, reactive oxygen species production, mitochondrial dysfunction and increased intracellular calcium concentration. We characterized genome-wide changes in gene expression caused by doxorubicin using RNA-seq, as well as electrophysiological abnormalities caused by doxorubicin with multi-electrode array technology. Finally, we show that CRISPR-Cas9-mediated disruption of TOP2B, a gene implicated in DIC in mouse studies, significantly reduces the sensitivity of hPSC-CMs to doxorubicin-induced double stranded DNA breaks and cell death. These data establish a human cellular model of DIC that recapitulates many of the cardinal features of this adverse drug reaction and could enable screening for protective agents against DIC as well as assessment of genetic variants involved in doxorubicin response. PMID:27142468

  15. Modeling Doxorubicin-Induced Cardiotoxicity in Human Pluripotent Stem Cell Derived-Cardiomyocytes.

    PubMed

    Maillet, Agnes; Tan, Kim; Chai, Xiaoran; Sadananda, Singh N; Mehta, Ashish; Ooi, Jolene; Hayden, Michael R; Pouladi, Mahmoud A; Ghosh, Sujoy; Shim, Winston; Brunham, Liam R

    2016-01-01

    Doxorubicin is a highly efficacious anti-cancer drug but causes cardiotoxicity in many patients. The mechanisms of doxorubicin-induced cardiotoxicity (DIC) remain incompletely understood. We investigated the characteristics and molecular mechanisms of DIC in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). We found that doxorubicin causes dose-dependent increases in apoptotic and necrotic cell death, reactive oxygen species production, mitochondrial dysfunction and increased intracellular calcium concentration. We characterized genome-wide changes in gene expression caused by doxorubicin using RNA-seq, as well as electrophysiological abnormalities caused by doxorubicin with multi-electrode array technology. Finally, we show that CRISPR-Cas9-mediated disruption of TOP2B, a gene implicated in DIC in mouse studies, significantly reduces the sensitivity of hPSC-CMs to doxorubicin-induced double stranded DNA breaks and cell death. These data establish a human cellular model of DIC that recapitulates many of the cardinal features of this adverse drug reaction and could enable screening for protective agents against DIC as well as assessment of genetic variants involved in doxorubicin response. PMID:27142468

  16. Nephron organoids derived from human pluripotent stem cells model kidney development and injury

    PubMed Central

    Morizane, Ryuji; Lam, Albert Q.; Freedman, Benjamin S.; Kishi, Seiji; Valerius, M. Todd; Bonventre, Joseph V.

    2015-01-01

    Kidney cells and tissues derived from human pluripotent stem cells (hPSCs) would enable organ regeneration, disease modeling, and drug screening in vitro. We established an efficient, chemically defined protocol for differentiating hPSCs into multipotent nephron progenitor cells (NPCs) that can form nephron-like structures. By recapitulating metanephric kidney development in vitro, we generate SIX2+SALL1+WT1+PAX2+ NPCs with 90% efficiency within 9 days of differentiation. The NPCs possess the developmental potential of their in vivo counterparts and form PAX8+LHX1+ renal vesicles that self-pattern into nephron structures. In both 2D and 3D culture, NPCs form kidney organoids containing epithelial nephron-like structures expressing markers of podocytes, proximal tubules, loops of Henle, and distal tubules in an organized, continuous arrangement that resembles the nephron in vivo. We also show that this organoid culture system can be used to study mechanisms of human kidney development and toxicity. PMID:26458176

  17. Human Umbilical Cord Blood Cells Ameliorates Motor Deficits In Rabbits In a Cerebral Palsy Model

    PubMed Central

    Drobyshevsky, A.; Cotten, C. M.; Shi, Z.; Luo, K.; Jiang, R.; Derrick, M.; Tracy, E. T.; Gentry, T.; Goldberg, R. N.; Kurtzberg, J.; Tan, S.

    2015-01-01

    Cerebral palsy (CP) has significant impact on both patients and society but therapy is limited. Human umbilical cord blood cells (HUCBC), containing various stem and progenitor cells, have been used to treat various brain genetic conditions. In small animal experiments, HUCBC have improved outcomes after hypoxic-ischemic injury. Clinical trials using HUCBC are underway testing feasibility, safety and efficacy for neonatal injury as well as CP. We tested HUCBC therapy in a validated rabbit model of CP after acute changes secondary to hypoxic-ischemic (H-I) injury had subsided. Following uterine ischemia at 70% gestation, we infused HUCBC to newborn rabbit kits with either mild or severe neurobehavioral changes. Infusion of high dose HUCBC, 5x106 cells, dramatically altered the natural history of the injury alleviating the abnormal phenotype including posture, righting reflex, locomotion, tone, and dystonia. Half the high dose showed lesser but still significant improvement. The swimming test however showed that joint function did not restore to naïve control function in either group. Tracing HUCBCs with either MRI biomarkers or PCR for human DNA found little penetration of HUCBC in the newborn brain in the immediate newborn period, suggesting that the beneficial effects were not due to cellular integration or direct proliferative effects but rather to paracrine signaling. This is the first study to show that HUCBC improve motor performance in a dose-dependent manner perhaps by improving compensatory repair processes. PMID:25791742

  18. Transplantation of human amniotic mesenchymal stem cells promotes neurological recovery in an intracerebral hemorrhage rat model.

    PubMed

    Zhou, Honglong; Zhang, Hongri; Yan, Zhongjie; Xu, Ruxiang

    2016-06-24

    Human amniotic membrane mesenchymal stem cells (hAMSCs) have recently been suggested as ideal candidate stem cells for cell-based therapy. Many studies have reported the therapeutic effects of hAMSCs in numerous disease models. However, no studies have used hAMSCs to treat intracerebral hemorrhage (ICH). In the present study, we examined the therapeutic potential of hAMSCs in a rat model of ICH, and characterized the possible mechanisms of action. Adult male Wistar rats were subjected to ICH by intrastriatal injection of VII collagenase, and then were intracerebrally administered hAMSCs, fibroblasts, or phosphate-buffered saline (PBS) at 24 h after ICH. Compared with the fibroblasts and the PBS control, hAMSCs treatment significantly promoted neurological recovery, and reduced the numbers of ED1(+) activated microglia, as well as myeloperoxidase (MPO(+)), and caspase-3(+) cells in the brain injury model. In addition, hAMSCs treatment significantly increased the expression of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) in the injured brain, and promoted neurogenesis and angiogenesis, compared with the fibroblasts and the PBS control. The transplanted hAMSCs survived for at least 27 days and were negative for β-tubulin III and glial fibrillary acidic protein (GFAP). Taken together, the results suggest that hAMSCs treatment significantly promotes neurological recovery in rats after ICH. The mechanism of action could be mediated by inhibition of inflammation and apoptosis, increasing neurotrophic factor expression, and promotion of neurogenesis and angiogenesis. Thus, hAMSCs are candidate stem cells for the treatment of ICH. PMID:27188654

  19. Effects of Modeled Microgravity on Expression Profiles of Micro RNA in Human Lymphoblastoid Cells

    NASA Technical Reports Server (NTRS)

    Mangala, Lingegowda S.; Emami, Kamal; Story, Michael; Ramesh, Govindarajan; Rohde, Larry; Wu, Honglu

    2010-01-01

    Among space radiation and other environmental factors, microgravity or an altered gravity is undoubtedly the most significant stress experienced by living organisms during flight. In comparison to the static 1g, microgravity has been shown to alter global gene expression patterns and protein levels in cultured cells or animals. Micro RNA (miRNA) has recently emerged as an important regulator of gene expression, possibly regulating as many as one-third of all human genes. miRNA represents a class of single-stranded noncoding regulatory RNA molecules ( 22 nt) that control gene expressions by inhibiting the translation of mRNA to proteins. However, very little is known on the effect of altered gravity on miRNA expression. We hypothesized that the miRNA expression profile will be altered in zero gravity resulting in regulation of the gene expression and functional changes of the cells. To test this hypothesis, we cultured TK6 human lymphoblastoid cells in Synthecon s Rotary cell culture system (bioreactors) for 72 h either in the rotating (10 rpm) to model the microgravity in space or in the static condition. The cell viability was determined before and after culturing the cells in the bioreactor using both trypan blue and guava via count. Expressions of a panel of 352 human miRNA were analyzed using the miRNA PCRarray. Out of 352 miRNAs, expressions of 75 were significantly altered by a change of greater than 1.5 folds and seven miRNAs were altered by a fold change greater than 2 under the rotating culture condition. Among these seven, miR-545 and miR-517a were down regulated by 2 folds, whereas miR-150, miR-302a, miR-139-3p, miR-515-3p and miR-564 were up regulated by 2 to 8 folds. To confirm whether this altered miRNA expression correlates with gene expression and functional changes of the cells, we performed DNA Illumina Microarray Analysis and validated the related genes using q-RT PCR.

  20. Using human induced pluripotent stem cells to model cerebellar disease: Hope and hype

    PubMed Central

    Wiethoff, Sarah; Arber, Charles; Li, Abi; Wray, Selina; Houlden, Henry; Patani, Rickie

    2015-01-01

    The cerebellum forms a highly ordered and indispensible component of motor function within the adult neuraxis, consisting of several distinct cellular subtypes. Cerebellar disease, through a variety of genetic and acquired causes, results in the loss of function of defined subclasses of neurons, and remains a significant and untreatable health care burden. The scarcity of therapies in this arena can partially be explained by unresolved disease mechanisms due to inaccessibility of human cerebellar neurons in a relevant experimental context where initiating disease mechanisms could be functionally elucidated, or drug screens conducted. In this review we discuss the potential promise of human induced pluripotent stem cells (hiPSCs) for regenerative neurology, with a particular emphasis on in vitro modelling of cerebellar degeneration. We discuss progress made thus far using hiPSC-based models of neurodegeneration, noting the relatively slower pace of discovery made in modelling cerebellar dysfunction. We conclude by speculating how strategies attempting cerebellar differentiation from hiPSCs can be refined to allow the generation of accurate disease models. This in turn will permit a greater understanding of cerebellar pathophysiology to inform mechanistically rationalised therapies, which are desperately needed in this field. PMID:25985846

  1. Human red blood cell-adapted Plasmodium knowlesi parasites: a new model system for malaria research

    PubMed Central

    Grüring, Christof; Moon, Robert W.; Lim, Caeul; Holder, Anthony A.; Blackman, Michael J.; Duraisingh, Manoj T.

    2014-01-01

    Summary Plasmodium knowlesi is a simian malaria parasite primarily infecting macaque species in Southeast Asia. Although its capacity to infect humans has been recognized since the early part of the last century, it has recently become evident that human infections are widespread and potentially life threatening. Historically, P. knowlesi has proven to be a powerful tool in early studies of malaria parasites, providing key breakthroughs in understanding many aspects of Plasmodium biology. However, the necessity to grow the parasite either in macaques or in vitro using macaque blood restricted research to laboratories with access to these resources. The recent adaptation of P. knowlesi to grow and proliferate in vitro in human red blood cells (RBCs) is therefore a substantial step towards revitalizing and expanding research on P. knowlesi. Furthermore, the development of a highly efficient transfection system to genetically modify the parasite makes P. knowlesi an ideal model to study parasite biology. In this review we elaborate on the importance of P. knowlesi in earlier phases of malaria research and highlight the future potential of the newly available human adapted P. knowlesi parasite lines. PMID:24506567

  2. Human red blood cell-adapted Plasmodium knowlesi parasites: a new model system for malaria research.

    PubMed

    Grüring, Christof; Moon, Robert W; Lim, Caeul; Holder, Anthony A; Blackman, Michael J; Duraisingh, Manoj T

    2014-05-01

    Plasmodium knowlesi is a simian malaria parasite primarily infecting macaque species in Southeast Asia. Although its capacity to infect humans has been recognized since the early part of the last century, it has recently become evident that human infections are widespread and potentially life threatening. Historically, P.  knowlesi has proven to be a powerful tool in early studies of malaria parasites, providing key breakthroughs in understanding many aspects of Plasmodium biology. However, the necessity to grow the parasite either in macaques or in vitro using macaque blood restricted research to laboratories with access to these resources. The recent adaptation of P.  knowlesi to grow and proliferate in vitro in human red blood cells (RBCs) is therefore a substantial step towards revitalizing and expanding research on P.  knowlesi. Furthermore, the development of a highly efficient transfection system to genetically modify the parasite makes P.  knowlesi an ideal model to study parasite biology. In this review, we elaborate on the importance of P.  knowlesi in earlier phases of malaria research and highlight the future potential of the newly available human adapted P.  knowlesi parasite lines. PMID:24506567

  3. Human mesenchymal stem cells suppress chronic airway inflammation in the murine ovalbumin asthma model.

    PubMed

    Bonfield, Tracey L; Koloze, Mary; Lennon, Donald P; Zuchowski, Brandon; Yang, Sung Eun; Caplan, Arnold I

    2010-12-01

    Allogeneic human mesenchymal stem cells (hMSCs) introduced intravenously can have profound anti-inflammatory activity resulting in suppression of graft vs. host disease as well as regenerative events in the case of stroke, infarct, spinal cord injury, meniscus regeneration, tendinitis, acute renal failure, and heart disease in human and animal models of these diseases. hMSCs produce bioactive factors that provide molecular cuing for: 1) immunosuppression of T cells; 2) antiscarring; 3) angiogenesis; 4) antiapoptosis; and 5) regeneration (i.e., mitotic for host-derived progenitor cells). Studies have shown that hMSCs have profound effects on the immune system and are well-tolerated and therapeutically active in immunocompetent rodent models of multiple sclerosis and stroke. Furthermore, intravenous administration of MSCs results in pulmonary localization. Asthma is a major debilitating pulmonary disease that impacts in excess of 150 million people in the world with uncontrolled asthma potentially leading to death. In addition, the socioeconomic impact of asthma-associated illnesses at the pediatric and adult level are in the millions of dollars in healthcare costs and lost days of work. hMSCs may provide a viable multiaction therapeutic for this inflammatory lung disease by secreting bioactive factors or directing cellular activity. Our studies show the effectiveness and specificity of the hMSCs on decreasing chronic airway inflammation associated with the murine ovalbumin model of asthma. In addition, the results from these studies verify the in vivo immunoeffectiveness of hMSCs in rodents and support the potential therapeutic use of hMSCs for the treatment of airway inflammation associated with chronic asthma. PMID:20817776

  4. In vivo inhibition of human CD19 targeted effector T cells by natural T regulatory cells in a xenotransplant murine model of B cell malignancy

    PubMed Central

    Lee, James; Hayman, Erik; Pegram, Hollie; Santos, Elmer; Heller, Glen; Sadelain, Michel; Brentjens, Renier J.

    2011-01-01

    Human T cells genetically modified to express chimeric antigen receptors (CARs) specific to the B cell tumor antigen CD19 can successfully eradicate systemic human CD19+ tumors in immunocompromised SCID-Beige mice. However, in the clinical setting, CD4+ CD25hi T regulatory cells (Tregs) present within the tumor microenvironment are potent suppressors of tumor-targeted effector T cells. In order to assess the impact of Tregs on CAR-modified T cells in the SCID-Beige xenotransplant model, we isolated, genetically targeted and expanded natural T regulatory cells (nTregs). In vitro, nTregs, modified to express CD19 targeted CARs efficiently inhibited the proliferation of activated human T cells, as well as the capacity of CD19-targeted 19-28z+ effector T cells to lyse CD19+ Raji tumor cells. Intravenous infusion of CD19-targeted nTregs into SCID-Beige mice with systemic Raji tumors traffic to sites of tumor and recapitulate a clinically relevant hostile tumor microenvironment. Anti-tumor efficacy of subsequently infused 19-28z+ effector T cells was fully abrogated as assessed by long-term survival of treated mice. Optimal suppression by genetically targeted nTregs was dependent on nTreg to effector T cell ratios and in vivo nTreg activation. Prior infusion of cyclophosphamide in the setting of this nTreg-mediated hostile microenvironment was able to restore the anti-tumor activity of subsequently infused 19-28z+ effector T cells through the eradication of tumor targeted nTregs. These findings have significant implications on the design of future clinical trials utilizing CAR-based adoptive T cell therapies of cancer. PMID:21487038

  5. The critical roles of tumor-initiating cells and the lymph node stromal microenvironment in human colorectal cancer extranodal metastasis using a unique humanized orthotopic mouse model.

    PubMed

    Margolin, David A; Myers, Tamara; Zhang, Xin; Bertoni, Danielle M; Reuter, Brian A; Obokhare, Izi; Borgovan, Theodor; Grimes, Chelsea; Green, Heather; Driscoll, Tiffany; Lee, Chung-Gi; Davis, Nancy K; Li, Li

    2015-08-01

    Colorectal cancer (CRC) is the second-most common cause of cancer-related mortality. The most important prognostic factors are lymph node (LN) involvement and extranodal metastasis. Our objective is to investigate the interactions between CD133(+)CXCR4(+) (CXC receptor 4) colorectal cancer tumor-initiating cells (Co-TICs) and the LN stromal microenvironment in human CRC extranodal metastasis. We established a unique humanized orthotopic xenograft model. Luciferase-tagged CRC cell lines and human cancer cells were injected intrarectally into nonobese diabetic/SCID mice. Mesenteric LN stromal cells, stromal cell line HK, or CXCL12 knockdown HK (HK-KD-A3) cells were coinoculated with CRC cells. Tumor growth and metastasis were monitored by bioluminescent imaging and immunohistochemistry. We found that this model mimics the human CRC metastatic pattern with CRC cell lines or patient specimens. Adding LN stromal cells promotes CRC tumor growth and extranodal metastasis (P < 0.001). Knocking down CXCL12 impaired HK cell support of CRC tumor formation and extranodal metastasis. When HK cells were added, sorted CD133(+)CXCR4(+) Co-TICs showed increased tumor formation and extranodal metastasis capacities compared to unseparated and non-Co-TIC populations. In conclusion, both Co-TIC and LN stromal factors play crucial roles in CRC metastasis through the CXCL12/CXCR4 axis. Blocking Co-TIC/LN-stromal interactions may lead to effective therapy to prevent extranodal metastasis. PMID:25962655

  6. Bioavailability of iron from spinach using an in vitro/human Caco-2 cell bioassay model

    NASA Technical Reports Server (NTRS)

    Rutzke, Corinne J.; Glahn, Raymond P.; Rutzke, Michael A.; Welch, Ross M.; Langhans, Robert W.; Albright, Louis D.; Combs, Gerald F Jr; Wheeler, Raymond M.

    2004-01-01

    Spinach (Spinacia oleracea) cv Whitney was tested for iron bioavailabilty using an in vitro human intestinal cell culture ferritin bioassay technique previously developed. Spinach was cultured in a growth chamber for 33 days, harvested, and freeze-dried. Total iron in the samples was an average of 71 micrograms/g dry weight. Spinach was digested in vitro (pepsin and 0.1 M HCl followed by pancreatin and 0.1 M NaHCO3) with and without the addition of supplemental ascorbic acid. Caco-2 cell cultures were used to determine iron bioavailability from the spinach mixtures. Production of the iron-binding protein ferritin in the Caco-2 cells showed the supplemental ascorbic acid doubled bioavailability of iron from spinach. The data show fresh spinach is a poor source of iron, and emphasize the importance of evaluation of whole meals rather than single food items. The data support the usefulness of the in vitro/Caco-2 cell ferritin bioassay model for prescreening of space flight diets for bioavailable iron.

  7. Human middle-ear model with compound eardrum and airway branching in mastoid air cells

    PubMed Central

    Keefe, Douglas H.

