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Sample records for human cell models

  1. Stem Cell Models of Human Brain Development.

    PubMed

    Kelava, Iva; Lancaster, Madeline A

    2016-06-01

    Recent breakthroughs in pluripotent stem cell technologies have enabled a new class of in vitro systems for functional modeling of human brain development. These advances, in combination with improvements in neural differentiation methods, allow the generation of in vitro systems that reproduce many in vivo features of the brain with remarkable similarity. Here, we describe advances in the development of these methods, focusing on neural rosette and organoid approaches, and compare their relative capabilities and limitations. We also discuss current technical hurdles for recreating the cell-type complexity and spatial architecture of the brain in culture and offer potential solutions.

  2. Modelling human disease with pluripotent stem cells

    PubMed Central

    Park, In-Hyun; Sullivan, Gareth J.

    2013-01-01

    Recent progress in the field of cellular reprogramming has opened up the doors to a new era of disease modelling, as pluripotent stem cells representing a myriad of genetic diseases can now be produced from patient tissue. These cells can be expanded and differentiated to produce a potentially limitless supply of the affected cell type, which can then be used as a tool to improve understanding of disease mechanisms and test therapeutic interventions. This process requires high levels of scrutiny and validation at every stage, but international standards for the characterisation of pluripotent cells and their progeny have yet to be established. Here we discuss the current state of the art with regard to modelling diseases affecting the ectodermal, mesodermal and endodermal lineages, focussing on studies which have demonstrated a disease phenotype in the tissue of interest. We also discuss the utility of pluripotent cell technology for the modelling of cancer and infectious disease. Finally, we spell out the technical and scientific challenges which must be addressed if the field is to deliver on its potential and produce improved patient outcomes in the clinic. PMID:23444871

  3. Induced pluripotent stem cells for modelling human diseases

    PubMed Central

    Unternaehrer, Juli J.; Daley, George Q.

    2011-01-01

    Research into the pathophysiological mechanisms of human disease and the development of targeted therapies have been hindered by a lack of predictive disease models that can be experimentally manipulated in vitro. This review describes the current state of modelling human diseases with the use of human induced pluripotent stem (iPS) cell lines. To date, a variety of neurodegenerative diseases, haematopoietic disorders, metabolic conditions and cardiovascular pathologies have been captured in a Petri dish through reprogramming of patient cells into iPS cells followed by directed differentiation of disease-relevant cells and tissues. However, realizing the true promise of iPS cells for advancing our basic understanding of disease and ultimately providing novel cell-based therapies will require more refined protocols for generating the highly specialized cells affected by disease, coupled with strategies for drug discovery and cell transplantation. PMID:21727133

  4. Chimeric animal models in human stem cell biology.

    PubMed

    Glover, Joel C; Boulland, Jean-Luc; Halasi, Gabor; Kasumacic, Nedim

    2009-01-01

    The clinical use of stem cells for regenerative medicine is critically dependent on preclinical studies in animal models. In this review we examine some of the key issues and challenges in the use of animal models to study human stem cell biology-experimental standardization, body size, immunological barriers, cell survival factors, fusion of host and donor cells, and in vivo imaging and tracking. We focus particular attention on the various imaging modalities that can be used to track cells in living animals, comparing their strengths and weaknesses and describing technical developments that are likely to lead to new opportunities for the dynamic assessment of stem cell behavior in vivo. We then provide an overview of some of the most commonly used animal models, their advantages and disadvantages, and examples of their use for xenotypic transplantation of human stem cells, with separate reviews of models involving rodents, ungulates, nonhuman primates, and the chicken embryo. As the use of human somatic, embryonic, and induced pluripotent stem cells increases, so too will the range of applications for these animal models. It is likely that increasingly sophisticated uses of human/animal chimeric models will be developed through advances in genetic manipulation, cell delivery, and in vivo imaging.