    2015-01-01

    An acoustical/mechanical model of normal adult human middle-ear function is described for forward and reverse transmission. The eardrum model included one component bound along the manubrium and another bound by the tympanic cleft. Eardrum components were coupled by a time-delayed impedance. The acoustics of the middle-ear cleft was represented by an acoustical transmission-line model for the tympanic cavity, aditus, antrum, and mastoid air cell system with variable amounts of excess viscothermal loss. Model parameters were fitted to published measurements of energy reflectance (0.25–13 kHz), equivalent input impedance at the eardrum (0.25–11 kHz), temporal-bone pressure in scala vestibuli and scala tympani (0.1–11 kHz), and reverse middle-ear impedance (0.25–8 kHz). Inner-ear fluid motion included cochlear and physiological third-window pathways. The two-component eardrum with time delay helped fit intracochlear pressure responses. A multi-modal representation of the eardrum and high-frequency modeling of the middle-ear cleft helped fit ear-canal responses. Input reactance at the eardrum was small at high frequencies due to multiple modal resonances. The model predicted the middle-ear efficiency between ear canal and cochlea, and the cochlear pressures at threshold. PMID:25994701

  8. Genetically engineered mouse models of human B-cell precursor leukemias

    PubMed Central

    Hauer, Julia; Borkhardt, Arndt; Sánchez-García, Isidro; Cobaleda, César

    2014-01-01

    B-cell precursor acute lymphoblastic leukemias (pB-ALLs) are the most frequent type of malignancies of the childhood, and also affect an important proportion of adult patients. In spite of their apparent homogeneity, pB-ALL comprises a group of diseases very different both clinically and pathologically, and with very diverse outcomes as a consequence of their biology, and underlying molecular alterations. Their understanding (as a prerequisite for their cure) will require a sustained multidisciplinary effort from professionals coming from many different fields. Among all the available tools for pB-ALL research, the use of animal models stands, as of today, as the most powerful approach, not only for the understanding of the origin and evolution of the disease, but also for the development of new therapies. In this review we go over the most relevant (historically, technically or biologically) genetically engineered mouse models (GEMMs) of human pB-ALLs that have been generated over the last 20 years. Our final aim is to outline the most relevant guidelines that should be followed to generate an “ideal” animal model that could become a standard for the study of human pB-ALL leukemia, and which could be shared among research groups and drug development companies in order to unify criteria for studies like drug testing, analysis of the influence of environmental risk factors, or studying the role of both low-penetrance mutations and cancer susceptibility alterations. PMID:25486471

  9. Bioluminescence imaging of transplanted human endothelial colony-forming cells in an ischemic mouse model.

    PubMed

    Ding, Jie; Zhao, Zhen; Wang, Chao; Wang, Cong-Xiao; Li, Pei-Cheng; Qian, Cheng; Teng, Gao-Jun

    2016-07-01

    Ischemic strokes are devastating events responsible for high mortality and morbidity worldwide each year. Endothelial colony-forming cell (ECFC) therapy holds promise for stroke treatment; however, grafted ECFCs need to be monitored better understand their biological behavior in vivo, so as to evaluate their safety and successful delivery. The objectives of this study are to visualize the fate of infused human cord blood derived ECFCs via bioluminescence imaging (BLI) in an ischemic stroke mouse model and to determine the therapeutic effects of ECFC transplantation. ECFCs derived from human umbilical cord blood were infected with lentivirus carrying enhanced green fluorescent protein (eGFP) and firefly luciferase (Luc2) double fusion reporter gene. Labeled ECFCs were grafted into a photothrombotic ischemic stroke mouse model via intra-arterial injection though the left cardiac ventricle. The homing of infused cells and functional recovery of stroke mice were evaluated using BLI, neurological scoring, and immunohistochemistry. Significantly, BLI signals were highest in the brain on day 1 and decreased steadily until day 14. GFP-positive cells were also found surrounding infarct border zones in brain sections using immunohistochemical staining, suggesting that ECFCs properly homed to the ischemic brain tissue. Using a modified neurological severity score assay and histological analysis of brain slices with CD31 immunostaining in brain tissue, double cortin analysis, and the TdT-mediated dUTP nick end labeling (TUNEL) assay, we demonstrated functional restoration, improved angiogenesis, neurogenesis, and decreased apoptosis in ischemic mice after ECFC infusion. Collectively, our data support that ECFCs may be a promising therapeutic agent for stroke. PMID:27038754

  10. Expression of P-glycoprotein in excised human nasal mucosa and optimized models of RPMI 2650 cells.

    PubMed

    Dolberg, Anne M; Reichl, Stephan

    2016-07-11

    To assess the transmucosal drug transport in the development of medications for intranasal administration, cellular in vitro models are preferred over the use of animal tissues due to inter-species variations and ethical concerns. With regard to the distribution of active agents and multidrug resistance, the ABC transporter P-glycoprotein plays a major role in several mammalian tissues. The present study compares the expression of this efflux pump in optimized in vitro models based on the human RPMI 2650 cell line with specimens of human turbinate mucosa. The presence of the ABCB1 gene was investigated at the mRNA and protein levels using RT-PCR and Western blot analysis in differently cultured RPMI 2650 cells and excised human nasal epithelium. Furthermore, the localization and activity of P-gp was examined by immunohistochemical staining and functionality assays using different substrates in both in vitro and ex vivo models. Both mRNA and protein expression of P-gp was found in all studied models. Furthermore, transporter functionality was detected in both RPMI 2650 cell culture models and excised human mucosa. The results demonstrated a highly promising comparability between RPMI 2650 models and explants of human nasal tissue concerning the influence of MDR1 on drug disposition. The RPMI 2650 cell line might become a useful tool in preclinical trials to improve reproducibility and achieve greater applicability to humans of experimental data regarding passive diffusion and active efflux of drug candidates. PMID:27155589

  11. Modeling Viral Infectious Diseases and Development of Antiviral Therapies Using Human Induced Pluripotent Stem Cell-Derived Systems

    PubMed Central

    Trevisan, Marta; Sinigaglia, Alessandro; Desole, Giovanna; Berto, Alessandro; Pacenti, Monia; Palù, Giorgio; Barzon, Luisa

    2015-01-01

    The recent biotechnology breakthrough of cell reprogramming and generation of induced pluripotent stem cells (iPSCs), which has revolutionized the approaches to study the mechanisms of human diseases and to test new drugs, can be exploited to generate patient-specific models for the investigation of host–pathogen interactions and to develop new antimicrobial and antiviral therapies. Applications of iPSC technology to the study of viral infections in humans have included in vitro modeling of viral infections of neural, liver, and cardiac cells; modeling of human genetic susceptibility to severe viral infectious diseases, such as encephalitis and severe influenza; genetic engineering and genome editing of patient-specific iPSC-derived cells to confer antiviral resistance. PMID:26184286

  12. Modeled Microgravity Disrupts Collagen I/Integrin Signaling During Osteoblastic Differentiation of Human Mesenchymal Stem Cells

    NASA Technical Reports Server (NTRS)

    Meyers, Valerie E.; Zayzafoon, Majd; Gonda, Steven R.; Gathings, William E.; McDonald, Jay M.

    2004-01-01

    Spaceflight leads to reduced bone mineral density in weight bearing bones that is primarily attributed to a reduction in bone formation. We have previously demonstrated severely reduced osteoblastogenesis of human mesenchymal stem cells (hMSC) following seven days culture in modeled microgravity. One potential mechanism for reduced osteoblastic differentiation is disruption of type I collagen-integrin interactions and reduced integrin signaling. Integrins are heterodimeric transmembrane receptors that bind extracellular matrix proteins and produce signals essential for proper cellular function, survival, and differentiation. Therefore, we investigated the effects of modeled microgravity on integrin expression and function in hMSC. We demonstrate that seven days of culture in modeled microgravity leads to reduced expression of the extracellular matrix protein, type I collagen (Col I). Conversely, modeled microgravity consistently increases Col I-specific alpha2 and beta1 integrin protein expression. Despite this increase in integrin sub-unit expression, autophosphorylation of adhesion-dependent kinases, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2), is significantly reduced. Activation of Akt is unaffected by the reduction in FAK activation. However, reduced downstream signaling via the Ras-MAPK pathway is evidenced by a reduction in Ras and ERK activation. Taken together, our findings indicate that modeled microgravity decreases integrin/MAPK signaling, which likely contributes to the observed reduction in osteoblastogenesis.

  13. In vitro co-culture model of medulloblastoma and human neural stem cells for drug delivery assessment.

    PubMed

    Ivanov, Delyan P; Parker, Terry L; Walker, David A; Alexander, Cameron; Ashford, Marianne B; Gellert, Paul R; Garnett, Martin C

    2015-07-10

    Physiologically relevant in vitro models can serve as biological analytical platforms for testing novel treatments and drug delivery systems. We describe the first steps in the development of a 3D human brain tumour co-culture model that includes the interplay between normal and tumour tissue along with nutrient gradients, cell-cell and cell-matrix interactions. The human medulloblastoma cell line UW228-3 and human foetal brain tissue were marked with two supravital fluorescent dyes (CDCFDASE, Celltrace Violet) and cultured together in ultra-low attachment 96-well plates to form reproducible single co-culture spheroids (d = 600 μm, CV% = 10%). Spheroids were treated with model cytotoxic drug etoposide (0.3-100 μM) and the viability of normal and tumour tissue quantified separately using flow cytometry and multiphoton microscopy. Etoposide levels of 10 μM were found to maximise toxicity to tumours (6.5% viability) while stem cells maintained a surviving fraction of 40%. The flexible cell marking procedure and high-throughput compatible protocol make this platform highly transferable to other cell types, primary tissues and personalised screening programs. The model's key anticipated use is for screening and assessment of drug delivery strategies to target brain tumours, and is ready for further developments, e.g. differentiation of stem cells to a range of cell types and more extensive biological validation. PMID:25592050

  14. In Silico Model-Driven Assessment of the Effects of Single Nucleotide Polymorphisms (SNPs) on Human Red Blood Cell Metabolism

    PubMed Central

    Jamshidi, Neema; Wiback, Sharon J.; Palsson, Bernhard Ø.

    2002-01-01

    The completion of the human genome project and the construction of single nucleotide polymorphism (SNP) maps have lead to significant efforts to find SNPs that can be linked to pathophysiology. In silico models of complete biochemical reaction networks relate a cell's individual reactions to the function of the entire network. Sequence variations can in turn be related to kinetic properties of individual enzymes, thus allowing an in silico model-driven assessment of the effects of defined SNPs on overall cellular functions. This process is applied to defined SNPs in two key enzymes of human red blood cell metabolism: glucose-6-phosphate dehydrogenase and pyruvate kinase. The results demonstrate the utility of in silico models in providing insight into differences between red cell function in patients with chronic and nonchronic anemia. In silico models of complex cellular processes are thus likely to aid in defining and understanding key SNPs in human pathophysiology. PMID:12421755

  15. Discrimination of Human Cell Lines by Infrared Spectroscopy and Mathematical Modeling.

    PubMed

    Zendehdel, Rezvan; H Shirazi, Farshad

    2015-01-01

    Variations in biochemical features are extensive among cells. Identification of marker that is specific for each cell is essential for following the differentiation of stem cell and metastatic growing. Fourier transform infrared spectroscopy (FTIR) as a biochemical analysis more focused on diagnosis of cancerous cells. In this study, commercially obtained cell lines such as Human ovarian carcinoma (A2780), Human lung adenocarcinoma (A549) and Human hepatocarcinoma (HepG2) cell lines in 20 individual samples for each cell lines were used for FTIR spectral measurements. Data dimension were reduced through principal component analysis (PCA) and then subjected to neural network and linear discrimination analysis to classify FTIR pattern in different cell lines. The results showed dramatic changes of FTIR spectra among different cell types. These appeared to be associated with changes in lipid bands from CH2 symmetric and asymmetric bands, as well as amide I and amid II bands of proteins. The PCA-ANN analysis provided over 90% accuracy for classifying the spectrum of lipid section in different cell lines. This work supports future study to establish the data bank of FTIR feature for different cells and move forward to tissues as more complex systems. PMID:26330868

  16. Characterization of Nipah virus infection in a model of human airway epithelial cells cultured at an air-liquid interface.

    PubMed

    Escaffre, Olivier; Borisevich, Viktoriya; Vergara, Leoncio A; Wen, Julie W; Long, Dan; Rockx, Barry

    2016-05-01

    Nipah virus (NiV) is an emerging paramyxovirus that can cause lethal respiratory illness in humans. No vaccine/therapeutic is currently licensed for humans. Human-to-human transmission was previously reported during outbreaks and NiV could be isolated from respiratory secretions, but the proportion of cases in Malaysia exhibiting respiratory symptoms was significantly lower than that in Bangladesh. Previously, we showed that primary human basal respiratory epithelial cells are susceptible to both NiV-Malaysia (M) and -Bangladesh (B) strains causing robust pro-inflammatory responses. However, the cells of the human respiratory epithelium that NiV targets are unknown and their role in NiV transmission and NiV-related lung pathogenesis is still poorly understood. Here, we characterized NiV infection of the human respiratory epithelium using a model of the human tracheal/bronchial (B-ALI) and small airway (S-ALI) epithelium cultured at an air-liquid interface. We show that NiV-M and NiV-B infect ciliated and secretory cells in B/S-ALI, and that infection of S-ALI, but not B-ALI, results in disruption of the epithelium integrity and host responses recruiting human immune cells. Interestingly, NiV-B replicated more efficiently in B-ALI than did NiV-M. These results suggest that the human tracheal/bronchial epithelium is favourable to NiV replication and shedding, while inducing a limited host response. Our data suggest that the small airways epithelium is prone to inflammation and lesions as well as constituting a point of virus entry into the pulmonary vasculature. The use of relevant models of the human respiratory tract, such as B/S-ALI, is critical for understanding NiV-related lung pathogenesis and identifying the underlying mechanisms allowing human-to-human transmission. PMID:26932515

  17. Generation of CD34+ cells from human embryonic stem cells using a clinically applicable methodology and engraftment in the fetal sheep model

    PubMed Central

    Kim, Jaehyup; Zanjani, Esmail D.; Jeanblanc, Christine M.; Goodrich, A. Daisy; Hematti, Peiman

    2013-01-01

    Until now, ex vivo generation of CD34+ hematopoietic stem cells (HSCs) from human embryonic stem cells (hESCs) mostly involved use of feeder cells of non-human origin. While they provided invaluable models to study hematopoiesis, in vivo engraftment of hESC-derived HSCs remains a challenging task. In this study, we used a novel coculture system comprised of human bone marrow derived mesenchymal stromal/stem cells (MSCs) and peripheral blood CD14+ monocyte-derived macrophages to generate CD34+ cells from hESCs in vitro. Human ESC-derived CD34+ cells generated using this method expressed surface makers associated with adult human HSCs and up-regulated hematopoietic stem cell genes compared to human bone marrow-derived CD34+ cells. Finally, transplantation of purified hESC-derived CD34+ cells into the pre-immune fetal sheep, primed with transplantation of MSCs derived from the same hESC line, demonstrated multi-lineage hematopoietic activity with graft presence up to 16 weeks post-transplantation. This in vivo demonstration of engraftment and robust multi-lineage hematopoietic activity by hESC-derived CD34+ cells lends credence to the translational value and potential clinical utility of this novel differentiation and transplantation protocol. PMID:23612043

  18. Malva sylvestris Inhibits Inflammatory Response in Oral Human Cells. An In Vitro Infection Model

    PubMed Central

    Benso, Bruna; Rosalen, Pedro Luiz; Alencar, Severino Matias; Murata, Ramiro Mendonça

    2015-01-01

    The aim of this study was to investigate the in vitro anti-inflammatory activity of Malva sylvestris extract (MSE) and fractions in a co-culture model of cells infected by Aggregatibacter actinomycetemcomitans. In addition, we evaluated the phytochemical content in the extract and fractions of M. sylvestris and demonstrated that polyphenols were the most frequent group in all samples studied. An in vitro dual-chamber model to mimic the periodontal structure was developed using a monolayer of epithelial keratinocytes (OBA-9) and a subepithelial layer of fibroblasts (HGF-1). The invasive periodontopathogen A. actinomycetemcomitans (D7S-1) was applied to migrate through the cell layers and induce the synthesis of immune factors and cytokines in the host cells. In an attempt to analyze the antimicrobial properties of MSE and fractions, a susceptibility test was carried out. The extract (MIC 175 μg/mL, MBC 500μg/mL) and chloroform fraction (MIC 150 μg/mL, MBC 250 μg/mL) were found to have inhibitory activity. The extract and all fractions were assessed using a cytotoxicity test and results showed that concentrations under 100 μg/mL did not significantly reduce cell viability compared to the control group (p > 0.05, viability > 90%). In order to analyze the inflammatory response, transcriptional factors and cytokines were quantified in the supernatant released from the cells. The chloroform fraction was the most effective in reducing the bacterial colonization (p< 0.05) and controlling inflammatory mediators, and promoted the down-regulation of genes including IL-1beta, IL-6, IL-10, CD14, PTGS, MMP-1 and FOS as well as the reduction of the IL-1beta, IL-6, IL-8 and GM-CSF protein levels (p< 0.05). Malva sylvestris and its chloroform fraction minimized the A. actinomycetemcomitans infection and inflammation processes in oral human cells by a putative pathway that involves important cytokines and receptors. Therefore, this natural product may be considered as a

  19. Malva sylvestris Inhibits Inflammatory Response in Oral Human Cells. An In Vitro Infection Model.

    PubMed

    Benso, Bruna; Rosalen, Pedro Luiz; Alencar, Severino Matias; Murata, Ramiro Mendonça

    2015-01-01

    The aim of this study was to investigate the in vitro anti-inflammatory activity of Malva sylvestris extract (MSE) and fractions in a co-culture model of cells infected by Aggregatibacter actinomycetemcomitans. In addition, we evaluated the phytochemical content in the extract and fractions of M. sylvestris and demonstrated that polyphenols were the most frequent group in all samples studied. An in vitro dual-chamber model to mimic the periodontal structure was developed using a monolayer of epithelial keratinocytes (OBA-9) and a subepithelial layer of fibroblasts (HGF-1). The invasive periodontopathogen A. actinomycetemcomitans (D7S-1) was applied to migrate through the cell layers and induce the synthesis of immune factors and cytokines in the host cells. In an attempt to analyze the antimicrobial properties of MSE and fractions, a susceptibility test was carried out. The extract (MIC 175 μg/mL, MBC 500μg/mL) and chloroform fraction (MIC 150 μg/mL, MBC 250 μg/mL) were found to have inhibitory activity. The extract and all fractions were assessed using a cytotoxicity test and results showed that concentrations under 100 μg/mL did not significantly reduce cell viability compared to the control group (p > 0.05, viability > 90%). In order to analyze the inflammatory response, transcriptional factors and cytokines were quantified in the supernatant released from the cells. The chloroform fraction was the most effective in reducing the bacterial colonization (p< 0.05) and controlling inflammatory mediators, and promoted the down-regulation of genes including IL-1beta, IL-6, IL-10, CD14, PTGS, MMP-1 and FOS as well as the reduction of the IL-1beta, IL-6, IL-8 and GM-CSF protein levels (p< 0.05). Malva sylvestris and its chloroform fraction minimized the A. actinomycetemcomitans infection and inflammation processes in oral human cells by a putative pathway that involves important cytokines and receptors. Therefore, this natural product may be considered as a

  20. 3D Human Adipose-Derived Stem Cell Clusters as a Model for In Vitro Fibrosis.

    PubMed

    Rajangam, Thanavel; Park, Min Hee; Kim, Sang-Heon

    2016-07-01

    Excessive extracellular matrix (ECM) deposition is a cause of progressive fibrosis, which ultimately leads to progressive organ dysfunction. The lack of an in vitro fibrosis model and in vitro drug screening tools limits the development of effective antifibrotic drugs. The profibrotic cytokine transforming growth factor-β1 (TGF-β1), which is secreted by a variety of cells under continuous hypoxic condition, correlates strongly with tissue fibrosis and is largely responsible for the observed increases in ECM deposition in fibrotic diseases. In this study, we established an in vitro fibrosis model in which human adipose-derived stem cells (hASCs) secrete TGF-β1 by engineering three-dimensional cell masses (3DCMs) of hASCs on a maltose-binding protein-basic fibroblast growth factor (MBP-FGF2)-immobilized substrate. We found that the hypoxic microenvironment created in the interior of 3DCMs during the early stages of culture leads to activation and synthesis of TGF-β1. The gene expression of fibrosis-related molecules such as TGF-β1, α-smooth muscle actin (αSMA), and collagen type I was upregulated in 3DCMs. As culture time increased, overexpression of TGF-β1 led to differentiation of hASCs into activated myofibroblasts, which accumulate excessive collagen type I and are characterized by αSMA expression. Furthermore, immunofluorescence data verified the increase in collagen type I synthesis in αSMA-positive cells. Scanning electron microscopy revealed rigid and compact 3DCMs, probably due to accumulation of ECM components and cross-linking of these components. The advantage of this TGF-β1-mediated 3D in vitro fibrosis model is that it opens up new avenues to understand the common mechanism of fibrosis, which will then facilitate the development of broadly effective antifibrotic compounds and the screening of existing antifibrotic agents. To the best of our knowledge, this is the first proper biomimetic 3D in vitro fibrosis model to be developed. PMID

  1. Chick embryo xenograft model reveals a novel perineural niche for human adipose-derived stromal cells

    PubMed Central

    Cordeiro, Ingrid R.; Lopes, Daiana V.; Abreu, José G.; Carneiro, Katia; Rossi, Maria I. D.; Brito, José M.

    2015-01-01

    ABSTRACT Human adipose-derived stromal cells (hADSC) are a heterogeneous cell population that contains adult multipotent stem cells. Although it is well established that hADSC have skeletal potential in vivo in adult organisms, in vitro assays suggest further differentiation capacity, such as into glia. Thus, we propose that grafting hADSC into the embryo can provide them with a much more instructive microenvironment, allowing the human cells to adopt diverse fates or niches. Here, hADSC spheroids were grafted into either the presumptive presomitic mesoderm or the first branchial arch (BA1) regions of chick embryos. Cells were identified without previous manipulations via human-specific Alu probes, which allows efficient long-term tracing of heterogeneous primary cultures. When grafted into the trunk, in contrast to previous studies, hADSC were not found in chondrogenic or osteogenic territories up to E8. Surprisingly, 82.5% of the hADSC were associated with HNK1+ tissues, such as peripheral nerves. Human skin fibroblasts showed a smaller tropism for nerves. In line with other studies, hADSC also adopted perivascular locations. When grafted into the presumptive BA1, 74.6% of the cells were in the outflow tract, the final goal of cardiac neural crest cells, and were also associated with peripheral nerves. This is the first study showing that hADSC could adopt a perineural niche in vivo and were able to recognize cues for neural crest cell migration of the host. Therefore, we propose that xenografts of human cells into chick embryos can reveal novel behaviors of heterogeneous cell populations, such as response to migration cues. PMID:26319582

  2. Comparative Analysis of Pain Behaviours in Humanized Mouse Models of Sickle Cell Anemia.

    PubMed

    Lei, Jianxun; Benson, Barbara; Tran, Huy; Ofori-Acquah, Solomon F; Gupta, Kalpna

    2016-01-01

    Pain is a hallmark feature of sickle cell anemia (SCA) but management of chronic as well as acute pain remains a major challenge. Mouse models of SCA are essential to examine the mechanisms of pain and develop novel therapeutics. To facilitate this effort, we compared humanized homozygous BERK and Townes sickle mice for the effect of gender and age on pain behaviors. Similar to previously characterized BERK sickle mice, Townes sickle mice show more mechanical, thermal, and deep tissue hyperalgesia with increasing age. Female Townes sickle mice demonstrate more hyperalgesia compared to males similar to that reported for BERK mice and patients with SCA. Mechanical, thermal and deep tissue hyperalgesia increased further after hypoxia/reoxygenation (H/R) treatment in Townes sickle mice. Together, these data show BERK sickle mice exhibit a significantly greater degree of hyperalgesia for all behavioral measures as compared to gender- and age-matched Townes sickle mice. However, the genetically distinct "knock-in" strategy of human α and β transgene insertion in Townes mice as compared to BERK mice, may provide relative advantage for further genetic manipulations to examine specific mechanisms of pain. PMID:27494522

  3. Comparative Analysis of Pain Behaviours in Humanized Mouse Models of Sickle Cell Anemia

    PubMed Central

    Lei, Jianxun; Benson, Barbara; Tran, Huy; Ofori-Acquah, Solomon F.; Gupta, Kalpna

    2016-01-01

    Pain is a hallmark feature of sickle cell anemia (SCA) but management of chronic as well as acute pain remains a major challenge. Mouse models of SCA are essential to examine the mechanisms of pain and develop novel therapeutics. To facilitate this effort, we compared humanized homozygous BERK and Townes sickle mice for the effect of gender and age on pain behaviors. Similar to previously characterized BERK sickle mice, Townes sickle mice show more mechanical, thermal, and deep tissue hyperalgesia with increasing age. Female Townes sickle mice demonstrate more hyperalgesia compared to males similar to that reported for BERK mice and patients with SCA. Mechanical, thermal and deep tissue hyperalgesia increased further after hypoxia/reoxygenation (H/R) treatment in Townes sickle mice. Together, these data show BERK sickle mice exhibit a significantly greater degree of hyperalgesia for all behavioral measures as compared to gender- and age-matched Townes sickle mice. However, the genetically distinct “knock-in” strategy of human α and β transgene insertion in Townes mice as compared to BERK mice, may provide relative advantage for further genetic manipulations to examine specific mechanisms of pain. PMID:27494522

  4. Neural Differentiation of Human Pluripotent Stem Cells for Nontherapeutic Applications: Toxicology, Pharmacology, and In Vitro Disease Modeling

    PubMed Central

    Yap, May Shin; Nathan, Kavitha R.; Yeo, Yin; Poh, Chit Laa; Richards, Mark; Lim, Wei Ling; Othman, Iekhsan; Heng, Boon Chin

    2015-01-01

    Human pluripotent stem cells (hPSCs) derived from either blastocyst stage embryos (hESCs) or reprogrammed somatic cells (iPSCs) can provide an abundant source of human neuronal lineages that were previously sourced from human cadavers, abortuses, and discarded surgical waste. In addition to the well-known potential therapeutic application of these cells in regenerative medicine, these are also various promising nontherapeutic applications in toxicological and pharmacological screening of neuroactive compounds, as well as for in vitro modeling of neurodegenerative and neurodevelopmental disorders. Compared to alternative research models based on laboratory animals and immortalized cancer-derived human neural cell lines, neuronal cells differentiated from hPSCs possess the advantages of species specificity together with genetic and physiological normality, which could more closely recapitulate in vivo conditions within the human central nervous system. This review critically examines the various potential nontherapeutic applications of hPSC-derived neuronal lineages and gives a brief overview of differentiation protocols utilized to generate these cells from hESCs and iPSCs. PMID:26089911

  5. A Recently Established Murine Model of Nasal Polyps Demonstrates Activation of B Cells, as Occurs in Human Nasal Polyps.

    PubMed

    Kim, Dong-Young; Lee, Sun Hye; Carter, Roderick G; Kato, Atsushi; Schleimer, Robert P; Cho, Seong H

    2016-08-01

    Animal model systems are invaluable for examining human diseases. Our laboratory recently established a mouse model of nasal polyps (NPs) and investigated similarities and differences between this mouse model and human NPs. We especially focus on the hypothesis that B cell activation occurs during NP generation in the murine model. After induction of ovalbumin-induced allergic rhinosinusitis, 6% ovalbumin and Staphylococcus aureus enterotoxin B (10 ng) were instilled into the nasal cavity of mice three times per week for 8 weeks. The development of structures that somewhat resemble NPs (which we will refer to as NPs) was confirmed by hematoxylin and eosin staining. The mRNA and protein levels of various inflammatory cell markers and mediators were measured by real-time PCR in nasal tissue and by ELISA in nasal lavage fluid (NLF), respectively. Total Ig isotype levels in NLF were also quantitated using the Mouse Ig Isotyping Multiplex kit (EMD Millipore, Billerica, MA) on a Luminex 200 instrument (Life Technologies, Grand Island, NY). Similar to human NPs, there were significant increases in gene expression of inflammatory cell markers, such as CD19, CD138, CD11c, and mast cell protease-6 in nasal tissue samples of the NP group compared with those of the control group. In further investigations of B cell activation, mRNA expressions of B cell activating factor and a proliferation-inducing ligand were found to be significantly increased in mouse NP tissue. B cell-activating factor protein concentration and IgA and IgG1 levels in NLF were significantly higher in the NP group compared with the control group. In this study, the NP mouse model demonstrated enhanced B cell responses, which are reminiscent of B cell responses in human NPs. PMID:27163839

  6. Human pulmonary artery endothelial cells in the model of mucopolysaccharidosis VI present a prohypertensive phenotype

    PubMed Central

    Golda, Adam; Jurecka, Agnieszka; Gajda, Karolina; Tylki-Szymańska, Anna; Lalik, Anna

    2015-01-01

    Background Mucopolysaccharidosis type VI (MPS VI) is an autosomal recessive lysosomal disorder caused by a deficient activity of N-acetylgalactosamine-4-sulfatase (ARSB). Pulmonary hypertension (PH) occurs in MPS VI patients and is a marker of bad prognosis. Malfunction of endothelium, which regulates vascular tonus and stimulates angiogenesis, can contribute to the occurrence of PH in MPS VI. Aim The aim of the study was to establish a human MPS VI cellular model of pulmonary artery endothelial cells (HPAECs) and evaluate how it affects factors that may trigger PH such as proliferation, apoptosis, expression of endothelial nitric oxide synthase (eNOS), natriuretic peptide type C (NPPC), and vascular endothelial growth factor A (VEGFA). Results Increasing concentrations of dermatan sulfate (DS) reduce the viability of the cells in both ARSB deficiency and controls, but hardly influence apoptosis. The expression of eNOS in HPAECs is reduced up to two thirds in the presence of DS. NPPC shows a biphasic expression reaction with an increase at 50 μg/mL DS and reduction at 0 and 100 μg/mL DS. The expression of VEGFA decreases with increasing DS concentrations and absence of elastin, and increases with increasing DS in the presence of elastin. Conclusion Our data suggest that MPS VI endothelium presents a prohypertensive phenotype due to the reduction of endothelium's proliferation ability and expression of vasorelaxing factors. PMID:26937388

  7. Generation of CD34+ cells from human embryonic stem cells using a clinically applicable methodology and engraftment in the fetal sheep model.

    PubMed

    Kim, Jaehyup; Zanjani, Esmail D; Jeanblanc, Christine M; Goodrich, A Daisy; Hematti, Peiman

    2013-08-01

    Until now, ex vivo generation of CD34(+) hematopoietic stem cells (HSCs) from human embryonic stem cells (hESCs) mostly involved use of feeder cells of nonhuman origin. Although they provided invaluable models to study hematopoiesis, in vivo engraftment of hESC-derived HSCs remains a challenging task. In this study, we used a novel coculture system composed of human bone marrow-derived mesenchymal stromal/stem cells (MSCs) and peripheral blood CD14(+) monocyte-derived macrophages to generate CD34(+) cells from hESCs in vitro. Human ESC-derived CD34(+) cells generated using this method expressed surface makers associated with adult human HSCs and upregulated hematopoietic stem cell genes comparable to human bone marrow-derived CD34(+) cells. Finally, transplantation of purified hESC-derived CD34(+) cells into the preimmune fetal sheep, primed with transplantation of MSCs derived from the same hESC line, demonstrated multilineage hematopoietic activity with graft presence up to 16 weeks after transplantation. This in vivo demonstration of engraftment and robust multilineage hematopoietic activity by hESC-derived CD34(+) cells lends credence to the translational value and potential clinical utility of this novel differentiation and transplantation protocol. PMID:23612043

  8. Natural Killer Cells Improve Hematopoietic Stem Cell Engraftment by Increasing Stem Cell Clonogenicity In Vitro and in a Humanized Mouse Model.

    PubMed

    Escobedo-Cousin, Michelle; Jackson, Nicola; Laza-Briviesca, Raquel; Ariza-McNaughton, Linda; Luevano, Martha; Derniame, Sophie; Querol, Sergio; Blundell, Michael; Thrasher, Adrian; Soria, Bernat; Cooper, Nichola; Bonnet, Dominique; Madrigal, Alejandro; Saudemont, Aurore

    2015-01-01

    Cord blood (CB) is increasingly used as a source of hematopoietic stem cells (HSC) for transplantation. Low incidence and severity of graft-versus-host disease (GvHD) and a robust graft-versus-leukemia (GvL) effect are observed following CB transplantation (CBT). However, its main disadvantages are a limited number of HSC per unit, delayed immune reconstitution and a higher incidence of infection. Unmanipulated grafts contain accessory cells that may facilitate HSC engraftment. Therefore, the effects of accessory cells, particularly natural killer (NK) cells, on human CB HSC (CBSC) functions were assessed in vitro and in vivo. CBSC cultured with autologous CB NK cells showed higher levels of CXCR4 expression, a higher migration index and a higher number of colony forming units (CFU) after short-term and long-term cultures. We found that CBSC secreted CXCL9 following interaction with CB NK cells. In addition, recombinant CXCL9 increased CBSC clonogenicity, recapitulating the effect observed of CB NK cells on CBSC. Moreover, the co-infusion of CBSC with CB NK cells led to a higher level of CBSC engraftment in NSG mouse model. The results presented in this work offer the basis for an alternative approach to enhance HSC engraftment that could improve the outcome of CBT. PMID:26465138

  9. Natural Killer Cells Improve Hematopoietic Stem Cell Engraftment by Increasing Stem Cell Clonogenicity In Vitro and in a Humanized Mouse Model

    PubMed Central

    Laza-Briviesca, Raquel; Ariza-McNaughton, Linda; Luevano, Martha; Derniame, Sophie; Querol, Sergio; Blundell, Michael; Thrasher, Adrian; Soria, Bernat; Cooper, Nichola; Bonnet, Dominique; Madrigal, Alejandro; Saudemont, Aurore

    2015-01-01

    Cord blood (CB) is increasingly used as a source of hematopoietic stem cells (HSC) for transplantation. Low incidence and severity of graft-versus-host disease (GvHD) and a robust graft-versus-leukemia (GvL) effect are observed following CB transplantation (CBT). However, its main disadvantages are a limited number of HSC per unit, delayed immune reconstitution and a higher incidence of infection. Unmanipulated grafts contain accessory cells that may facilitate HSC engraftment. Therefore, the effects of accessory cells, particularly natural killer (NK) cells, on human CB HSC (CBSC) functions were assessed in vitro and in vivo. CBSC cultured with autologous CB NK cells showed higher levels of CXCR4 expression, a higher migration index and a higher number of colony forming units (CFU) after short-term and long-term cultures. We found that CBSC secreted CXCL9 following interaction with CB NK cells. In addition, recombinant CXCL9 increased CBSC clonogenicity, recapitulating the effect observed of CB NK cells on CBSC. Moreover, the co-infusion of CBSC with CB NK cells led to a higher level of CBSC engraftment in NSG mouse model. The results presented in this work offer the basis for an alternative approach to enhance HSC engraftment that could improve the outcome of CBT. PMID:26465138

  10. Early pathogenesis of Duchenne muscular dystrophy modelled in patient-derived human induced pluripotent stem cells

    PubMed Central

    Shoji, Emi; Sakurai, Hidetoshi; Nishino, Tokiko; Nakahata, Tatsutoshi; Heike, Toshio; Awaya, Tomonari; Fujii, Nobuharu; Manabe, Yasuko; Matsuo, Masafumi; Sehara-Fujisawa, Atsuko

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a progressive and fatal muscle degenerating disease caused by a dystrophin deficiency. Effective suppression of the primary pathology observed in DMD is critical for treatment. Patient-derived human induced pluripotent stem cells (hiPSCs) are a promising tool for drug discovery. Here, we report an in vitro evaluation system for a DMD therapy using hiPSCs that recapitulate the primary pathology and can be used for DMD drug screening. Skeletal myotubes generated from hiPSCs are intact, which allows them to be used to model the initial pathology of DMD in vitro. Induced control and DMD myotubes were morphologically and physiologically comparable. However, electric stimulation of these myotubes for in vitro contraction caused pronounced calcium ion (Ca2+) influx only in DMD myocytes. Restoration of dystrophin by the exon-skipping technique suppressed this Ca2+ overflow and reduced the secretion of creatine kinase (CK) in DMD myotubes. These results suggest that the early pathogenesis of DMD can be effectively modelled in skeletal myotubes induced from patient-derived iPSCs, thereby enabling the development and evaluation of novel drugs. PMID:26290039

  11. Human iPS cell models of Jervell and Lange-Nielsen syndrome

    PubMed Central

    Bellin, Milena; Greber, Boris

    2015-01-01

    Recessive mutations in the ion channel-encoding KCNQ1 gene may cause Jervell and Lange-Nielsen syndrome (JLNS), a fatal cardiac disease leading to arrhythmia and sudden cardiac death in young patients. Mutations in KCNQ1 may also cause a milder and dominantly inherited form of the disease, long QT syndrome 1 (LQT1). However, why some mutations cause LQT1 and others cause JLNS can often not be understood a priori. In a recent study,1 we have generated human induced pluripotent stem cell (hiPSC) models of JLNS. Our work mechanistically revealed how distinct classes of JLNS-causing genetic lesions, namely, missense and splice-site mutations, may promote the typical severe features of the disease at the cellular level. Interestingly, the JLNS models also displayed highly sensitive responses to pro-arrhythmic stresses. We hence propose JLNS hiPSCs as a powerful system for evaluating both phenotype-correcting as well as cardiotoxicity-causing drug effects. PMID:26481773

  12. Sulforhodamine 101 selectively labels human astrocytoma cells in an animal model of glioblastoma.

    PubMed

    Georges, Joseph F; Martirosyan, Nikolay L; Eschbacher, Jennifer; Nichols, Joshua; Tissot, Maya; Preul, Mark C; Feuerstein, Burt; Anderson, Trent; Spetzler, Robert F; Nakaji, Peter

    2014-05-01

    Sulforhodamine 101 (SR101) is a useful tool for immediate staining of astrocytes. We hypothesized that if the selectivity of SR101was maintained in astrocytoma cells, it could prove useful for glioma research. Cultured astrocytoma cells and acute slices from orthotopic human glioma (n=9) and lymphoma (n=6) xenografts were incubated with SR101 and imaged with confocal microscopy. A subset of slices (n=18) were counter-immunostained with glial fibrillary acidic protein and CD20 for stereological assessment of SR101 co-localization. SR101 differentiated astrocytic tumor cells from lymphoma cells. In acute slices, SR101 labeled 86.50% (±1.86; p<0.0001) of astrocytoma cells and 2.19% (±0.47; p<0.0001) of lymphoma cells. SR101-labeled astrocytoma cells had a distinct morphology when compared with in vivo astrocytes. Immediate imaging of human astrocytoma cells in vitro and in ex vivo rodent xenograft tissue labeled with SR101 can identify astrocytic tumor cells and help visualize the tumor margin. These features are useful in studying astrocytoma in the laboratory and may have clinical applications. PMID:24666692

  13. Human organotypic retinal cultures (HORCs) as a chronic experimental model for investigation of retinal ganglion cell degeneration.

    PubMed

    Osborne, Andrew; Hopes, Marina; Wright, Phillip; Broadway, David C; Sanderson, Julie

    2016-02-01

    There is a growing need for models of human diseases that utilise native, donated human tissue in order to model disease processes and develop novel therapeutic strategies. In this paper we assessed the suitability of adult human retinal explants as a potential model of chronic retinal ganglion cell (RGC) degeneration. Our results confirmed that RGC markers commonly used in rodent studies (NeuN, βIII Tubulin and Thy-1) were appropriate for labelling human RGCs and followed the expected differential expression patterns across, as well as throughout, the macular and para-macular regions of the retina. Furthermore, we showed that neither donor age nor post-mortem time (within 24 h) significantly affected the initial expression levels of RGC markers. In addition, the feasibility of using human post mortem donor tissue as a long-term model of RGC degeneration was determined with RGC protein being detectable up to 4 weeks in culture with an associated decline in RGC mRNA and significant, progressive, apoptotic labelling of NeuN(+) cells. Differences in RGC apoptosis might have been influenced by medium compositions indicating that media constituents could play a role in supporting axotomised RGCs. We propose that using ex vivo human explants may prove to be a useful model for testing the effectiveness of neuroprotective strategies. PMID:26432917

  14. Human B-cell cancer cell lines as a preclinical model for studies of drug effect in diffuse large B-cell lymphoma and multiple myeloma.

    PubMed

    Laursen, Maria Bach; Falgreen, Steffen; Bødker, Julie Støve; Schmitz, Alexander; Kjeldsen, Malene Krag; Sørensen, Suzette; Madsen, Jakob; El-Galaly, Tarec Christoffer; Bøgsted, Martin; Dybkær, Karen; Johnsen, Hans Erik

    2014-11-01

    Drug resistance in cancer refers to recurrent or primary refractory disease following drug therapy. At the cellular level, it is a consequence of molecular functions that ultimately enable the cell to resist cell death-one of the classical hallmarks of cancer. Thus, drug resistance is a fundamental aspect of the cancer cell phenotype, in parallel with sustained proliferation, immortality, angiogenesis, invasion, and metastasis. Here we present a preclinical model of human B-cell cancer cell lines used to identify genes involved in specific drug resistance. This process includes a standardized technical setup for specific drug screening, analysis of global gene expression, and the statistical considerations required to develop resistance gene signatures. The state of the art is illustrated by the first-step classical drug screen (including the CD20 antibody rituximab, the DNA intercalating topoisomerase II inhibitor doxorubicin, the mitotic inhibitor vincristine, and the alkylating agents cyclophosphamide and melphalan) along with the generation of gene lists predicting the chemotherapeutic outcome as validated retrospectively in clinical trial datasets. This B-cell lineage-specific preclinical model will allow us to initiate a range of laboratory studies, with focus on specific gene functions involved in molecular resistance mechanisms. PMID:25072621

  15. Importance of B cell co-stimulation in CD4(+) T cell differentiation: X-linked agammaglobulinaemia, a human model.

    PubMed

    Martini, H; Enright, V; Perro, M; Workman, S; Birmelin, J; Giorda, E; Quinti, I; Lougaris, V; Baronio, M; Warnatz, K; Grimbacher, B

    2011-06-01

    We were interested in the question of whether the congenital lack of B cells actually had any influence on the development of the T cell compartment in patients with agammaglobulinaemia. Sixteen patients with X-linked agammaglobulinaemia (XLA) due to mutations in Btk, nine patients affected by common variable immune deficiency (CVID) with <2% of peripheral B cells and 20 healthy volunteers were enrolled. The T cell phenotype was determined with FACSCalibur and CellQuest Pro software. Mann-Whitney two-tailed analysis was used for statistical analysis. The CD4 T cell memory compartment was reduced in patients with XLA of all ages. This T cell subset encompasses both CD4(+)CD45RO(+) and CD4(+)CD45RO(+)CXCR5(+) cells and both subsets were decreased significantly when compared to healthy controls: P = 0·001 and P < 0·0001, respectively. This observation was confirmed in patients with CVID who had <2% B cells, suggesting that not the lack of Bruton's tyrosine kinase but the lack of B cells is most probably the cause of the impaired CD4 T cell maturation. We postulate that this defect is a correlate of the observed paucity of germinal centres in XLA. Our results support the importance of the interplay between B and T cells in the germinal centre for the activation of CD4 T cells in humans. PMID:21488866

  16. Importance of B cell co-stimulation in CD4+ T cell differentiation: X-linked agammaglobulinaemia, a human model

    PubMed Central

    Martini, H; Enright, V; Perro, M; Workman, S; Birmelin, J; Giorda, E; Quinti, I; Lougaris, V; Baronio, M; Warnatz, K; Grimbacher, B

    2011-01-01

    We were interested in the question of whether the congenital lack of B cells actually had any influence on the development of the T cell compartment in patients with agammaglobulinaemia. Sixteen patients with X-linked agammaglobulinaemia (XLA) due to mutations in Btk, nine patients affected by common variable immune deficiency (CVID) with <2% of peripheral B cells and 20 healthy volunteers were enrolled. The T cell phenotype was determined with FACSCalibur and CellQuest Pro software. Mann–Whitney two-tailed analysis was used for statistical analysis. The CD4 T cell memory compartment was reduced in patients with XLA of all ages. This T cell subset encompasses both CD4+CD45RO+ and CD4+CD45RO+CXCR5+ cells and both subsets were decreased significantly when compared to healthy controls: P = 0·001 and P < 0·0001, respectively. This observation was confirmed in patients with CVID who had <2% B cells, suggesting that not the lack of Bruton's tyrosine kinase but the lack of B cells is most probably the cause of the impaired CD4 T cell maturation. We postulate that this defect is a correlate of the observed paucity of germinal centres in XLA. Our results support the importance of the interplay between B and T cells in the germinal centre for the activation of CD4 T cells in humans. PMID:21488866

  17. Predictivity of dog co-culture model, primary human hepatocytes and HepG2 cells for the detection of hepatotoxic drugs in humans

    SciTech Connect

    Atienzar, Franck A.; Novik, Eric I.; Gerets, Helga H.; Parekh, Amit; Delatour, Claude; Cardenas, Alvaro; MacDonald, James; Yarmush, Martin L.; Dhalluin, Stéphane

    2014-02-15

    Drug Induced Liver Injury (DILI) is a major cause of attrition during early and late stage drug development. Consequently, there is a need to develop better in vitro primary hepatocyte models from different species for predicting hepatotoxicity in both animals and humans early in drug development. Dog is often chosen as the non-rodent species for toxicology studies. Unfortunately, dog in vitro models allowing long term cultures are not available. The objective of the present manuscript is to describe the development of a co-culture dog model for predicting hepatotoxic drugs in humans and to compare the predictivity of the canine model along with primary human hepatocytes and HepG2 cells. After rigorous optimization, the dog co-culture model displayed metabolic capacities that were maintained up to 2 weeks which indicates that such model could be also used for long term metabolism studies. Most of the human hepatotoxic drugs were detected with a sensitivity of approximately 80% (n = 40) for the three cellular models. Nevertheless, the specificity was low approximately 40% for the HepG2 cells and hepatocytes compared to 72.7% for the canine model (n = 11). Furthermore, the dog co-culture model showed a higher superiority for the classification of 5 pairs of close structural analogs with different DILI concerns in comparison to both human cellular models. Finally, the reproducibility of the canine system was also satisfactory with a coefficient of correlation of 75.2% (n = 14). Overall, the present manuscript indicates that the dog co-culture model may represent a relevant tool to perform chronic hepatotoxicity and metabolism studies. - Highlights: • Importance of species differences in drug development. • Relevance of dog co-culture model for metabolism and toxicology studies. • Hepatotoxicity: higher predictivity of dog co-culture vs HepG2 and human hepatocytes.

  18. Neural Stem Cell or Human Induced Pluripotent Stem Cell-Derived GABA-ergic Progenitor Cell Grafting in an Animal Model of Chronic Temporal Lobe Epilepsy.

    PubMed

    Upadhya, Dinesh; Hattiangady, Bharathi; Shetty, Geetha A; Zanirati, Gabriele; Kodali, Maheedhar; Shetty, Ashok K

    2016-01-01

    Grafting of neural stem cells (NSCs) or GABA-ergic progenitor cells (GPCs) into the hippocampus could offer an alternative therapy to hippocampal resection in patients with drug-resistant chronic epilepsy, which afflicts >30% of temporal lobe epilepsy (TLE) cases. Multipotent, self-renewing NSCs could be expanded from multiple regions of the developing and adult brain, human embryonic stem cells (hESCs), and human induced pluripotent stem cells (hiPSCs). On the other hand, GPCs could be generated from the medial and lateral ganglionic eminences of the embryonic brain and from hESCs and hiPSCs. To provide comprehensive methodologies involved in testing the efficacy of transplantation of NSCs and GPCs in a rat model of chronic TLE, NSCs derived from the rat medial ganglionic eminence (MGE) and MGE-like GPCs derived from hiPSCs are taken as examples in this unit. The topics comprise description of the required materials, reagents and equipment, methods for obtaining rat MGE-NSCs and hiPSC-derived MGE-like GPCs in culture, generation of chronically epileptic rats, intrahippocampal grafting procedure, post-grafting evaluation of the effects of grafts on spontaneous recurrent seizures and cognitive and mood impairments, analyses of the yield and the fate of graft-derived cells, and the effects of grafts on the host hippocampus. © 2016 by John Wiley & Sons, Inc. PMID:27532817

  19. Androgen regulation of the human FERM domain encoding gene EHM2 in a cell model of steroid-induced differentiation

    PubMed Central

    Chauhan, Sanjay; Pandey, Ritu; Way, Jeffrey F.; Sroka, Thomas C.; Demetriou, Manolis C.; Kunz, Susan; Cress, Anne E.; Mount, David W.; Miesfeld, Roger L.

    2009-01-01

    We have developed a cell model to investigate steroid control of differentiation using a subline of HT1080 cells (HT-AR1) that have been engineered to express the human androgen receptor. Dihydrotestosterone (DHT) treatment of HT-AR1 cells induced growth arrest and cytoskeletal reorganization that was associated with the expression of fibronectin and the neuroendocrine markers chromogranin A and neuron-specific enolase. Expression profiling analysis identified the human FERM domain-encoding gene EHM2 as uniquely induced in HT-AR1 cells as compared to 16 other FERM domain containing genes. Since FERM domain proteins control cytoskeletal functions in differentiating cells, and the human EHM2 gene has not been characterized, we investigated EHM2 steroid-regulation, genomic organization, and sequence conservation. We found that DHT, but not dexamethasone, induced the expression of a 3.8 kb transcript in HT-AR1 cells encoding a 504 amino acid protein, and moreover, that human brain tissue contains a 5.8 kb transcript encoding a 913 amino acid isoform. Construction of an unrooted phylogenetic tree using 98 FERM domain proteins revealed that the human EHM2 gene is a member of a distinct subfamily consisting of nine members, all of which contain a highly conserved 325 amino acid FERM domain. PMID:14521927

  20. Androgen regulation of the human FERM domain encoding gene EHM2 in a cell model of steroid-induced differentiation.

    PubMed

    Chauhan, Sanjay; Pandey, Ritu; Way, Jeffrey F; Sroka, Thomas C; Demetriou, Manolis C; Kunz, Susan; Cress, Anne E; Mount, David W; Miesfeld, Roger L

    2003-10-17

    We have developed a cell model to investigate steroid control of differentiation using a subline of HT1080 cells (HT-AR1) that have been engineered to express the human androgen receptor. Dihydrotestosterone (DHT) treatment of HT-AR1 cells induced growth arrest and cytoskeletal reorganization that was associated with the expression of fibronectin and the neuroendocrine markers chromogranin A and neuron-specific enolase. Expression profiling analysis identified the human FERM domain-encoding gene EHM2 as uniquely induced in HT-AR1 cells as compared to 16 other FERM domain containing genes. Since FERM domain proteins control cytoskeletal functions in differentiating cells, and the human EHM2 gene has not been characterized, we investigated EHM2 steroid-regulation, genomic organization, and sequence conservation. We found that DHT, but not dexamethasone, induced the expression of a 3.8 kb transcript in HT-AR1 cells encoding a 504 amino acid protein, and moreover, that human brain tissue contains a 5.8 kb transcript encoding a 913 amino acid isoform. Construction of an unrooted phylogenetic tree using 98 FERM domain proteins revealed that the human EHM2 gene is a member of a distinct subfamily consisting of nine members, all of which contain a highly conserved 325 amino acid FERM domain. PMID:14521927

  1. Human Embryonic Stem Cells: A Model for the Study of Neural Development and Neurological Diseases

    PubMed Central

    Prajumwongs, Piya; Weeranantanapan, Oratai; Jaroonwitchawan, Thiranut; Noisa, Parinya

    2016-01-01

    Although the mechanism of neurogenesis has been well documented in other organisms, there might be fundamental differences between human and those species referring to species-specific context. Based on principles learned from other systems, it is found that the signaling pathways required for neural induction and specification of human embryonic stem cells (hESCs) recapitulated those in the early embryo development in vivo at certain degree. This underscores the usefulness of hESCs in understanding early human neural development and reinforces the need to integrate the principles of developmental biology and hESC biology for an efficient neural differentiation. PMID:27239201

  2. Use of human embryonic stem cells to model pediatric gliomas with H3.3K27M histone mutation.

    PubMed

    Funato, Kosuke; Major, Tamara; Lewis, Peter W; Allis, C David; Tabar, Viviane

    2014-12-19

    Over 70% of diffuse intrinsic pediatric gliomas, an aggressive brainstem tumor, harbor heterozygous mutations that create a K27M amino acid substitution (methionine replaces lysine 27) in the tail of histone H3.3. The role of the H3.3K27M mutation in tumorigenesis is not fully understood. Here, we use a human embryonic stem cell system to model this tumor. We show that H3.3K27M expression synergizes with p53 loss and PDGFRA activation in neural progenitor cells derived from human embryonic stem cells, resulting in neoplastic transformation. Genome-wide analyses indicate a resetting of the transformed precursors to a developmentally more primitive stem cell state, with evidence of major modifications of histone marks at several master regulator genes. Drug screening assays identified a compound targeting the protein menin as an inhibitor of tumor cell growth in vitro and in mice. PMID:25525250

  3. Use of human embryonic stem cells to model pediatric gliomas with H3.3K27M histone mutation

    PubMed Central

    Funato, Kosuke; Major, Tamara; Lewis, Peter W.; Allis, C. David; Tabar, Viviane

    2016-01-01

    Over 70% of diffuse intrinsic pediatric gliomas, an aggressive brainstem tumor, harbor heterozygous mutations that create a K27M amino acid substitution (methionine replaces lysine 27) in the tail of histone H3.3. The role of the H3.3K27M mutation in tumorigenesis not fully understood. Here, we use a human embryonic stem cell system to model this tumor. We show that H3.3K27M expression synergizes with p53 loss and PDGFRA activation in neural progenitor cells derived from human embryonic stem cells, resulting in neoplastic transformation. Genome-wide analyses indicate a resetting of the transformed precursors to a developmentally more primitive stem cell state, with evidence of major modifications of histone marks at several master regulator genes. Drug screening assays identified a compound targeting the protein menin as an inhibitor of tumor cell growth in vitro and in mice. PMID:25525250

  4. [Primary cultures of human umbilical chord vein endothelial cells: a biological model for studying enterococcal infection mechanisms].

    PubMed

    Chiriboga, Carlos Andrés; Fontanilla, Marta Raquel

    2004-12-01

    Although enterococcus bacteria are normal human intestinal flora, they rank as the third most common pathogen involved in hospital acquired infections. Generally, these bacteria are considered extracellular pathogens; however, an increasing number of reports indicate invasiveness to epithelial cell lines and macrophages. Despite their importance as nosocomial infection agents in patients suffering bacteremias and endocarditis, their interaction with endothelial cells has not been fully described. Herein, the nosocomial Enterococcus faecalis isolate Ef2890 from a hospitalized patient was exposed to cultured human venous endothelial cells from the umbilical chord. When the primary cell cultures were inoculated with Ef2890 and treated with bactericidal antibiotics to kill extracellular and adhered bacteria, intracellular bacteria were recovered and plated 4 h post-infection. These observations indicate that cell cultures provide a valuable biological model to study interactions between endothelium and enterococci. PMID:15678808

  5. Induced pluripotent stem cell-derived neuron as a human model for testing environmentally induced developmental neurotoxicity

    EPA Science Inventory

    Induced pluripotent stem cell-derived neurons as a human model for testing environmentally induced developmental neurotoxicity Ingrid L. Druwe1, Timothy J. Shafer2, Kathleen Wallace2, Pablo Valdivia3 ,and William R. Mundy2. 1University of North Carolina, Curriculum in Toxicology...

  6. Naltrindole inhibits human multiple myeloma cell proliferation in vitro and in a murine xenograft model in vivo.

    PubMed

    Mundra, Jyoti Joshi; Terskiy, Alexandra; Howells, Richard D

    2012-08-01

    It has been demonstrated previously that immune cell activation and proliferation were sensitive to the effects of naltrindole, a nonpeptidic δ-opioid receptor-selective antagonist; therefore, we hypothesized that human multiple myeloma (MM) would be a valuable model for studying potential antineoplastic properties of naltrindole. [(3)H]naltrindole exhibited saturable, low-affinity binding to intact human MM cells; however, the pharmacological profile of the binding site differed considerably from the properties of δ-, κ-, and μ-opioid receptors, and opioid receptor mRNA was not detected in MM cells by reverse transcriptase-polymerase chain reaction. Naltrindole inhibited the proliferation of cultured human U266 MM cells in a time- and dose-dependent manner with an EC(50) of 16 μM. The naltrindole-induced inhibition of U266 cell proliferation was not blocked by a 10-fold molar excess of naltrexone, a nonselective opioid antagonist. Additive inhibition of MM cell proliferation was observed when using a combination of naltrindole with the histone deacetylase inhibitor sodium valproate, the proteasome inhibitor bortezomib, the glucocorticoid receptor agonist dexamethasone, and the HMG CoA reductase inhibitor simvastatin. Treatment of U266 cells with naltrindole significantly decreased the level of the active, phosphorylated form of the kinases, extracellular signal-regulated kinase and Akt, which may be related to its antiproliferative activity. The antiproliferative activity of naltrindole toward MM cells was maintained in cocultures of MM and bone marrow-derived stromal cells, mimicking the bone marrow microenvironment. In vivo, naltrindole significantly decreased tumor cell volumes in human MM cell xenografts in severe combined immunodeficient mice. We hypothesize that naltrindole inhibits the proliferation of MM cells through a nonopioid receptor-dependent mechanism. PMID:22537770

  7. Pyrogallol induces G2-M arrest in human lung cancer cells and inhibits tumor growth in an animal model.

    PubMed

    Yang, Chih-Jen; Wang, Chuan-Sheng; Hung, Jen-Yu; Huang, Hurng-Wern; Chia, Yi-Chen; Wang, Pei-Hui; Weng, Ching-Feng; Huang, Ming-Shyan

    2009-11-01

    Pyrogallol, a catechin compound, is an active component of Emblica officinalis extracts and has an anti-proliferative effect on some human cancer cell lines. In our preliminary study, pyrogallol had highly cytotoxic effect on human lung cancer cell lines and less effect on human bronchial epithelium cell line. This study was performed to investigate the beneficial effect of pyrogallol on human lung cancer cell lines - H441 (lung adenocarcinoma) and H520 (lung squamous cell carcinoma). The MTT (cytotoxic) data showed the inhibition growth of lung cancer cells followed pyrogallol treatment. The cell cycle of lung cancer cells was arrested in G2/M phase using flow cytometry. Using Western blot analysis, the cell cycle related proteins - cyclin B1 and Cdc25c were decreased in a time-dependent manner and the phosphorylated Cdc2 (Thr14) was increased within 4h pyrogallol treatment. Moreover, the higher cleavage of poly (ADP)-ribose polymerase (PARP), the increased of Bax concurrent with the decreased of Bcl-2 indicated that pyrogallol treatment resulted in apoptosis of lung cancer cells. The cell apoptosis was also directly demonstrated using Annexin V-FITC and TUNEL stain. Additionally, the tumoricidal effect of pyrogallol was measured using a xenograft nude mice model. After 5 weeks of pyrogallol treatment could cause the regression of tumor. Taken in vitro and in vivo studies together, these results suggest that pyrogallol can be developed as a promising anti-lung cancer drug particular for the non-small cell lung cancer (NSCLC). PMID:19233505

  8. Naltrindole Inhibits Human Multiple Myeloma Cell Proliferation In Vitro and in a Murine Xenograft Model In Vivo

    PubMed Central

    Mundra, Jyoti Joshi; Terskiy, Alexandra

    2012-01-01

    It has been demonstrated previously that immune cell activation and proliferation were sensitive to the effects of naltrindole, a nonpeptidic δ-opioid receptor-selective antagonist; therefore, we hypothesized that human multiple myeloma (MM) would be a valuable model for studying potential antineoplastic properties of naltrindole. [3H]naltrindole exhibited saturable, low-affinity binding to intact human MM cells; however, the pharmacological profile of the binding site differed considerably from the properties of δ-, κ-, and μ-opioid receptors, and opioid receptor mRNA was not detected in MM cells by reverse transcriptase-polymerase chain reaction. Naltrindole inhibited the proliferation of cultured human U266 MM cells in a time- and dose-dependent manner with an EC50 of 16 μM. The naltrindole-induced inhibition of U266 cell proliferation was not blocked by a 10-fold molar excess of naltrexone, a nonselective opioid antagonist. Additive inhibition of MM cell proliferation was observed when using a combination of naltrindole with the histone deacetylase inhibitor sodium valproate, the proteasome inhibitor bortezomib, the glucocorticoid receptor agonist dexamethasone, and the HMG CoA reductase inhibitor simvastatin. Treatment of U266 cells with naltrindole significantly decreased the level of the active, phosphorylated form of the kinases, extracellular signal-regulated kinase and Akt, which may be related to its antiproliferative activity. The antiproliferative activity of naltrindole toward MM cells was maintained in cocultures of MM and bone marrow-derived stromal cells, mimicking the bone marrow microenvironment. In vivo, naltrindole significantly decreased tumor cell volumes in human MM cell xenografts in severe combined immunodeficient mice. We hypothesize that naltrindole inhibits the proliferation of MM cells through a nonopioid receptor-dependent mechanism. PMID:22537770

  9. Model of human immunodeficiency virus budding and self-assembly: Role of the cell membrane

    NASA Astrophysics Data System (ADS)

    Zhang, Rui; Nguyen, Toan T.

    2008-11-01

    Budding from the plasma membrane of the host cell is an indispensable step in the life cycle of the human immunodeficiency virus (HIV), which belongs to a large family of enveloped RNA viruses, retroviruses. Unlike regular enveloped viruses, retrovirus budding happens concurrently with the self-assembly of the main retrovirus protein subunits (called Gag protein after the name of the genetic material that codes for this protein: Group-specific AntiGen) into spherical virus capsids on the cell membrane. Led by this unique budding and assembly mechanism, we study the free energy profile of retrovirus budding, taking into account the Gag-Gag attraction energy and the membrane elastic energy. We find that if the Gag-Gag attraction is strong, budding always proceeds to completion. During early stage of budding, the zenith angle of partial budded capsids, α , increases with time as α∝t1/2 . However, if the Gag-Gag attraction is weak, a metastable state of partial budding appears. The zenith angle of these partially spherical capsids is given by α0≃(τ2/κσ)1/4 in a linear approximation, where κ and σ are the bending modulus and the surface tension of the membrane, and τ is a line tension of the capsid proportional to the strength of Gag-Gag attraction. Numerically, we find α0<0.3π without any approximations. Using experimental parameters, we show that HIV budding and assembly always proceed to completion in normal biological conditions. On the other hand, by changing Gag-Gag interaction strength or membrane rigidity, it is relatively easy to tune it back and forth between complete budding and partial budding. Our model agrees reasonably well with experiments observing partial budding of retroviruses including HIV.

  10. STAT3 signaling is activated preferentially in tumor-initiating cells in claudin-low models of human breast cancer.

    PubMed

    Wei, Wei; Tweardy, David J; Zhang, Mei; Zhang, Xiaomei; Landua, John; Petrovic, Ivana; Bu, Wen; Roarty, Kevin; Hilsenbeck, Susan G; Rosen, Jeffrey M; Lewis, Michael T

    2014-10-01

    In breast cancer, a subset of tumor-initiating cells (TIC) or "cancer stem cells" are thought to be responsible for tumor maintenance, treatment resistance, and disease recurrence. While current breast cancer stem cell markers (e.g., CD44(high) /CD24(low/neg) , ALDH positive) have allowed enrichment for such cells, they are not universally expressed and may actually identify distinct TIC subpopulations in the same tumor. Thus, additional markers of functional stem cells are needed. The STAT3 pathway is a critical regulator of the function of normal stem cells, and evidence is accumulating for its important role in breast cancer stem cells. However, due to the lack of a method for separating live cells based on their level of STAT3 activity, it remains unknown whether STAT3 functions in the cancer stem cells themselves, or in surrounding niche cells, or in both. To approach this question, we constructed a series of lentiviral fluorescent (enhanced green fluorescent protein, EGFP) reporters that enabled flow cytometric enrichment of cells differing in STAT3-mediated transcriptional activity, as well as in vivo/in situ localization of STAT3 responsive cells. Using in vivo claudin-low cell line xenograft models of human breast cancer, we found that STAT3 signaling reporter activity (EGFP(+) ) is associated with a subpopulation of cancer cells enriched for mammosphere-forming efficiency, as well as TIC function in limiting dilution transplantation assays compared to negative or unsorted populations. Our results support STAT3 signaling activity as another functional marker for human breast cancer stem cells thus making it an attractive therapeutic target for stem-cell-directed therapy in some breast cancer subtypes. PMID:24891218

  11. An Overview on Human Umbilical Cord Blood Stem Cell-Based Alternative In Vitro Models for Developmental Neurotoxicity Assessment.

    PubMed

    Singh, Abhishek Kumar; Kashyap, Mahendra Pratap

    2016-07-01

    The developing brain is found highly vulnerable towards the exposure of different environmental chemicals/drugs, even at concentrations, those are generally considered safe in mature brain. The brain development is a very complex phenomenon which involves several processes running in parallel such as cell proliferation, migration, differentiation, maturation and synaptogenesis. If any step of these cellular processes hampered due to exposure of any xenobiotic/drug, there is almost no chance of recovery which could finally result in a life-long disability. Therefore, the developmental neurotoxicity (DNT) assessment of newly discovered drugs/molecules is a very serious concern among the neurologists. Animal-based DNT models have their own limitations such as ethical concerns and lower sensitivity with less predictive values in humans. Furthermore, non-availability of human foetal brain tissues/cells makes job more difficult to understand about mechanisms involve in DNT in human beings. Although, the use of cell culture have been proven as a powerful tool for DNT assessment, but many in vitro models are currently utilizing genetically unstable cell lines. The interpretation of data generated using such terminally differentiated cells is hard to extrapolate with in vivo situations. However, human umbilical cord blood stem cells (hUCBSCs) have been proposed as an excellent tool for alternative DNT testing because neuronal development from undifferentiated state could exactly mimic the original pattern of neuronal development in foetus when hUCBSCs differentiated into neuronal cells. Additionally, less ethical concern, easy availability and high plasticity make them an attractive source for establishing in vitro model of DNT assessment. In this review, we are focusing towards recent advancements on hUCBSCs-based in vitro model to understand DNTs. PMID:26041658

  12. The modeling of Alzheimer's disease by the overexpression of mutant Presenilin 1 in human embryonic stem cells.

    PubMed

    Honda, Makoto; Minami, Itsunari; Tooi, Norie; Morone, Nobuhiro; Nishioka, Hisae; Uemura, Kengo; Kinoshita, Ayae; Heuser, John E; Nakatsuji, Norio; Aiba, Kazuhiro

    2016-01-15

    Cellular disease models are useful tools for Alzheimer's disease (AD) research. Pluripotent stem cells, including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), are promising materials for creating cellular models of such diseases. In the present study, we established cellular models of AD in hESCs that overexpressed the mutant Presenilin 1 (PS1) gene with the use of a site-specific gene integration system. The overexpression of PS1 did not affect the undifferentiated status or the neural differentiation ability of the hESCs. We found increases in the ratios of amyloid-β 42 (Aβ42)/Aβ40 and Aβ43/Aβ40. Furthermore, synaptic dysfunction was observed in a cellular model of AD that overexpressed mutant PS1. These results suggest that the AD phenotypes, in particular, the electrophysiological abnormality of the synapses in our AD models might be useful for AD research and drug discovery. PMID:26687948

  13. Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption.

    PubMed

    Sagrillo-Fagundes, Lucas; Clabault, Hélène; Laurent, Laetitia; Hudon-Thibeault, Andrée-Anne; Salustiano, Eugênia Maria Assunção; Fortier, Marlène; Bienvenue-Pariseault, Josianne; Wong Yen, Philippe; Sanderson, J Thomas; Vaillancourt, Cathy

    2016-01-01

    This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation. PMID:27500522

  14. Transplantation of human induced cerebellar granular-like cells improves motor functions in a novel mouse model of cerebellar ataxia

    PubMed Central

    Zhu, Tongming; Tang, Hailiang; Shen, Yiwen; Tang, Qisheng; Chen, Luping; Wang, Zhifu; Zhou, Ping; Xu, Feng; Zhu, Jianhong

    2016-01-01

    Stem cell-based reparative approaches have been applied to cerebellum-related disorders during the last two decades. Direct lineage reprogramming of human fibroblasts into functional granular neurons holds great promise for biomedical applications such as cerebellum regeneration and cellbased disease modeling. In the present study, we showed that a combination of Ascl1, Sox2 and OCT4, in a culture subsequently treated with secreted factors (BMP4, Wnt3a and FGF8b), was capable of converting human fibroblasts from the scalp tissue of patients with traumatic brain injury (TBI) into functional human induced cerebellar granular-like cells (hiCGCs). Morphological analysis, immunocytochemistry, gene expression and electrophysiological analysis were performed to identify the similarity of induced neuronal cells to human cerebellum granular cells. Our strategy improved the efficiency for hiCGCs induction, which gave the highest conversion efficiency 12.30±0.88%, and Ath1+/Tuj1+ double positive cells to 5.56±0.80%. We transplanted hiCGCs into the cerebellum of NmycTRE/TRE: tTS mice, a novel mouse model of cerebellar ataxia, and demonstrated that the hiCGCs were able to survive, migrate, proliferate and promote mild functional recovery after been grafted into cerebellum. PMID:27158363

  15. An in vitro-differentiated human cell line as a model system to study the interaction of Neisseria gonorrhoeae with phagocytic cells.

    PubMed Central

    Hauck, C R; Lorenzen, D; Saas, J; Meyer, T F

    1997-01-01

    The extreme host specificity of pathogenic neisseriae limits investigations aimed at the analysis of bacterial-host interactions almost completely to the use of in vitro models. Although permanent epithelial and endothelial cell lines are already indispensable tools with respect to initial infection processes, studies concerning the interaction of neisseriae with phagocytic cells have been confined to primary human blood cells. We investigated the use of human leukemia-derived monocytic and myelomonocytic cell lines that can be differentiated in vitro towards phagocytic cells by a panel of chemical and biological reagents including cytokines, vitamin analogs, and antileukemia drugs. Whereas tumor necrosis factor alpha, gamma interferon, bufalin, or granulocyte-macrophage colony-stimulating factor only marginally increased the ability of monocytic MonoMac-6 and myelomonocytic JOSK-M cells to interact with the bacteria, retinoic acid and vitamin D3 treatment for 2 to 4 days led to highly phagocytic cells that internalized gonococci in an Opa protein-specific manner. This is comparable to the phagocytosis by primary monocytes from human blood, where more than 80% of cells are infected with intracellular bacteria. The increased phagocytic activity of JOSK-M cells following in vitro differentiation was paralleled by enhanced oxidative burst capacity. Whereas undifferentiated cells responded to neither phorbol 12-myristate 13-acetate nor other known soluble and particulate stimuli, cells incubated with retinoic acid and bufalin showed the same pattern and the same intensity of oxidative burst activity in response to Neisseria gonorrhoeae as primary cells: Opa-expressing gonococci elicited an oxidative burst, whereas Opa- gonococci did not. The surface expression of major histocompatibility complex (MHC) class II molecules was only slightly changed after retinoic acid treatment. Also, phagocytosis of gonococci had no influence on MHC class II surface expression. Taken

  16. Mineralized human primary osteoblast matrices as a model system to analyse interactions of prostate cancer cells with the bone microenvironment.

    PubMed

    Reichert, Johannes C; Quent, Verena M C; Burke, Leslie J; Stansfield, Scott H; Clements, Judith A; Hutmacher, Dietmar W

    2010-11-01

    Prostate cancer metastasis is reliant on the reciprocal interactions between cancer cells and the bone niche/micro-environment. The production of suitable matrices to study metastasis, carcinogenesis and in particular prostate cancer/bone micro-environment interaction has been limited to specific protein matrices or matrix secreted by immortalised cell lines that may have undergone transformation processes altering signaling pathways and modifying gene or receptor expression. We hypothesize that matrices produced by primary human osteoblasts are a suitable means to develop an in vitro model system for bone metastasis research mimicking in vivo conditions. We have used a decellularized matrix secreted from primary human osteoblasts as a model for prostate cancer function in the bone micro-environment. We show that this collagen I rich matrix is of fibrillar appearance, highly mineralized, and contains proteins, such as osteocalcin, osteonectin and osteopontin, and growth factors characteristic of bone extracellular matrix (ECM). LNCaP and PC3 cells grown on this matrix, adhere strongly, proliferate, and express markers consistent with a loss of epithelial phenotype. Moreover, growth of these cells on the matrix is accompanied by the induction of genes associated with attachment, migration, increased invasive potential, Ca(2+) signaling and osteolysis. In summary, we show that growth of prostate cancer cells on matrices produced by primary human osteoblasts mimics key features of prostate cancer bone metastases and thus is a suitable model system to study the tumor/bone micro-environment interaction in this disease. PMID:20688384

  17. PAZ6 Cells Constitute a Representative Model for Human Brown Pre-Adipocytes

    PubMed Central

    Kazantzis, Melissa; Takahashi, Virginia; Hinkle, Jessica; Kota, Smitha; Zilberfarb, Vladimir; Issad, Tarik; Abdelkarim, Mouaadh; Chouchane, Lotfi

    2012-01-01

    The role of brown adipose tissue (BAT) in human metabolism and its potential as an anti-obesity target organ have recently received much renewed attention. Following radiological detection of substantial amounts of BAT in adults by several independent research groups, an increasing number of studies are now dedicated to uncover BAT’s genetic, developmental, and environmental determinants. In contrast to murine BAT, human BAT is not present as a single major fat depot in a well-defined location. The distribution of BAT in several areas in the body significantly limits its availability to research. A human brown adipocyte cell line is therefore critical in broadening the options available to researchers in the field. The human BAT-cell line PAZ6 was created to address such a need and has been well characterized by several research groups around the world. In the present review, we discuss their findings and propose potential applications of the PAZ6 cells in addressing the relevant questions in the BAT field, namely for future use in therapeutic applications. PMID:22649407

  18. Logic-Based and Cellular Pharmacodynamic Modeling of Bortezomib Responses in U266 Human Myeloma Cells.

    PubMed

    Chudasama, Vaishali L; Ovacik, Meric A; Abernethy, Darrell R; Mager, Donald E

    2015-09-01

    Systems models of biological networks show promise for informing drug target selection/qualification, identifying lead compounds and factors regulating disease progression, rationalizing combinatorial regimens, and explaining sources of intersubject variability and adverse drug reactions. However, most models of biological systems are qualitative and are not easily coupled with dynamical models of drug exposure-response relationships. In this proof-of-concept study, logic-based modeling of signal transduction pathways in U266 multiple myeloma (MM) cells is used to guide the development of a simple dynamical model linking bortezomib exposure to cellular outcomes. Bortezomib is a commonly used first-line agent in MM treatment; however, knowledge of the signal transduction pathways regulating bortezomib-mediated cell cytotoxicity is incomplete. A Boolean network model of 66 nodes was constructed that includes major survival and apoptotic pathways and was updated using responses to several chemical probes. Simulated responses to bortezomib were in good agreement with experimental data, and a reduction algorithm was used to identify key signaling proteins. Bortezomib-mediated apoptosis was not associated with suppression of nuclear factor κB (NFκB) protein inhibition in this cell line, which contradicts a major hypothesis of bortezomib pharmacodynamics. A pharmacodynamic model was developed that included three critical proteins (phospho-NFκB, BclxL, and cleaved poly (ADP ribose) polymerase). Model-fitted protein dynamics and cell proliferation profiles agreed with experimental data, and the model-predicted IC50 (3.5 nM) is comparable to the experimental value (1.5 nM). The cell-based pharmacodynamic model successfully links bortezomib exposure to MM cellular proliferation via protein dynamics, and this model may show utility in exploring bortezomib-based combination regimens. PMID:26163548

  19. Human hematopoietic cell lines: a model system for study of minimal residual disease detection technique in acute leukemia.

    PubMed

    Koníková, E; Kusenda, J; Babusíková, O; Glasová, M

    1995-01-01

    these surface and cytoplasmic marker combinations minor neoplastic cell populations could be detected. Human hematopoietic cell lines could serve as a reliable model system for monitoring minimal residual disease in acute leukemia patients. PMID:8552200

  20. MUTZ-3, a human cell line model for the cytokine-induced differentiation of dendritic cells from CD34+ precursors.

    PubMed

    Masterson, Allan J; Sombroek, Claudia C; De Gruijl, Tanja D; Graus, Yvo M F; van der Vliet, Hans J J; Lougheed, Sinéad M; van den Eertwegh, Alfons J M; Pinedo, Herbert M; Scheper, Rik J

    2002-07-15

    Many human myeloid leukemia-derived cell lines possess the ability to acquire a dendritic cell (DC) phenotype. However, cytokine responsiveness is generally poor, requiring direct manipulation of intracellular signaling mechanisms for differentiation. In contrast, the CD34+ human acute myeloid leukemia cell line MUTZ-3 responds to granulocyte macrophage- colony-stimulating factor (GM-CSF), interleukin 4 (IL-4), and tumor necrosis factor alpha (TNFalpha), cytokines known to be pivotal both in vivo and in vitro for DC generation from monocytes and CD34+ stem cells. In all respects, MUTZ-3 cells behave as the immortalized equivalent of CD34+ DC precursors. Upon stimulation with specific cytokine cocktails, they acquire a phenotype consistent with either interstitial- or Langerhans-like DCs and upon maturation (mDC), express CD83. MUTZ-3 DC display the full range of functional antigen processing and presentation pathways. These findings demonstrate the unique suitability of MUTZ-3 cells as an unlimited source of CD34+ DC progenitors for the study of cytokine-induced DC differentiation. PMID:12091369

  1. Sodium orthovanadate inhibits growth of human hepatocellular carcinoma cells in vitro and in an orthotopic model in vivo.

    PubMed

    Wu, Yaohua; Ma, Yong; Xu, Zhilin; Wang, Dawei; Zhao, Baolei; Pan, Huayang; Wang, Jizhou; Xu, Dongsheng; Zhao, Xiaoyang; Pan, Shangha; Liu, Lianxin; Dai, Wenjie; Jiang, Hongchi

    2014-08-28

    The transition metal vanadium is widely distributed in the environment and exhibits various biological and physiological effects in the human body. As a well known vanadium compound, sodium orthovanadate (SOV) has shown promising antineoplastic activity in several human cancers. However, the effects of SOV on liver cancer are still unknown. In this study, for the first time, we showed that SOV could effectively suppress proliferation, induce G2/M cell cycle arrest and apoptosis, and diminish the mitochondrial membrane potential (MMP) of HCC cells in vitro. In addition, our in vitro results were recapitulated in vivo, showing that SOV exhibited a dose-dependent inhibition of growth of human HCC in an orthotopic model, evidenced by the reduction in tumor size, proliferation index and microvessel density, and increase in cell apoptosis. Most important, we found that SOV could inhibit autophagy in HCC cells in vitro and in vivo, which plays a prodeath role. Thus, our findings suggest that SOV could effectively suppress the growth of human HCC through the regulations of proliferation, cell cycle, apoptosis and autophagy, and thus may act as a potential therapeutic agent in HCC treatment. PMID:24858025

  2. Three-dimensional model of angiogenesis: coculture of human retinal cells with bovine aortic endothelial cells in the NASA bioreactor.

    PubMed

    Dutt, Kamla; Sanford, Gary; Harris-Hooker, Sandra; Brako, Lawrence; Kumar, Ravindra; Sroufe, Angela; Melhado, Caroline

    2003-10-01

    Ocular angiogenesis is the leading cause of blindness and is associated with diabetic retinopathy and age-related macular degeneration. We describe, in this report, our preliminary studies using a horizontally rotating bioreactor (HRB), developed by the National Aeronautics and Space Administration (NASA), to explore growth and differentiation-associated events in the early phase of ocular angiogenesis. Human retinal (HRet) cells and bovine endothelial cells (ECs) were cocultured on laminin-coated Cytodex-3 microcarrier beads in an HRB for 1-36 days. Endothelial cells grown alone in the HRB remained cuboidal and were well differentiated. However, when HRet cells were cocultured with ECs, cordlike structures formed as early as 18-36 h and were positive for von Willebrand factor. In addition to the formation of cords and capillary-like structures, ECs showed the beginning of sprouts. The HRB seems not only to promote accelerated capillary formation, but also to enhance differentiation of retinal precursor cells. This leads to the formation of rosette-like structures (which may be aggregates of photoreceptors that were positive for rhodopsin). Upregulation of vascular endothelial growth factor and basic fibroblast growth factor was seen in retinal cells grown in the HRB as compared with monolayers and could be one of the factors responsible for accelerated capillary formation. Hence, the HRB promotes three-dimensional assembly and differentiation, possibly through promoting cell-to-cell interaction and/or secretion of growth and differentiation factors. PMID:14633374

  3. Potential large animal models for gene therapy of human genetic diseases of immune and blood cell systems.

    PubMed

    Bauer, Thomas R; Adler, Rima L; Hickstein, Dennis D

    2009-01-01

    Genetic mutations involving the cellular components of the hematopoietic system--red blood cells, white blood cells, and platelets--manifest clinically as anemia, infection, and bleeding. Although gene targeting has recapitulated many of these diseases in mice, these murine homologues are limited as translational models by their small size and brief life span as well as the fact that mutations induced by gene targeting do not always faithfully reflect the clinical manifestations of such mutations in humans. Many of these limitations can be overcome by identifying large animals with genetic diseases of the hematopoietic system corresponding to their human disease counterparts. In this article, we describe human diseases of the cellular components of the hematopoietic system that have counterparts in large animal species, in most cases carrying mutations in the same gene (CD18 in leukocyte adhesion deficiency) or genes in interacting proteins (DNA cross-link repair 1C protein and protein kinase, DNA-activated catalytic polypeptide in radiation-sensitive severe combined immunodeficiency). Furthermore, we describe the potential of these animal models to serve as disease-specific preclinical models for testing the efficacy and safety of clinical interventions such as hematopoietic stem cell transplantation or gene therapy before their use in humans with the corresponding disease. PMID:19293460

  4. Potential Large Animal Models for Gene Therapy of Human Genetic Diseases of Immune and Blood Cell Systems

    PubMed Central

    Bauer, Thomas R.; Adler, Rima L.; Hickstein, Dennis D.

    2009-01-01

    Genetic mutations involving the cellular components of the hematopoietic system—red blood cells, white blood cells, and platelets—manifest clinically as anemia, infection, and bleeding. Although gene targeting has recapitulated many of these diseases in mice, these murine homologues are limited as translational models by their small size and brief life span as well as the fact that mutations induced by gene targeting do not always faithfully reflect the clinical manifestations of such mutations in humans. Many of these limitations can be overcome by identifying large animals with genetic diseases of the hematopoietic system corresponding to their human disease counterparts. In this article, we describe human diseases of the cellular components of the hematopoietic system that have counterparts in large animal species, in most cases carrying mutations in the same gene (CD18 in leukocyte adhesion deficiency) or genes in interacting proteins (DNA cross-link repair 1C protein and protein kinase, DNA-activated, catalytic polypeptide in radiation-sensitive severe combined immunodeficiency). Furthermore, we describe the potential of these animal models to serve as disease-specific, preclinical models for testing the efficacy and safety of clinical interventions such as hematopoietic stem cell transplantation or gene therapy approaches before their use in humans with the corresponding disease. PMID:19293460

  5. Mucin production by human colonic carcinoma cells correlates with their metastatic potential in animal models of colon cancer metastasis.

    PubMed Central

    Bresalier, R S; Niv, Y; Byrd, J C; Duh, Q Y; Toribara, N W; Rockwell, R W; Dahiya, R; Kim, Y S

    1991-01-01

    Patients with mucinous colorectal cancers characteristically present with advanced disease, however, the relationship between mucin production by colon cancer cells and their metastatic potential remains unclear. We therefore sought to define the relationship between mucin production by human colon cancer cells and metastatic ability by employing animal models of colon cancer metastasis. LS LiM 6, a colon carcinoma cell line with high liver metastasizing ability during cecal growth in nude mice produced twofold more metabolically labeled intracellular mucin and secreted four- to fivefold more mucin into the culture medium compared to poorly metastatic parental line LS174T. This was accompanied by a similar elevation in poly(A)+ RNA detected by blot hybridization with a human intestinal mucin cDNA probe, and increases in mucin core carbohydrate antigens determined immunohistochemically. Variants of LS174T selected for high (HM 7) or low (LM 12) mucin synthesizing capacity also yielded metastases after cecal growth and colonized the liver after splenic-portal injection in proportion to their ability to produce mucin. Inhibition of mucin glycosylation by the arylglycoside benzyl-alpha-N-acetyl-galactosamine greatly reduced liver colonization after splenic-portal injection of the tumor cells. These data suggest that mucin production by human colon cancer cells correlates with their metastatic potential and affects their ability to colonize the liver in experimental model systems. Images PMID:1999484

  6. Efficient derivation of cortical glutamatergic neurons from human pluripotent stem cells: a model system to study neurotoxicity in Alzheimer's disease.

    PubMed

    Vazin, Tandis; Ball, K Aurelia; Lu, Hui; Park, Hyungju; Ataeijannati, Yasaman; Head-Gordon, Teresa; Poo, Mu-ming; Schaffer, David V

    2014-02-01

    Alzheimer's disease (AD) is among the most prevalent forms of dementia affecting the aging population, and pharmacological therapies to date have not been successful in preventing disease progression. Future therapeutic efforts may benefit from the development of models that enable basic investigation of early disease pathology. In particular, disease-relevant models based on human pluripotent stem cells (hPSCs) may be promising approaches to assess the impact of neurotoxic agents in AD on specific neuronal populations and thereby facilitate the development of novel interventions to avert early disease mechanisms. We implemented an efficient paradigm to convert hPSCs into enriched populations of cortical glutamatergic neurons emerging from dorsal forebrain neural progenitors, aided by modulating Sonic hedgehog (Shh) signaling. Since AD is generally known to be toxic to glutamatergic circuits, we exposed glutamatergic neurons derived from hESCs to an oligomeric pre-fibrillar forms of Aβ known as "globulomers", which have shown strong correlation with the level of cognitive deficits in AD. Administration of such Aβ oligomers yielded signs of the disease, including cell culture age-dependent binding of Aβ and cell death in the glutamatergic populations. Furthermore, consistent with previous findings in postmortem human AD brain, Aβ-induced toxicity was selective for glutamatergic rather than GABAeric neurons present in our cultures. This in vitro model of cortical glutamatergic neurons thus offers a system for future mechanistic investigation and therapeutic development for AD pathology using human cell types specifically affected by this disease. PMID:24055772

  7. Establishment and characterization of intraperitoneal xenograft models by co-injection of human tumor cells and extracellular matrix gel

    PubMed Central

    YAO, YUQIN; ZHOU, YONGJUN; SU, XIAOLAN; DAI, LEI; YU, LIN; DENG, HONGXIN; GOU, LANTU; YANG, JINLIANG

    2015-01-01

    Establishing a feasible intraperitoneal (i.p.) xenograft model in nude mice is a good strategy to evaluate the antitumor effect of drugs in vivo. However, the manipulation of human cancer cells in establishing a stable peritoneal carcinomatosis model in nude mice is problematic. In the present study, the ovarian and colorectal peritoneal tumor models were successfully established in nude mice by co-injection of human tumor cells and extracellular matrix gel. In ovarian tumor models, the mean number tumor nodes was significantly higher in the experimental group (intraperitoneal tumor cell co-injection with ECM gel) compared with the PBS control group on the 30th day (21.0±3.0 vs. 3.6±2.5; P<0.05). The same results were observed in the colorectal peritoneal tumor models on the 28th day. The colorectal peritoneal tumor model was further used to evaluate the chemotherapy effect of irinotecan (CPT-11). The mean weight of peritoneal tumor nodes in CPT-11 treatment group was significantly less than that of the control group (0.81±0.16 vs. 2.18±0.21 g; P<0.05). The results confirmed the value of these i.p. xenograft models in nude mice as efficient and feasible tools for preclinical evaluation. PMID:26788149

  8. Genomic analysis of head and neck squamous cell carcinoma cell lines and human tumors: a rational approach to preclinical model selection

    PubMed Central

    Li, Hua; Wawrose, John S.; Gooding, William E.; Garraway, Levi A.; Lui, Vivian Wai Yan; Peyser, Noah D.; Grandis, Jennifer R.

    2014-01-01

    Head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide. The increasing amount of genomic information on human tumors and cell lines provides more biological data to design preclinical studies. We and others previously reported whole exome sequencing data of 106 HNSCC primary tumors. In 2012, high throughput genomic data and pharmacological profiling of anticancer drugs of hundreds of cancer cell lines were reported. Here we compared the genomic data of 39 HNSCC cell lines with the genomic findings in 106 HNSCC tumors. Amplification of eight genes (PIK3CA, EGFR, CCND2, KDM5A, ERBB2, PMS1, FGFR1 and WHSCIL1) and deletion of five genes (CDKN2A, SMAD4, NOTCH2, NRAS and TRIM33) were found in both HNSCC cell lines and tumors. Seventeen genes were only mutated in HNSCC cell lines (>10%) suggesting that these mutations may arise through immortalization in tissue culture. Conversely, 11 genes were only mutated in >10% of human HNSCC tumors. Several mutant genes in the EGFR pathway are shared both in cell lines and in tumors. Pharmacological profiling of eight anticancer agents in six HNSCC cell lines suggested that PIK3CA mutation may serve as a predictive biomarker for the drugs targeting the EGFR/PI3K pathway. These findings suggest that a correlation of gene mutations between HNSCC cell lines and human tumors may be used to guide the selection of preclinical models for translational research. PMID:24425785

  9. Prediction model for aneuploidy in early human embryo development revealed by single-cell analysis

    PubMed Central

    Vera-Rodriguez, Maria; Chavez, Shawn L.; Rubio, Carmen; Pera, Renee A. Reijo; Simon, Carlos

    2015-01-01

    Aneuploidies are prevalent in the human embryo and impair proper development, leading to cell cycle arrest. Recent advances in imaging and molecular and genetic analyses are postulated as promising strategies to unveil the mechanisms involved in aneuploidy generation. Here we combine time-lapse, complete chromosomal assessment and single-cell RT–qPCR to simultaneously obtain information from all cells that compose a human embryo until the approximately eight-cell stage (n=85). Our data indicate that the chromosomal status of aneuploid embryos (n=26), including those that are mosaic (n=3), correlates with significant differences in the duration of the first mitotic phase when compared with euploid embryos (n=28). Moreover, gene expression profiling suggests that a subset of genes is differentially expressed in aneuploid embryos during the first 30 h of development. Thus, we propose that the chromosomal fate of an embryo is likely determined as early as the pronuclear stage and may be predicted by a 12-gene transcriptomic signature. PMID:26151134

  10. Global Transcriptomic Analysis of Model Human Cell Lines Exposed to Surface-Modified Gold Nanoparticles: The Effect of Surface Chemistry

    PubMed Central

    Grzincic, E. M.; Yang, J. A.; Drnevich, J.; Falagan-Lotsch, P.; Murphy, C. J.

    2015-01-01

    Gold nanoparticles (Au NPs) are attractive for biomedical applications not only for their remarkable physical properties, but also for the ease of which their surface chemistry can be manipulated. Many applications involve functionalization of the Au NP surface in order to improve biocompatibility, attach targeting ligands or carry drugs. However, changes in cells exposed to Au NPs of different surface chemistries have been observed, and little is known about how Au NPs and their surface coatings may impact cellular gene expression. The gene expression of two model human cell lines, human dermal fibroblasts (HDF) and prostate cancer cells (PC3) was interrogated by microarray analysis of over 14,000 human genes. The cell lines were exposed to four differently functionalized Au NPs: citrate, poly(allylamine hydrochloride) (PAH), and lipid coatings combined with alkanethiols or PAH. Gene functional annotation categories and weighted gene correlation network analysis were used in order to connect gene expression changes to common cellular functions and to elucidate expression patterns between Au NP samples. Coated Au NPs affect genes implicated in proliferation, angiogenesis, and metabolism in HDF cells, and inflammation, angiogenesis, proliferation apoptosis regulation, survival and invasion in PC3 cells. Subtle changes in surface chemistry, such as the initial net charge, lability of the ligand, and underlying layers greatly influence the degree of expression change and the type of cellular pathway affected. PMID:25491924

  11. Reliable and versatile immortal muscle cell models from healthy and myotonic dystrophy type 1 primary human myoblasts.

    PubMed

    Pantic, Boris; Borgia, Doriana; Giunco, Silvia; Malena, Adriana; Kiyono, Tohru; Salvatori, Sergio; De Rossi, Anita; Giardina, Emiliano; Sangiuolo, Federica; Pegoraro, Elena; Vergani, Lodovica; Botta, Annalisa

    2016-03-01

    Primary human skeletal muscle cells (hSkMCs) are invaluable tools for deciphering the basic molecular mechanisms of muscle-related biological processes and pathological alterations. Nevertheless, their use is quite restricted due to poor availability, short life span and variable purity of the cells during in vitro culture. Here, we evaluate a recently published method of hSkMCs immortalization, relying on ectopic expression of cyclin D1 (CCND1), cyclin-dependent kinase 4 (CDK4) and telomerase (TERT) in myoblasts from healthy donors (n=3) and myotonic dystrophy type 1 (DM1) patients (n=2). The efficacy to maintain the myogenic and non-transformed phenotype, as well as the main pathogenetic hallmarks of DM1, has been assessed. Combined expression of the three genes i) maintained the CD56(NCAM)-positive myoblast population and differentiation potential; ii) preserved the non-transformed phenotype and iii) maintained the CTG repeat length, amount of nuclear foci and aberrant alternative splicing in immortal muscle cells. Moreover, immortal hSkMCs displayed attractive additional features such as structural maturation of sarcomeres, persistence of Pax7-positive cells during differentiation and complete disappearance of nuclear foci following (CAG)7 antisense oligonucleotide (ASO) treatment. Overall, the CCND1, CDK4 and TERT immortalization yields versatile, reliable and extremely useful human muscle cell models to investigate the basic molecular features of human muscle cell biology, to elucidate the molecular pathogenetic mechanisms and to test new therapeutic approaches for DM1 in vitro. PMID:26905645

  12. LOSS OF P130 ACCELERATES TUMOR DEVELOPMENT IN A MOUSE MODEL FOR HUMAN SMALL CELL LUNG CARCINOMA

    PubMed Central

    Schaffer, Bethany E.; Park, Kwon-Sik; Yiu, Gloria; Conklin, Jamie F.; Lin, Chenwei; Burkhart, Deborah L.; Karnezis, Anthony N.; Sweet-Cordero, Alejandro; Sage, Julien

    2010-01-01

    Small cell lung carcinoma (SCLC) is a neuroendocrine subtype of lung cancer. While SCLC patients often initially respond to therapy, tumors nearly always recur, resulting in a 5-year survival rate of less than 10%. A mouse model has been developed based on the fact that the RB and p53 tumor suppressor genes are mutated in more than 90% of human SCLCs. Emerging evidence in patients and mouse models suggests that p130, a gene related to RB, may act as a tumor suppressor in SCLC cells. To test this idea, we used conditional mutant mice to delete p130 in combination with Rb and p53 in adult lung epithelial cells. We found that loss of p130 resulted in increased proliferation and significant acceleration of SCLC development in this triple knockout mouse model. The histopathological features of the triple mutant mouse tumors closely resembled that of human SCLC. Genome-wide expression profiling experiments further showed that Rb/p53/p130 mutant mouse tumors were similar to human SCLC. These findings indicate that p130 plays a key tumor suppressor role in SCLC. Rb/p53/p130 mutant mice provide a novel pre-clinical mouse model to identify novel therapeutic targets against SCLC. PMID:20406986

  13. Human olfactory bulb neural stem cells mitigate movement disorders in a rat model of Parkinson's disease.

    PubMed

    Marei, Hany E S; Lashen, Samah; Farag, Amany; Althani, Asmaa; Afifi, Nahla; A, Abd-Elmaksoud; Rezk, Shaymaa; Pallini, Roberto; Casalbore, Patrizia; Cenciarelli, Carlo

    2015-07-01

    Parkinson's disease (PD) is a neurological disorder characterized by the loss of midbrain dopaminergic (DA) neurons. Neural stem cells (NSCs) are multipotent stem cells that are capable of differentiating into different neuronal and glial elements. The production of DA neurons from NSCs could potentially alleviate behavioral deficits in Parkinsonian patients; timely intervention with NSCs might provide a therapeutic strategy for PD. We have isolated and generated highly enriched cultures of neural stem/progenitor cells from the human olfactory bulb (OB). If NSCs can be obtained from OB, it would alleviate ethical concerns associated with the use of embryonic tissue, and provide an easily accessible cell source that would preclude the need for invasive brain surgery. Following isolation and culture, olfactory bulb neural stem cells (OBNSCs) were genetically engineered to express hNGF and GFP. The hNFG-GFP-OBNSCs were transplanted into the striatum of 6-hydroxydopamin (6-OHDA) Parkinsonian rats. The grafted cells survived in the lesion environment for more than eight weeks after implantation with no tumor formation. The grafted cells differentiated in vivo into oligodendrocyte-like (25 ± 2.88%), neuron-like (52.63 ± 4.16%), and astrocyte -like (22.36 ± 1.56%) lineages, which we differentiated based on morphological and immunohistochemical criteria. Transplanted rats exhibited a significant partial correction in stepping and placing in non-pharmacological behavioral tests, pole and rotarod tests. Taken together, our data encourage further investigations of the possible use of OBNSCs as a promising cell-based therapeutic strategy for Parkinson's disease. PMID:25536543

  14. Surface-induced modulation of human mesenchymal progenitor cells. An in vitro model for early implant integration.

    PubMed

    Baschong, Werner; Jaquiery, Claude; Martin, Ivan; Lambrecht, Thomas J

    2007-01-01

    Clinical experience indicates that the surface architecture of dental implants has an important impact on their integration. This has been related to the finding that differentially treated substrates can modulate the expression of osteogenic markers in various bone-related cell lines and primary cells. Here, we investigated the influence of surface architecture on the differentiation of human mesenchymal progenitor cells (HMPC) from adult bone marrow, i. e. the cells likely involved in initial bone synthesis at the bone-implant interface. Cells were seeded on machine surfaced (MS) or sandblasted/acid etched (SE) titanium discs in agarose-coated dishes, and on polystyrene (PS) controls. On all substrates cell densities did not change between days 7 and 14. Cell numbers were higher on SE, likely due to increased attachment to the rougher material. Alkaline phosphatase activity (ALP) was similar on all substrates, whereas mRNA expression of bone sialoprotein (BSP) at day 14 was about tenfold higher on SE (p < 0.05%). The SE-related increase of BSP in progenitor cells indicates an earlier differentiation of immigrated cells and could thus explain earlier implant integration and shorter time to functional loading observed in the clinic. The in vitro model and BSP quantification could be used to screen for changes in osteogenic cell differentiation induced by specific implant surfaces, with potential relevance on the prediction of bone-implant integration. PMID:17966928

  15. AML cells are differentially sensitive to chemotherapy treatment in a human xenograft model.

    PubMed

    Wunderlich, Mark; Mizukawa, Benjamin; Chou, Fu-Sheng; Sexton, Christina; Shrestha, Mahesh; Saunthararajah, Yogen; Mulloy, James C

    2013-03-21

    As acute myeloid leukemia (AML) xenograft models improve, the potential for using them to evaluate novel therapeutic strategies becomes more appealing. Currently, there is little information on using standard chemotherapy regimens in AML xenografts. Here we have characterized the immunodeficient mouse response to combined Ara-C (cytarabine) and doxorubicin treatment. We observed significant toxicity associated with doxorubicin that required optimization of the route of injection as well as the maximum-tolerated dose for immunodeficient strains. Mice treated with an optimized 5-day induction protocol showed transient weight loss, short-term reduction of peripheral blood cell and platelet counts, and slight anemia. Considerable cytotoxicity was observed in the bone marrow (BM), with primitive LSK cells having a significant survival advantage relative to more mature cells, consistent with the idea of chemotherapy targeting actively growing cells. Treated leukemic mice demonstrated reduced disease burden and increased survival, demonstrating efficacy. AML cells showed significantly increased sensitivity to doxorubicin-containing therapy compared with murine BM cells. Although early treatment could result in some cures, mice with significant leukemia grafts were not cured by using induction therapy alone. Overall, the data show that this model system is useful for the evaluation of novel chemotherapies in combination with standard induction therapy. PMID:23349390

  16. Mouse Models of Human T Lymphotropic Virus Type-1–Associated Adult T-Cell Leukemia/Lymphoma

    PubMed Central

    Zimmerman, B.; Niewiesk, S.; Lairmore, M. D.

    2011-01-01

    Human T-lymphotropic virus type-1 (HTLV-1), the first human retrovirus discovered, is the causative agent of adult T-cell leukemia/lymphoma (ATL) and a number of lymphocyte-mediated inflammatory conditions including HTLV-1–associated myelopathy/tropical spastic paraparesis. Development of animal models to study the pathogenesis of HTLV-1–associated diseases has been problematic. Mechanisms of early infection and cell-to-cell transmission can be studied in rabbits and nonhuman primates, but lesion development and reagents are limited in these species. The mouse provides a cost-effective, highly reproducible model in which to study factors related to lymphoma development and the preclinical efficacy of potential therapies against ATL. The ability to manipulate transgenic mice has provided important insight into viral genes responsible for lymphocyte transformation. Expansion of various strains of immunodeficient mice has accelerated the testing of drugs and targeted therapy against ATL. This review compares various mouse models to illustrate recent advances in the understanding of HTLV-1–associated ATL development and how improvements in these models are critical to the future development of targeted therapies against this aggressive T-cell lymphoma. PMID:20442421

  17. Establishment of human iPSC-based models for the study and targeting of glioma initiating cells

    PubMed Central

    Sancho-Martinez, Ignacio; Nivet, Emmanuel; Xia, Yun; Hishida, Tomoaki; Aguirre, Aitor; Ocampo, Alejandro; Ma, Li; Morey, Robert; Krause, Marie N.; Zembrzycki, Andreas; Ansorge, Olaf; Vazquez-Ferrer, Eric; Dubova, Ilir; Reddy, Pradeep; Lam, David; Hishida, Yuriko; Wu, Min-Zu; Esteban, Concepcion Rodriguez; O'Leary, Dennis; Wahl, Geoffrey M.; Verma, Inder M.; Laurent, Louise C.; Izpisua Belmonte, Juan Carlos

    2016-01-01

    Glioma tumour-initiating cells (GTICs) can originate upon the transformation of neural progenitor cells (NPCs). Studies on GTICs have focused on primary tumours from which GTICs could be isolated and the use of human embryonic material. Recently, the somatic genomic landscape of human gliomas has been reported. RTK (receptor tyrosine kinase) and p53 signalling were found dysregulated in ∼90% and 86% of all primary tumours analysed, respectively. Here we report on the use of human-induced pluripotent stem cells (hiPSCs) for modelling gliomagenesis. Dysregulation of RTK and p53 signalling in hiPSC-derived NPCs (iNPCs) recapitulates GTIC properties in vitro. In vivo transplantation of transformed iNPCs leads to highly aggressive tumours containing undifferentiated stem cells and their differentiated derivatives. Metabolic modulation compromises GTIC viability. Last, screening of 101 anti-cancer compounds identifies three molecules specifically targeting transformed iNPCs and primary GTICs. Together, our results highlight the potential of hiPSCs for studying human tumourigenesis. PMID:26899176

  18. Transplantation of human neural stem cells restores cognition in an immunodeficient rodent model of traumatic brain injury.

    PubMed

    Haus, Daniel L; López-Velázquez, Luci; Gold, Eric M; Cunningham, Kelly M; Perez, Harvey; Anderson, Aileen J; Cummings, Brian J

    2016-07-01

    Traumatic brain injury (TBI) in humans can result in permanent tissue damage and has been linked to cognitive impairment that lasts years beyond the initial insult. Clinically effective treatment strategies have yet to be developed. Transplantation of human neural stem cells (hNSCs) has the potential to restore cognition lost due to injury, however, the vast majority of rodent TBI/hNSC studies to date have evaluated cognition only at early time points, typically <1month post-injury and cell transplantation. Additionally, human cell engraftment and long-term survival in rodent models of TBI has been difficult to achieve due to host immunorejection of the transplanted human cells, which confounds conclusions pertaining to transplant-mediated behavioral improvement. To overcome these shortfalls, we have developed a novel TBI xenotransplantation model that utilizes immunodeficient athymic nude (ATN) rats as the host recipient for the post-TBI transplantation of human embryonic stem cell (hESC) derived NSCs and have evaluated cognition in these animals at long-term (≥2months) time points post-injury. We report that immunodeficient ATN rats demonstrate hippocampal-dependent spatial memory deficits (Novel Place, Morris Water Maze), but not non-spatial (Novel Object) or emotional/anxiety-related (Elevated Plus Maze, Conditioned Taste Aversion) deficits, at 2-3months post-TBI, confirming that ATN rats recapitulate some of the cognitive deficits found in immunosufficient animal strains. Approximately 9-25% of transplanted hNSCs survived for at least 5months post-transplantation and differentiated into mature neurons (NeuN, 18-38%), astrocytes (GFAP, 13-16%), and oligodendrocytes (Olig2, 11-13%). Furthermore, while this model of TBI (cortical impact) targets primarily cortex and the underlying hippocampus and generates a large lesion cavity, hNSC transplantation facilitated cognitive recovery without affecting either lesion volume or total spared cortical or hippocampal

  19. Modeled microgravity suppressed invasion and migration of human glioblastoma U87 cells through downregulating store-operated calcium entry

    SciTech Connect

    Shi, Zi-xuan; Rao, Wei; Wang, Huan; Wang, Nan-ding; Si, Jing-Wen; Zhao, Jiao; Li, Jun-chang; Wang, Zong-ren

    2015-02-13

    Glioblastoma is the most common brain tumor and is characterized with robust invasion and migration potential resulting in poor prognosis. Previous investigations have demonstrated that modeled microgravity (MMG) could decline the cell proliferation and attenuate the metastasis potential in several cell lines. In this study, we studied the effects of MMG on the invasion and migration potentials of glioblastoma in human glioblastoma U87 cells. We found that MMG stimulation significantly attenuated the invasion and migration potentials, decreased thapsigargin (TG) induced store-operated calcium entry (SOCE) and downregulated the expression of Orai1 in U87 cells. Inhibition of SOCE by 2-APB or stromal interaction molecule 1 (STIM1) downregulation both mimicked the effects of MMG on the invasion and migration potentials in U87 cells. Furthermore, upregulation of Orai1 significantly weakened the effects of MMG on the invasion and migration potentials in U87 cells. Therefore, these findings indicated that MMG stimulation inhibited the invasion and migration potentials of U87 cells by downregulating the expression of Orai1 and sequentially decreasing the SOCE, suggesting that MMG might be a new potential therapeutic strategy in glioblastoma treatment in the future. - Highlights: • Modeled microgravity (MMG) suppressed migration and invasion in U87 cells. • MMG downregulated the SOCE and the expression of Orai1. • SOCE inhibition mimicked the effects of MMG on migration and invasion potentials. • Restoration of SOCE diminished the effects of MMG on migration and invasion.

  20. CD38 and Airway hyperresponsiveness: Studies on human airway smooth muscle cells and mouse models

    PubMed Central

    Guedes, Alonso GP; Deshpande, Deepak A; Dileepan, Mythili; Walseth, Timothy F; Panettieri, Reynold A; Subramanian, Subbaya; Kannan, Mathur S

    2015-01-01

    Asthma is an inflammatory disease in which altered calcium regulation, contractility and airway smooth muscle (ASM) proliferation contribute to airway hyperresponsiveness and airway wall remodeling. The enzymatic activity of CD38, a cell-surface protein expressed in human ASM cells, generates calcium mobilizing second messenger molecules such as cyclic ADP-ribose. CD38 expression in human ASM cells is augmented by cytokines (e.g. TNF-α) that requires activation of MAP kinases and the transcription factors, NF-ƙB and AP-1 and post-transcriptionally regulated by miR-140-3p and miR-708 by binding to 3’ Untranslated Region of CD38 as well as by modulating the activation of signaling mechanisms involved in its regulation. Mice deficient in CD38 exhibit reduced airway responsiveness to inhaled methacholine relative to response in wild-type mice. Intranasal challenge of CD38 deficient mice with TNF-α or IL-13, or the environmental fungus Alternaria alternata, causes significantly attenuated methacholine responsiveness compared to wild-type mice, with comparable airway inflammation. Reciprocal bone marrow transfer studies revealed partial restoration of airway hyperresponsiveness to inhaled methacholine in the Cd38 deficient mice. These studies provide evidence for CD38 involvement in the development of airway hyperresponsiveness, a hallmark feature of asthma. Future studies aimed at drug discovery and delivery targeting CD38 expression and/or activity are warranted. PMID:25594684

  1. Encapsulated human hepatocellular carcinoma cells by alginate gel beads as an in vitro metastasis model

    SciTech Connect

    Xu, Xiao-xi; Liu, Chang; Liu, Yang; Li, Nan; Guo, Xin; Wang, Shu-jun; Sun, Guang-wei; Wang, Wei; Ma, Xiao-jun

    2013-08-15

    Hepatocellular carcinoma (HCC) is the most common primary liver cancer and often forms metastases, which are the most important prognostic factors. For further elucidation of the mechanism underlying the progression and metastasis of HCC, a culture system mimicking the in vivo tumor microenvironment is needed. In this study, we investigated the metastatic ability of HCC cells cultured within alginate gel (ALG) beads. In the culture system, HCC cells formed spheroids by proliferation and maintained in nuclear abnormalities. The gene and protein expression of metastasis-related molecules was increased in ALG beads, compared with the traditional adhesion culture. Furthermore, several gene expression levels in ALG bead culture system were even closer to liver cancer tissues. More importantly, in vitro invasion assay showed that the invasion cells derived from ALG beads was 7.8-fold higher than adhesion cells. Our results indicated that the in vitro three-dimensional (3D) model based on ALG beads increased metastatic ability compared with adhesion culture, even partly mimicked the in vivo tumor tissues. Moreover, due to the controllable preparation conditions, steady characteristics and production at large-scale, the 3D ALG bead model would become an important tool used in the high-throughput screening of anti-metastasis drugs and the metastatic mechanism research. -- Highlights: •We established a 3D metastasis model mimicking the metastatic ability in vivo. •The invasion ability of cells derived from our model was increased significantly. •The model is easy to reproduce, convenient to handle, and amenable for large-scale.

  2. Human breast cancer bone metastasis in vitro and in vivo: a novel 3D model system for studies of tumour cell-bone cell interactions.

    PubMed

    Holen, I; Nutter, F; Wilkinson, J M; Evans, C A; Avgoustou, P; Ottewell, Penelope D

    2015-10-01

    Bone is established as the preferred site of breast cancer metastasis. However, the precise mechanisms responsible for this preference remain unidentified. In order to improve outcome for patients with advanced breast cancer and skeletal involvement, we need to better understand how this process is initiated and regulated. As bone metastasis cannot be easily studied in patients, researchers have to date mainly relied on in vivo xenograft models. A major limitation of these is that they do not contain a human bone microenvironment, increasingly considered to be an important component of metastases. In order to address this shortcoming, we have developed a novel humanised bone model, where 1 × 10(5) luciferase-expressing MDA-MB-231 or T47D human breast tumour cells are seeded on viable human subchaodral bone discs in vitro. These discs contain functional osteoclasts 2-weeks after in vitro culture and positive staining for calcine 1-week after culture demonstrating active bone resorption/formation. In vitro inoculation of MDA-MB-231 or T47D cells colonised human bone cores and remained viable for <4 weeks, however, use of matrigel to enhance adhesion or a moving platform to increase diffusion of nutrients provided no additional advantage. Following colonisation by the tumour cells, bone discs pre-seeded with MDA-MB-231 cells were implanted subcutaneously into NOD SCID mice, and tumour growth monitored using in vivo imaging for up to 6 weeks. Tumour growth progressed in human bone discs in 80 % of the animals mimicking the later stages of human bone metastasis. Immunohistochemical and PCR analysis revealed that growing MDA-MB-231 cells in human bone resulted in these cells acquiring a molecular phenotype previously associated with breast cancer bone metastases. MDA-MB-231 cells grown in human bone discs showed increased expression of IL-1B, HRAS and MMP9 and decreased expression of S100A4, whereas, DKK2 and FN1 were unaltered compared with the same cells grown in

  3. Mössbauer Study and Modeling of Iron Import and Trafficking in Human Jurkat Cells

    PubMed Central

    Jhurry, Nema D.; Chakrabarti, Mrinmoy; McCormick, Sean P.; Gohil, Vishal M.; Lindahl, Paul A.

    2014-01-01

    The Fe content of Jurkat cells grown on transferrin-bound iron (TBI) and FeIII citrate (FC) was characterized using Mössbauer, EPR, and UV-vis spectroscopies, electron microscopy, and ICP-MS. Isolated mitochondria were similarly characterized. Fe-limited cells contained ∼ 100 μM of essential Fe, mainly as mitochondrial Fe and non-mitochondrial nonheme high-spin (NHHS) FeII. Fe-replete cells also contained ferritin-bound Fe and FeIII oxyhydroxide nanoparticles. Only 400 ± 100 Fe ions were loaded per ferritin complex, regardless of the growth medium Fe concentration. Ferritin regulation thus appears more complex than is commonly assumed. The magnetic/structural properties of Jurkat nanoparticles differed from those in yeast mitochondria. They were smaller and may be located in the cytosol. The extent of nanoparticle formation scaled nonlinearly with the concentration of Fe in the medium. Nanoparticle formation was not strongly correlated with ROS damage. Cells could utilize nanoparticle Fe, converting such aggregates into essential Fe forms. Cells grown on galactose rather than glucose respired faster, grew slower, exhibited more ROS damage and generally contained more nanoparticles. Cells grown with TBI rather than FC contained lower Fe concentrations, more ferritin and fewer nanoparticles. Cells in which transferrin receptor expression was increased contained more ferritin Fe. Frataxin-deficient cells contained more nanoparticles than comparable WT cells. Data were analyzed by a chemically-based mathematical model. Although simple, it captured essential features of Fe import, trafficking and regulation. TBI import was highly regulated but FC import was not. Nanoparticle formation was not regulated but the rate was third-order in cytosolic Fe. PMID:24180611

  4. Agent-based model of human alveoli predicts chemotactic signaling by epithelial cells during early Aspergillus fumigatus infection.

    PubMed

    Pollmächer, Johannes; Figge, Marc Thilo

    2014-01-01

    Aspergillus fumigatus is one of the most important human fungal pathogens, causing life-threatening diseases. Since humans inhale hundreds to thousands of fungal conidia every day, the lower respiratory tract is the primary site of infection. Current interaction networks of the innate immune response attribute fungal recognition and detection to alveolar macrophages, which are thought to be the first cells to get in contact with the fungus. At present, these networks are derived from in vitro or in situ assays, as the peculiar physiology of the human lung makes in vivo experiments, including imaging on the cell-level, hard to realize. We implemented a spatio-temporal agent-based model of a human alveolus in order to perform in silico experiments of a virtual infection scenario, for an alveolus infected with A. fumigatus under physiological conditions. The virtual analog captures the three-dimensional alveolar morphology consisting of the two major alveolar epithelial cell types and the pores of Kohn as well as the dynamic process of respiration. To the best of our knowledge this is the first agent-based model of a dynamic human alveolus in the presence of respiration. A key readout of our simulations is the first-passage-time of alveolar macrophages, which is the period of time that elapses until the first physical macrophage-conidium contact is established. We tested for random and chemotactic migration modes of alveolar macrophages and varied their corresponding parameter sets. The resulting first-passage-time distributions imply that randomly migrating macrophages fail to find the conidium before the start of germination, whereas guidance by chemotactic signals derived from the alveolar epithelial cell associated with the fungus enables a secure and successful discovery of the pathogen in time. PMID:25360787

  5. A Cell Kinetic Model of Granulocytopoiesis Under Radiation Exposure: Extension from Murines to Canines and Humans

    NASA Technical Reports Server (NTRS)

    Hu, Shaowen; Cucinotta, Francis A.

    2009-01-01

    Space radiation poses significant challenges to space travel, and it is essential to understand the possible adverse effects from space radiation exposure to the radiosensitive organ systems that are important for immediate survival of human, e.g., the hematopoietic system. In this presentation a biomathematical model of granulocytopoiesis is described and used to analyze the blood granulocyte changes seen in the blood of mammalians under continuous and acute radiation exposure. This is one of a set of hematopoietic models that have been successfully utilized to simulate and interpret the experimental data of acute and chronic radiation on rodents. We discuss the underlying implicit regulation mechanism and the biological relevance of the kinetic parameters estimation method. Extension of the model to predictions in dogs and humans systems indicates that the modeling results are consistent with the cumulative experimental and empirical data from various sources. This implies the potential to integrate the models into one united system for monitoring the hematopoietic response of various species under irradiation. Based on the evidence of threshold responses of dogs to extended periods of low daily dose exposures, we discuss the potential health risks of the space traveler under chronic stress of low-dose irradiation and the possibly encountered Solar Particle Events.

  6. Space Radiation Effects on Human Cells: Modeling DNA Breakage, DNA Damage Foci Distribution, Chromosomal Aberrations and Tissue Effects

    NASA Technical Reports Server (NTRS)

    Ponomarev, A. L.; Huff, J. L.; Cucinotta, F. A.

    2011-01-01

    Future long-tem space travel will face challenges from radiation concerns as the space environment poses health risk to humans in space from radiations with high biological efficiency and adverse post-flight long-term effects. Solar particles events may dramatically affect the crew performance, while Galactic Cosmic Rays will induce a chronic exposure to high-linear-energy-transfer (LET) particles. These types of radiation, not present on the ground level, can increase the probability of a fatal cancer later in astronaut life. No feasible shielding is possible from radiation in space, especially for the heavy ion component, as suggested solutions will require a dramatic increase in the mass of the mission. Our research group focuses on fundamental research and strategic analysis leading to better shielding design and to better understanding of the biological mechanisms of radiation damage. We present our recent effort to model DNA damage and tissue damage using computational models based on the physics of heavy ion radiation, DNA structure and DNA damage and repair in human cells. Our particular area of expertise include the clustered DNA damage from high-LET radiation, the visualization of DSBs (DNA double strand breaks) via DNA damage foci, image analysis and the statistics of the foci for different experimental situations, chromosomal aberration formation through DSB misrepair, the kinetics of DSB repair leading to a model-derived spectrum of chromosomal aberrations, and, finally, the simulation of human tissue and the pattern of apoptotic cell damage. This compendium of theoretical and experimental data sheds light on the complex nature of radiation interacting with human DNA, cells and tissues, which can lead to mutagenesis and carcinogenesis later in human life after the space mission.

  7. Human bronchial epithelial BEAS-2B cells, an appropriate in vitro model to study heavy metals induced carcinogenesis.

    PubMed

    Park, Youn-Hee; Kim, Donghern; Dai, Jin; Zhang, Zhuo

    2015-09-15

    Occupational and environmental exposure to arsenic (III) and chromium VI (Cr(VI)) have been confirmed to cause lung cancer. Mechanisms of these metals carcinogenesis are still under investigation. Selection of cell lines to be used is essential for the studies. Human bronchial epithelial BEAS-2B cells are the cells to be utilized by most of scientists. However, due to p53 missense mutation (CCG→TCG) at codon 47 and the codon 72 polymorphism (CGC→CCC) in BEAS-2B cells, its usage has frequently been questioned. The present study has examined activity and expression of 53 and its downstream target protein p21 upon acute or chronic exposure of BEAS-2B cells to arsenic and Cr(VI). The results show that short-term exposure of BEAS-2B cells to arsenic or Cr(VI) was able to activate both p53 and p21. Chronic exposure of BEAS-2B cells to these two metals caused malignant cell transformation and tumorigenesis. In arsenic-transformed BEAS-2B cells reductions in p53 promoter activity, mRNA expression, and phosphorylation of p53 at Ser392 were observed, while the total p53 protein level remained the same compared to those in passage-matched parent ones. p21 promoter activity and expression were decreased in arsenic-transformed cells. Cr(VI)-transformed cells exhibit elevated p53 promoter activity, mRNA expression, and phosphorylation at Ser15, but reduced phosphorylation at Ser392 and total p53 protein level compared to passage-matched parent ones. p21 promoter activity and expression were elevated in Cr(VI)-transformed cells. These results demonstrate that p53 is able to respond to exposure of arsenic or Cr(VI), suggesting that BEAS-2B cells are an appropriate in vitro model to investigate arsenic or Cr(VI) induced lung cancer. PMID:26091798

  8. Anti-Leukemia Activity of In Vitro-Expanded Human Gamma Delta T Cells in a Xenogeneic Ph+ Leukemia Model

    PubMed Central

    Siegers, Gabrielle M.; Felizardo, Tania C.; Mathieson, A. Mark; Kosaka, Yoko; Wang, Xing-Hua; Medin, Jeffrey A.; Keating, Armand

    2011-01-01

    Gamma delta T cells (GDTc) lyse a variety of hematological and solid tumour cells in vitro and in vivo, and are thus promising candidates for cellular immunotherapy. We have developed a protocol to expand human GDTc in vitro, yielding highly cytotoxic Vgamma9/Vdelta2 CD27/CD45RA double negative effector memory cells. These cells express CD16, CD45RO, CD56, CD95 and NKG2D. Flow cytometric, clonogenic, and chromium release assays confirmed their specific cytotoxicity against Ph+ cell lines in vitro. We have generated a fluorescent and bioluminescent Ph+ cell line, EM-2eGFPluc, and established a novel xenogeneic leukemia model. Intravenous injection of EM-2eGFPluc into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice resulted in significant dose-dependent bone marrow engraftment; lower levels engrafted in blood, lung, liver and spleen. In vitro-expanded human GDTc injected intraperitoneally were found at higher levels in blood and organs compared to those injected intravenously; GDTc survived at least 33 days post-injection. In therapy experiments, we documented decreased bone marrow leukemia burden in mice treated with GDTc. Live GDTc were found in spleen and bone marrow at endpoint, suggesting the potential usefulness of this therapy. PMID:21304898

  9. Distinct Splice Variants and Pathway Enrichment in the Cell Line Models of Aggressive Human Breast Cancer Subtypes

    PubMed Central

    Menon, Rajasree; Im, Hogune; Zhang, Emma (Yue); Wu, Shiaw-Lin; Chen, Rui; Snyder, Michael; Hancock, William S.; Omenn, Gilbert S.

    2013-01-01

    This study was conducted as a part of the Chromosome-Centric Human Proteome Project (C-HPP) of the Human Proteome Organization. The United States team of C-HPP is focused on characterizing the protein-coding genes in chromosome 17. Despite its small size, chromosome 17 is rich in protein-coding genes, it contains many cancer-associated genes, including BRCA1, ERBB2 (Her2/neu), and TP53. The goal of this study was to examine the splice variants expressed in three ERBB2 expressed breast cancer cell line models of hormone receptor negative breast cancers by integrating RNA-Seq and proteomic mass spectrometry data. The cell-lines represent distinct phenotypic variations subtype: SKBR3 (ERBB2+ (over-expression)/ ER−/PR−; adenocarcinoma), SUM190 (ERBB2+ (over-expression)/ER−/PR−; inflammatory breast cancer) and SUM149 (ERBB2 (low expression) ER−/PR −; inflammatory breast cancer). We identified more than one splice variant for 1167 genes expressed in at least one of the three cancer cell lines. We found multiple variants of genes that are in the signaling pathways downstream of ERBB2 along with variants specific to one cancer cell line compared to the other two cancer cell lines and to normal mammary cells. The overall transcript profiles based on read counts indicated more similarities between SKBR3 and SUM190. The top-ranking Gene Ontology and BioCarta pathways for the cell-line specific variants pointed to distinct key mechanisms including: amino sugar metabolism, caspase activity, and endocytosis in SKBR3; different aspects of metabolism, especially of lipids in SUM190; cell- to-cell adhesion, integrin and ERK1/ERK2 signaling, and translational control in SUM149. The analyses indicated an enrichment in the electron transport chain processes in the ERBB2 over-expressed cell line models; and an association of nucleotide binding, RNA splicing and translation processes with the IBC models, SUM190 and SUM149. Detailed experimental studies on the distinct

  10. Transplantation of neural differentiated human mesenchymal stem cells into the cochlea of an auditory-neuropathy guinea pig model.

    PubMed

    Cho, Yong-Bum; Cho, Hyong-Ho; Jang, Sujeong; Jeong, Han-Seong; Park, Jong-Seong

    2011-04-01

    The aim of this study was to determine the effects of transplanted neural differentiated human mesenchymal stem cells (hMSCs) in a guinea pig model of auditory neuropathy. In this study, hMSCs were pretreated with a neural-induction protocol and transplanted into the scala tympani of the guinea pig cochlea 7 days after ouabain injury. A control model was made by injection of Hanks balanced salt solution alone into the scala tympani of the guinea pig cochlea 7 days after ouabain injury. We established the auditory neuropathy guinea pig model using 1 mM ouabain application to the round window niche. After application of ouabain to the round window niche, degeneration of most spiral ganglion neurons (SGNs) without the loss of hair cells within the organ of Corti and increasing the auditory brain responses (ABR) threshold were found. After transplantation of neural differentiated hMSCs, the number of SGNs was increased, and some of the SGNs expressed immunoreactivity with human nuclear antibody under confocal laser scanning microscopy. ABR results showed mild hearing recovery after transplantation. Based on an auditory neuropathy animal model, these findings suggest that it may be possible to replace degenerated SGNs by grafting stem cells into the scala tympani. PMID:21468255

  11. Escherichia coli uropathogenesis in vitro: invasion, cellular escape, and secondary infection analyzed in a human bladder cell infection model.

    PubMed

    Andersen, Thomas E; Khandige, Surabhi; Madelung, Michelle; Brewer, Jonathan; Kolmos, Hans J; Møller-Jensen, Jakob

    2012-05-01

    Uropathogenic Escherichia coli (UPEC) strains are capable of invading bladder epithelial cells (BECs) on the bladder luminal surface. Based primarily on studies in mouse models, invasion is proposed to trigger an intracellular uropathogenic cascade involving intracellular bacterial proliferation followed by escape of elongated, filamentous bacteria from colonized BECs. UPEC filaments on the mouse bladder epithelium are able to revert to rod-shaped bacteria, which are believed to invade neighboring cells to initiate new rounds of intracellular colonization. So far, however, these late-stage infection events have not been replicated in vitro. We have established an in vitro model of human bladder cell infection by the use of a flow chamber (FC)-based culture system, which allows investigation of steps subsequent to initial invasion. Short-term bacterial colonization on the FC-BEC layer led to intracellular colonization. Exposing invaded BECs to a flow of urine, i.e., establishing conditions similar to those faced by UPEC reemerging on the bladder luminal surface, led to outgrowth of filamentous bacteria similar to what has been reported to occur in mice. These filaments were capable of reverting to rods that could invade other BECs. Hence, under growth conditions established to resemble those present in vivo, the elements of the proposed uropathogenic cascade were inducible in a human BEC model system. Here, we describe the model and show how these characteristics are reproduced in vitro. PMID:22354025

  12. Global transcriptomic analysis of model human cell lines exposed to surface-modified gold nanoparticles: the effect of surface chemistry

    NASA Astrophysics Data System (ADS)

    Grzincic, E. M.; Yang, J. A.; Drnevich, J.; Falagan-Lotsch, P.; Murphy, C. J.

    2015-01-01

    Gold nanoparticles (Au NPs) are attractive for biomedical applications not only for their remarkable physical properties, but also for the ease of which their surface chemistry can be manipulated. Many applications involve functionalization of the Au NP surface in order to improve biocompatibility, attach targeting ligands or carry drugs. However, changes in cells exposed to Au NPs of different surface chemistries have been observed, and little is known about how Au NPs and their surface coatings may impact cellular gene expression. The gene expression of two model human cell lines, human dermal fibroblasts (HDF) and prostate cancer cells (PC3) was interrogated by microarray analysis of over 14 000 human genes. The cell lines were exposed to four differently functionalized Au NPs: citrate, poly(allylamine hydrochloride) (PAH), and lipid coatings combined with alkanethiols or PAH. Gene functional annotation categories and weighted gene correlation network analysis were used in order to connect gene expression changes to common cellular functions and to elucidate expression patterns between Au NP samples. Coated Au NPs affect genes implicated in proliferation, angiogenesis, and metabolism in HDF cells, and inflammation, angiogenesis, proliferation apoptosis regulation, survival and invasion in PC3 cells. Subtle changes in surface chemistry, such as the initial net charge, lability of the ligand, and underlying layers greatly influence the degree of expression change and the type of cellular pathway affected.Gold nanoparticles (Au NPs) are attractive for biomedical applications not only for their remarkable physical properties, but also for the ease of which their surface chemistry can be manipulated. Many applications involve functionalization of the Au NP surface in order to improve biocompatibility, attach targeting ligands or carry drugs. However, changes in cells exposed to Au NPs of different surface chemistries have been observed, and little is known about how

  13. A cell kinetic model of granulopoiesis under radiation exposure: extension from rodents to canines and humans.

    PubMed

    Hu, Shaowen; Cucinotta, Francis A

    2011-02-01

    As significant ionising radiation exposure will occur during prolonged space travel in future, it is essential to understand their adverse effects on the radiosensitive organ systems that are important for immediate survival of humans, e.g. the haematopoietic system. In this paper, a biomathematical model of granulopoiesis is used to analyse the granulocyte changes seen in the blood of mammalians under acute and continuous radiation exposure. This is one of a set of haematopoietic models that have been successfully utilised to simulate and interpret the experimental data of acute and chronic radiation on rodents. Extension to canine and human systems indicates that the results of the model are consistent with the cumulative experimental and empirical data from various sources, implying the potential to integrate them into one united model system to monitor the haematopoietic response of various species under irradiation. The suppression of granulocytes' level of a space traveller under chronic stress of low-dose irradiation as well as the granulopoietic response when encountering a historically large solar particle event is also discussed. PMID:21196459

  14. Human Adrenocortical Carcinoma Cell Lines

    PubMed Central

    Wang, Tao; Rainey, William E.

    2011-01-01

    Summary The human adrenal cortex secretes mineralocorticoids, glucocorticoids and adrenal androgens. These steroids are produced from unique cell types located within the three distinct zones of the adrenal cortex. Disruption of adrenal steroid production results in a variety of diseases that can lead to hypertension, metabolic syndrome, infertility and androgen excess. The adrenal cortex is also a common site for the development of adenomas, and rarely the site for the development of carcinomas. The adenomas can lead to diseases associated with adrenal steroid excess, while the carcinomas are particularly aggressive and have a poor prognosis. In vitro cell culture models provide an important tool to examine molecular and cellular mechanisms controlling both the normal and pathologic function of the adrenal cortex. Herein we discuss the human adrenocortical cell lines and their use as model systems for adrenal studies. PMID:21924324

  15. Therapeutic Potential of Stem Cells from Human Exfoliated Deciduous Teeth in Models of Acute Kidney Injury

    PubMed Central

    Hattori, Yuka; Kim, Hangsoo; Tsuboi, Naotake; Yamamoto, Akihito; Akiyama, Shinichi; Shi, Yiqin; Katsuno, Takayuki; Kosugi, Tomoki; Ueda, Minoru; Matsuo, Seiichi; Maruyama, Shoichi

    2015-01-01

    Background Acute kidney injury (AKI) is a critical condition associated with high mortality. However, the available treatments for AKI are limited. Stem cells from human exfoliated deciduous teeth (SHED) have recently gained attention as a novel source of stem cells. The purpose of this study was to clarify whether SHED have a therapeutic effect on AKI induced by ischemia-reperfusion injury. Methods The left renal artery and vein of the mice were clamped for 20 min to induce ischemia. SHED, bone marrow derived mesenchymal stem cells (BMMSC) or phosphate-buffered saline (control) were administered into the subrenal capsule. To confirm the potency of SHED in vitro, H2O2 stimulation assays and scratch assays were performed. Results The serum creatinine and blood urea nitrogen levels of the SHED group were significantly lower than those of the control group, while BMMSC showed no therapeutic effect. Infiltration of macrophages and neutrophils in the kidney was significantly attenuated in mice treated with SHED. Cytokine levels (MIP-2, IL-1β, and MCP-1) in mice kidneys were significantly reduced in the SHED group. In in vitro experiments, SHED significantly decreased MCP-1 secretion in tubular epithelial cells (TEC) stimulated with H2O2. In addition, SHED promoted wound healing in the scratch assays, which was blunted by anti-HGF antibodies. Discussion SHED attenuated the levels of inflammatory cytokines and improved kidney function in AKI induced by IRI. SHED secreted factors reduced MCP-1 and increased HGF expression, which promoted wound healing. These results suggest that SHED might provide a novel stem cell resource, which can be applied for the treatment of ischemic kidney injury. PMID:26509261

  16. Expression profiles of genes involved in xenobiotic metabolism and disposition in human renal tissues and renal cell models

    SciTech Connect

    Van der Hauwaert, Cynthia; Savary, Grégoire; Buob, David; Leroy, Xavier; Aubert, Sébastien; Flamand, Vincent; Hennino, Marie-Flore; Perrais, Michaël; and others

    2014-09-15

    Numerous xenobiotics have been shown to be harmful for the kidney. Thus, to improve our knowledge of the cellular processing of these nephrotoxic compounds, we evaluated, by real-time PCR, the mRNA expression level of 377 genes encoding xenobiotic-metabolizing enzymes (XMEs), transporters, as well as nuclear receptors and transcription factors that coordinate their expression in eight normal human renal cortical tissues. Additionally, since several renal in vitro models are commonly used in pharmacological and toxicological studies, we investigated their metabolic capacities and compared them with those of renal tissues. The same set of genes was thus investigated in HEK293 and HK2 immortalized cell lines in commercial primary cultures of epithelial renal cells and in proximal tubular cell primary cultures. Altogether, our data offers a comprehensive description of kidney ability to process xenobiotics. Moreover, by hierarchical clustering, we observed large variations in gene expression profiles between renal cell lines and renal tissues. Primary cultures of proximal tubular epithelial cells exhibited the highest similarities with renal tissue in terms of transcript profiling. Moreover, compared to other renal cell models, Tacrolimus dose dependent toxic effects were lower in proximal tubular cell primary cultures that display the highest metabolism and disposition capacity. Therefore, primary cultures appear to be the most relevant in vitro model for investigating the metabolism and bioactivation of nephrotoxic compounds and for toxicological and pharmacological studies. - Highlights: • Renal proximal tubular (PT) cells are highly sensitive to xenobiotics. • Expression of genes involved in xenobiotic disposition was measured. • PT cells exhibited the highest similarities with renal tissue.

  17. Effect of Citrus bergamia juice on human neuroblastoma cells in vitro and in metastatic xenograft models.

    PubMed

    Navarra, M; Ursino, M R; Ferlazzo, N; Russo, M; Schumacher, U; Valentiner, U

    2014-06-01

    Neuroblastoma is the most common extracranial pediatric solid tumor with poor prognosis in children with disseminated stage of disease. A number of studies show that molecules largely distributed in commonly consumed fruits and vegetables may have anti-tumor activity. In this study we evaluate the effect of Citrus bergamia (bergamot) juice (BJ) in vitro and in a spontaneous metastatic neuroblastoma SCID mouse model. Qualitative and quantitative characterizations of BJ flavonoid fractions were performed by RP-HPLC/PDA/MS. We show that BJ significantly affects SK-N-SH and LAN-1 cell proliferation in vitro, but fails to reduce primary tumor weight in vivo. Moreover, BJ reduced cell adhesiveness and invasion of LAN-1 and SK-N-SH cells in vitro and the number of pulmonary metastases under consideration of the number of tumor cells in the blood in mice inoculated with LAN-1 cells in vivo. These effects without any apparent sign of systemic toxicity confirm the potential clinical interest of BJ and lay the basis for further investigation in cancer. PMID:24594241

  18. Induction of Hepatic and Endothelial Differentiation by Perfusion in a Three-Dimensional Cell Culture Model of Human Fetal Liver

    PubMed Central

    Pekor, Christopher; Gerlach, Jörg C.; Nettleship, Ian

    2015-01-01

    The development of functional engineered tissue constructs depends on high cell densities and appropriate vascularization. In this study we implemented a four-compartment three-dimensional perfusion bioreactor culture model for studying the effects of medium perfusion on endothelial, hepatic, and hematopoietic cell populations of primary human fetal liver in an in vivo-like environment. Human fetal liver cells were cultured in bioreactors configured to provide either perfusion or diffusion conditions. Metabolic activities of the cultures were monitored daily by measuring glucose consumption and lactate production. Cell viability during culture was analyzed by lactate dehydrogenase activity. Hepatic functionality was determined by the release of albumin and alpha-fetoprotein (AFP) in culture medium samples. After 4 days of culture, cells were analyzed for the expression of a variety of endothelial, hepatic, and hematopoietic genes, as well as the surface marker expression of CD31 and CD34 in flow cytometry. We found that medium perfusion increased the gene expression of endothelial markers such as CD31, von Willebrand factor (vWF), CD140b, CD309, and CD144 while decreasing the gene expression of the erythrocyte-surface marker CD235a. Hepatic differentiation was promoted under perfusion conditions as demonstrated by lower AFP and higher albumin secretion compared with cultures not exposed to medium perfusion. Additionally, cultures exposed to medium perfusion gave higher rates of glucose consumption and lactate production, indicating increased metabolic activity. In conclusion, high-density bioreactors configured to provide constant medium perfusion significantly induced hepatic and endothelial cell differentiation and provided improved conditions for the culture of human fetal liver cells compared with cultures without perfusion. PMID:25559936

  19. Induction of Hepatic and Endothelial Differentiation by Perfusion in a Three-Dimensional Cell Culture Model of Human Fetal Liver.

    PubMed

    Pekor, Christopher; Gerlach, Jörg C; Nettleship, Ian; Schmelzer, Eva

    2015-07-01

    The development of functional engineered tissue constructs depends on high cell densities and appropriate vascularization. In this study we implemented a four-compartment three-dimensional perfusion bioreactor culture model for studying the effects of medium perfusion on endothelial, hepatic, and hematopoietic cell populations of primary human fetal liver in an in vivo-like environment. Human fetal liver cells were cultured in bioreactors configured to provide either perfusion or diffusion conditions. Metabolic activities of the cultures were monitored daily by measuring glucose consumption and lactate production. Cell viability during culture was analyzed by lactate dehydrogenase activity. Hepatic functionality was determined by the release of albumin and alpha-fetoprotein (AFP) in culture medium samples. After 4 days of culture, cells were analyzed for the expression of a variety of endothelial, hepatic, and hematopoietic genes, as well as the surface marker expression of CD31 and CD34 in flow cytometry. We found that medium perfusion increased the gene expression of endothelial markers such as CD31, von Willebrand factor (vWF), CD140b, CD309, and CD144 while decreasing the gene expression of the erythrocyte-surface marker CD235a. Hepatic differentiation was promoted under perfusion conditions as demonstrated by lower AFP and higher albumin secretion compared with cultures not exposed to medium perfusion. Additionally, cultures exposed to medium perfusion gave higher rates of glucose consumption and lactate production, indicating increased metabolic activity. In conclusion, high-density bioreactors configured to provide constant medium perfusion significantly induced hepatic and endothelial cell differentiation and provided improved conditions for the culture of human fetal liver cells compared with cultures without perfusion. PMID:25559936

  20. Human induced pluripotent stem cell-derived hepatic cell lines as a new model for host interaction with hepatitis B virus

    PubMed Central

    Kaneko, Shun; Kakinuma, Sei; Asahina, Yasuhiro; Kamiya, Akihide; Miyoshi, Masato; Tsunoda, Tomoyuki; Nitta, Sayuri; Asano, Yu; Nagata, Hiroko; Otani, Satoshi; Kawai-Kitahata, Fukiko; Murakawa, Miyako; Itsui, Yasuhiro; Nakagawa, Mina; Azuma, Seishin; Nakauchi, Hiromitsu; Nishitsuji, Hironori; Ujino, Saneyuki; Shimotohno, Kunitada; Iwamoto, Masashi; Watashi, Koichi; Wakita, Takaji; Watanabe, Mamoru

    2016-01-01

    Hepatitis B virus (HBV) is not eradicated by current antiviral therapies due to persistence of HBV covalently closed circular DNA (cccDNA) in host cells, and thus development of novel culture models for productive HBV infection is urgently needed, which will allow the study of HBV cccDNA eradication. To meet this need, we developed culture models of HBV infection using human induced pluripotent stem cell-derived hepatocyte lineages, including immature proliferating hepatic progenitor-like cell lines (iPS-HPCs) and differentiated hepatocyte-like cells (iPS-Heps). These cells were susceptible to HBV infection, produced HBV particles, and maintained innate immune responses. The infection efficiency of HBV in iPS-HPCs predominantly depended on the expression levels of sodium taurocholate cotransporting polypeptide (NTCP), and was low relative to iPS-Heps: however, long-term culture of iPS-Heps was difficult. To provide a model for HBV persistence, iPS-HPCs overexpressing NTCP were established. The long-term persistence of HBV cccDNA was detected in iPS-HPCs overexpressing NTCP, and depended on the inhibition of the Janus-kinase signaling pathway. In conclusion, this study provides evidence that iPS-derived hepatic cell lines can be utilized for novel HBV culture models with genetic variation to investigate the interactions between HBV and host cells and the development of anti-HBV strategies. PMID:27386799

  1. Proinsulin multi-peptide immunotherapy induces antigen-specific regulatory T cells and limits autoimmunity in a humanized model.

    PubMed

    Gibson, V B; Nikolic, T; Pearce, V Q; Demengeot, J; Roep, B O; Peakman, M

    2015-12-01

    Peptide immunotherapy (PIT) is a targeted therapeutic approach, involving administration of disease-associated peptides, with the aim of restoring antigen-specific immunological tolerance without generalized immunosuppression. In type 1 diabetes, proinsulin is a primary antigen targeted by the autoimmune response, and is therefore a strong candidate for exploitation via PIT in this setting. To elucidate the optimal conditions for proinsulin-based PIT and explore mechanisms of action, we developed a preclinical model of proinsulin autoimmunity in a humanized HLA-DRB1*0401 transgenic HLA-DR4 Tg mouse. Once proinsulin-specific tolerance is broken, HLA-DR4 Tg mice develop autoinflammatory responses, including proinsulin-specific T cell proliferation, interferon (IFN)-γ and autoantibody production. These are preventable and quenchable by pre- and post-induction treatment, respectively, using intradermal proinsulin-PIT injections. Intradermal proinsulin-PIT enhances proliferation of regulatory [forkhead box protein 3 (FoxP3(+))CD25(high) ] CD4 T cells, including those capable of proinsulin-specific regulation, suggesting this as its main mode of action. In contrast, peptide delivered intradermally on the surface of vitamin D3-modulated (tolerogenic) dendritic cells, controls autoimmunity in association with proinsulin-specific IL-10 production, but no change in regulatory CD4 T cells. These studies define a humanized, translational model for in vivo optimization of PIT to control autoimmunity in type 1 diabetes and indicate that dominant mechanisms of action differ according to mode of peptide delivery. PMID:26206289

  2. Enhanced Efficacy of Human Brain-Derived Neural Stem Cells by Transplantation of Cell Aggregates in a Rat Model of Parkinson's Disease

    PubMed Central

    Shin, Eun Sil; Hwang, Onyou; Hwang, Yu-Shik; Suh, Jun-Kyo Francis; Chun, Young Il

    2014-01-01

    Objective Neural tissue transplantation has been a promising strategy for the treatment of Parkinson's disease (PD). However, transplantation has the disadvantages of low-cell survival and/or development of dyskinesia. Transplantation of cell aggregates has the potential to overcome these problems, because the cells can extend their axons into the host brain and establish synaptic connections with host neurons. In this present study, aggregates of human brain-derived neural stem cells (HB-NSC) were transplanted into a PD animal model and compared to previous report on transplantation of single-cell suspensions. Methods Rats received an injection of 6-OHDA into the right medial forebrain bundle to generate the PD model and followed by injections of PBS only, or HB-NSC aggregates in PBS into the ipsilateral striatum. Behavioral tests, multitracer (2-deoxy-2-[18F]-fluoro-D-glucose ([18F]-FDG) and [18F]-N-(3-fluoropropyl)-2-carbomethoxy-3-(4-iodophenyl)nortropane ([18F]-FP-CIT) microPET scans, as well as immunohistochemical (IHC) and immunofluorescent (IF) staining were conducted to evaluate the results. Results The stepping test showed significant improvement of contralateral forelimb control in the HB-NSC group from 6-10 weeks compared to the control group (p<0.05). [18F]-FP-CIT microPET at 10 weeks posttransplantation demonstrated a significant increase in uptake in the HB-NSC group compared to pretransplantation (p<0.05). In IHC and IF staining, tyrosine hydroxylase and human β2 microglobulin (a human cell marker) positive cells were visualized at the transplant site. Conclusion These results suggest that the HB-NSC aggregates can survive in the striatum and exert therapeutic effects in a PD model by secreting dopamine. PMID:25535514

  3. Human bronchial epithelial BEAS-2B cells, an appropriate in vitro model to study heavy metals induced carcinogenesis

    SciTech Connect

    Park, Youn-hee; Kim, Donghern; Dai, Jin; Zhang, Zhuo

    2015-09-15

    Occupational and environmental exposure to arsenic (III) and chromium VI (Cr(VI)) have been confirmed to cause lung cancer. Mechanisms of these metals carcinogenesis are still under investigation. Selection of cell lines to be used is essential for the studies. Human bronchial epithelial BEAS-2B cells are the cells to be utilized by most of scientists. However, due to p53 missense mutation (CCG → TCG) at codon 47 and the codon 72 polymorphism (CGC → CCC) in BEAS-2B cells, its usage has frequently been questioned. The present study has examined activity and expression of 53 and its downstream target protein p21 upon acute or chronic exposure of BEAS-2B cells to arsenic and Cr(VI). The results show that short-term exposure of BEAS-2B cells to arsenic or Cr(VI) was able to activate both p53 and p21. Chronic exposure of BEAS-2B cells to these two metals caused malignant cell transformation and tumorigenesis. In arsenic-transformed BEAS-2B cells reductions in p53 promoter activity, mRNA expression, and phosphorylation of p53 at Ser392 were observed, while the total p53 protein level remained the same compared to those in passage-matched parent ones. p21 promoter activity and expression were decreased in arsenic-transformed cells. Cr(VI)-transformed cells exhibit elevated p53 promoter activity, mRNA expression, and phosphorylation at Ser15, but reduced phosphorylation at Ser392 and total p53 protein level compared to passage-matched parent ones. p21 promoter activity and expression were elevated in Cr(VI)-transformed cells. These results demonstrate that p53 is able to respond to exposure of arsenic or Cr(VI), suggesting that BEAS-2B cells are an appropriate in vitro model to investigate arsenic or Cr(VI) induced lung cancer. - Highlights: • Short-term exposure of BEAS-2B cells to arsenic or Cr(VI) activates p53 and p21. • Chronic exposure of BEAS-2B cells to arsenic or Cr(VI) causes cell transformation and tumorigenesis. • Arsenic-transformed cells exhibit

  4. Human ovarian tumor cell interactions with extracellular matrix: development of a model to study tumor cell invasion

    SciTech Connect

    Niedbala, M.J.

    1986-01-01

    In order to investigate the mechanisms involved in ovarian carcinoma cell implantation and the associated tumor cell-host interactions, a model system was developed employing a mesothelial cell line grown on bovine corneal endothelial cell extracellular matrix (ECM), in an attempt to reconstruct the mesothelium in vitro. Morphologic alterations of the reconstructed mesothelium induced by OCC were observed using immunohistochemical staining, light and electron microscopy. A relationship was observed between extracellular ..beta..-N-acetylhexosaminidase activity and (1) the ability of OCC to morphologically degrade ECM; (2) the capacity of OCC to degrade (/sup 3/H)-glucosamine radiolabelled ECM. The rate of accumulation of extracellular hexosaminidase in cell free-conditioned medium was progressive and closely paralleled the rate of OCC mediated release of (/sup 3/H)-glucosamine from ECM. Purified hexosaminidase (placental and/or OCC) was observed to directly hydrolzye (/sup 3/H)-glucosamine radiolabelled structurally intact ECM (up to 70% radiolabel) and resulted in the cumulative release of free (/sup 3/H)-N-acetylglucosamine.

  5. Cardiac repair in a porcine model of acute myocardial infarction with human induced pluripotent stem cell-derived cardiovascular cell populations

    PubMed Central

    Ye, Lei; Chang, Ying-Hua; Xiong, Qiang; Zhang, Pengyuan; Zhang, Liying; Somasundaram, Porur; Lepley, Mike; Swingen, Cory; Su, Liping; Wendel, Jacqueline S.; Guo, Jing; Jang, Albert; Rosenbush, Daniel; Greder, Lucas; Dutton, James R.; Zhang, Jianhua; Kamp, Timothy J.; Kaufman, Dan S.; Ge, Ying; Zhang, Jianyi

    2014-01-01

    Summary Human induced pluripotent stem cells (hiPSCs) hold promise for myocardial repair following injury, but preclinical studies in large animal models are required to determine optimal cell preparation and delivery strategies to maximize functional benefits and to evaluate safety. Here, we utilized a porcine model of acute myocardial infarction (MI) to investigate the functional impact of intramyocardial transplantation of hiPSC-derived cardiomyocytes, endothelial cells, and smooth muscle cells, in combination with a 3D fibrin patch loaded with insulin growth factor (IGF)-encapsulated microspheres. hiPSC-derived cardiomyocytes integrated into host myocardium and generated organized sarcomeric structures, and endothelial and smooth muscle cells contributed to host vasculature. Tri-lineage cell transplantation significantly improved left ventricular function, myocardial metabolism, and arteriole density, while reducing infarct size, ventricular wall stress and apoptosis without inducing ventricular arrhythmias. These findings in a large animal MI model highlight the potential of utilizing hiPSC-derived cells for cardiac repair. PMID:25479750

  6. Immortalized human cerebral microvascular endothelial cells maintain the properties of primary cells in an in vitro model of immune migration across the blood brain barrier

    PubMed Central

    Daniels, Brian P.; Cruz-Orengo, Lillian; Pasieka, Tracy Jo; Couraud, Pierre-Olivier; Romero, Ignacio A.; Weksler, Babette; Cooper, John A.; Doering, Tamara L.; Klein, Robyn S.

    2012-01-01

    The immortalized human cerebral microvascular endothelial cell line HCMEC/D3 presents a less expensive and more logistically feasible alternative to primary human brain microvascular endothelial cells (HBMEC’s) for use in constructing in vitro models of the blood brain barrier (BBB). However, the fidelity of the HCMEC/D3 cell line to primary HBMEC’s in studies of immune transmigration has yet to be established. Flow cytometric analysis of primary human leukocyte migration across in vitro BBB’s generated with either HCMEC/D3 or primary HBMEC’s revealed that HCMEC/D3 maintains the immune barrier properties of primary HBMEC’s. Leukocyte migration responses and inflammatory cytokine production were statistically indistinguishable between both endothelial cell types, and both cell types responded similarly to astrocyte coculture, stimulation of leukocytes with phorbol myristate acetate (PMA) and ionomycin, and inflammatory cytokine treatment. This report is the first to validate the HCMEC/D3 cell line in a neuroimmunological experimental system via direct comparison to primary HBMEC’s, demonstrating remarkable fidelity in terms of barrier resistance, immune migration profiles, and responsiveness to inflammatory cytokines. Moreover, we report novel findings demonstrating that interaction effects between immune cells and resident CNS cells are preserved in HCMEC/D3, suggesting that important characteristics of neuroimmune interactions during CNS inflammation are preserved in systems utilizing this cell line. Together, these findings demonstrate that HCMEC/D3 is a valid and powerful tool for less expensive and higher throughput in vitro investigations of immune migration at the BBB. PMID:23068604

  7. Therapeutic Effects of CUR-Activated Human Umbilical Cord Mesenchymal Stem Cells on 1-Methyl-4-phenylpyridine-Induced Parkinson's Disease Cell Model

    PubMed Central

    Jinfeng, Li; Yunliang, Wang; Xinshan, Liu; Yutong, Wang; Shanshan, Wang; Peng, Xue; Xiaopeng, Yang; Zhixiu, Xu; Qingshan, Lu; Honglei, Yin; Xia, Cao; Hongwei, Wang; Bingzhen, Cao

    2016-01-01

    The purpose of this study is to evaluate the therapeutic effects of human umbilical cord-derived mesenchymal stem cells (hUC-MSC) activated by curcumin (CUR) on PC12 cells induced by 1-methyl-4-phenylpyridinium ion (MPP+), a cell model of Parkinson's disease (PD). The supernatant of hUC-MSC and hUC-MSC activated by 5 µmol/L CUR (hUC-MSC-CUR) were collected in accordance with the same concentration. The cell proliferation and differentiation potential to dopaminergic neuronal cells and antioxidation were observed in PC12 cells after being treated with the above two supernatants and 5 µmol/L CUR. The results showed that the hUC-MSC-CUR could more obviously promote the proliferation and the expression of tyrosine hydroxylase (TH) and microtubule associated protein-2 (MAP2) and significantly decreased the expression of nitric oxide (NO) and inducible nitric oxide synthase (iNOS) in PC12 cells. Furtherly, cytokines detection gave a clue that the expression of IL-6, IL-10, and NGF was significantly higher in the group treated with the hUC-MSC-CUR compared to those of other two groups. Therefore, the hUC-MSC-CUR may be a potential strategy to promote the proliferation and differentiation of PD cell model, therefore providing new insights into a novel therapeutic approach in PD. PMID:27340670

  8. Transcriptome Profiling Reveals Degree of Variability in Induced Pluripotent Stem Cell Lines: Impact for Human Disease Modeling.

    PubMed

    Schuster, Jens; Halvardson, Jonatan; Pilar Lorenzo, Laureanne; Ameur, Adam; Sobol, Maria; Raykova, Doroteya; Annerén, Göran; Feuk, Lars; Dahl, Niklas

    2015-10-01

    Induced pluripotent stem cell (iPSC) technology has become an important tool for disease modeling. Insufficient data on the variability among iPSC lines derived from a single somatic parental cell line have in practice led to generation and analysis of several, usually three, iPSC sister lines from each parental cell line. We established iPSC lines from a human fibroblast line (HDF-K1) and used transcriptome sequencing to investigate the variation among three sister lines (iPSC-K1A, B, and C). For comparison, we analyzed the transcriptome of an iPSC line (iPSC-K5B) derived from a different fibroblast line (HDF-K5), a human embryonic stem cell (ESC) line (ESC-HS181), as well as the two parental fibroblast lines. All iPSC lines fulfilled stringent criteria for pluripotency. In an unbiased cluster analysis, all stem cell lines (four iPSCs and one ESC) clustered together as opposed to the parental fibroblasts. The transcriptome profiles of the three iPSC sister lines were indistinguishable from each other, and functional pathway analysis did not reveal any significant hits. In contrast, the expression profiles of the ESC line and the iPSC-K5B line were distinct from that of the sister lines iPSC-K1A, B, and C. Differentiation to embryoid bodies and subsequent analysis of germ layer markers in the five stem cell clones confirmed that the distribution of their expression profiles was retained. Taken together, our observations stress the importance of using iPSCs of different parental origin rather than several sister iPSC lines to distinguish disease-associated mechanisms from genetic background effects in disease modeling. PMID:26348590

  9. Feasibility of Human Amniotic Fluid Derived Stem Cells in Alleviation of Neuropathic Pain in Chronic Constrictive Injury Nerve Model

    PubMed Central

    Chiang, Chien-Yi; Liu, Shih-An; Sheu, Meei-Ling; Chen, Fu-Chou; Chen, Chun-Jung; Su, Hong-Lin; Pan, Hung-Chuan

    2016-01-01

    Purpose The neurobehavior of neuropathic pain by chronic constriction injury (CCI) of sciatic nerve is very similar to that in humans, and it is accompanied by a profound local inflammation response. In this study, we assess the potentiality of human amniotic fluid derived mesenchymal stem cells (hAFMSCs) for alleviating the neuropathic pain in a chronic constriction nerve injury model. Methods and Methods This neuropathic pain animal model was conducted by four 3–0 chromic gut ligatures loosely ligated around the left sciatic nerve in Sprague—Dawley rats. The intravenous administration of hAFMSCs with 5x105 cells was conducted for three consecutive days. Results The expression IL-1β, TNF-α and synaptophysin in dorsal root ganglion cell culture was remarkably attenuated when co-cultured with hAFMSCs. The significant decrease of PGP 9.5 in the skin after CCI was restored by administration of hAFMSCs. Remarkably increased expression of CD 68 and TNF-α and decreased S-100 and neurofilament expression in injured nerve were rescued by hAFMSCs administration. Increases in synaptophysin and TNF-α over the dorsal root ganglion were attenuated by hAFMSCs. Significant expression of TNF-α and OX-42 over the dorsal spinal cord was substantially attenuated by hAFMSCs. The increased amplitude of sensory evoked potential as well as expression of synaptophysin and TNF-α expression was alleviated by hAFMSCs. Human AFMSCs significantly improved the threshold of mechanical allodynia and thermal hyperalgesia as well as various parameters of CatWalk XT gait analysis. Conclusion Human AFMSCs administration could alleviate the neuropathic pain demonstrated in histomorphological alteration and neurobehavior possibly through the modulation of the inflammatory response. PMID:27441756

  10. Cell motion predicts human epidermal stemness

    PubMed Central

    Toki, Fujio; Tate, Sota; Imai, Matome; Matsushita, Natsuki; Shiraishi, Ken; Sayama, Koji; Toki, Hiroshi; Higashiyama, Shigeki

    2015-01-01

    Image-based identification of cultured stem cells and noninvasive evaluation of their proliferative capacity advance cell therapy and stem cell research. Here we demonstrate that human keratinocyte stem cells can be identified in situ by analyzing cell motion during their cultivation. Modeling experiments suggested that the clonal type of cultured human clonogenic keratinocytes can be efficiently determined by analysis of early cell movement. Image analysis experiments demonstrated that keratinocyte stem cells indeed display a unique rotational movement that can be identified as early as the two-cell stage colony. We also demonstrate that α6 integrin is required for both rotational and collective cell motion. Our experiments provide, for the first time, strong evidence that cell motion and epidermal stemness are linked. We conclude that early identification of human keratinocyte stem cells by image analysis of cell movement is a valid parameter for quality control of cultured keratinocytes for transplantation. PMID:25897083