  5. Using Human Induced Pluripotent Stem Cells to Model Skeletal Diseases.

    PubMed

    Barruet, Emilie; Hsiao, Edward C

    2016-01-01

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

  6. Modeling neurodevelopmental disorders using human pluripotent stem cells.

    PubMed

    Telias, Michael; Ben-Yosef, Dalit

    2014-08-01

    Neurodevelopmental disorders (NDs) are impairments that affect the development and growth of the brain and the central nervous system during embryonic and early postnatal life. Genetically manipulated animals have contributed greatly to the advancement of ND research, but many of them differ considerably from the human phenotype. Cellular in vitro models are also valuable, but the availability of human neuronal cells is limited and their lifespan in culture is short. Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, comprise a powerful tool for studying developmentally regulated diseases, including NDs. We reviewed all recent studies in which hPSCs were used as in vitro models for diseases and syndromes characterized by impairment of neurogenesis or synaptogenesis leading to intellectual disability and delayed neurodevelopment. We analyzed their methodology and results, focusing on the data obtained following in vitro neural differentiation and gene expression and profiling of the derived neurons. Electrophysiological recording of action potentials, synaptic currents and response to neurotransmitters is pivotal for validation of the neuronal fate as well as for assessing phenotypic dysfunctions linked to the disease in question. We therefore focused on the studies which included electrophysiological recordings on the in vitro-derived neurons. Finally, we addressed specific issues that are critical for the advancement of this area of research, specifically in providing a reliable human pre-clinical research model and drug screening platform. PMID:24728983

  7. 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.

  8. 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.

  9. An Optimised Human Cell Culture Model for Alveolar Epithelial Transport

    PubMed Central

    Birch, Nigel P.; Suresh, Vinod

    2016-01-01

    Robust and reproducible in vitro models are required for investigating the pathways involved in fluid homeostasis in the human alveolar epithelium. We performed functional and phenotypic characterisation of ion transport in the human pulmonary epithelial cell lines NCI-H441 and A549 to determine their similarity to primary human alveolar type II cells. NCI-H441 cells exhibited high expression of junctional proteins ZO-1, and E-cadherin, seal-forming claudin-3, -4, -5 and Na+-K+-ATPase while A549 cells exhibited high expression of pore-forming claudin-2. Consistent with this phenotype NCI-H441, but not A549, cells formed a functional barrier with active ion transport characterised by higher electrical resistance (529 ± 178 Ω cm2 vs 28 ± 4 Ω cm2), lower paracellular permeability ((176 ± 42) ×10−8 cm/s vs (738 ± 190) ×10−8 cm/s) and higher transepithelial potential difference (11.9 ± 4 mV vs 0 mV). Phenotypic and functional properties of NCI-H441 cells were tuned by varying cell seeding density and supplement concentrations. The cells formed a polarised monolayer typical of in vivo epithelium at seeding densities of 100,000 cells per 12-well insert while higher densities resulted in multiple cell layers. Dexamethasone and insulin-transferrin-selenium supplements were required for the development of high levels of electrical resistance, potential difference and expression of claudin-3 and Na+-K+-ATPase. Treatment of NCI-H441 cells with inhibitors and agonists of sodium and chloride channels indicated sodium absorption through ENaC under baseline and forskolin-stimulated conditions. Chloride transport was not sensitive to inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) under either condition. Channels inhibited by 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB) contributed to chloride secretion following forskolin stimulation, but not at baseline. These data precisely define experimental conditions for the application of NCI

  10. Acrylamide induces accelerated endothelial aging in a human cell model.

    PubMed

    Sellier, Cyril; Boulanger, Eric; Maladry, François; Tessier, Frédéric J; Lorenzi, Rodrigo; Nevière, Rémi; Desreumaux, Pierre; Beuscart, Jean-Baptiste; Puisieux, François; Grossin, Nicolas

    2015-09-01

    Acrylamide (AAM) has been recently discovered in food as a Maillard reaction product. AAM and glycidamide (GA), its metabolite, have been described as probably carcinogenic to humans. It is widely established that senescence and carcinogenicity are closely related. In vitro, endothelial aging is characterized by replicative senescence in which primary cells in culture lose their ability to divide. Our objective was to assess the effects of AAM and GA on human endothelial cell senescence. Human umbilical vein endothelial cells (HUVECs) cultured in vitro were used as model. HUVECs were cultured over 3 months with AAM or GA (1, 10 or 100 μM) until growth arrest. To analyze senescence, β-galactosidase activity and telomere length of HUVECs were measured by cytometry and semi-quantitative PCR, respectively. At all tested concentrations, AAM or GA reduced cell population doubling compared to the control condition (p < 0.001). β-galactosidase activity in endothelial cells was increased when exposed to AAM (≥10 μM) or GA (≥1 μM) (p < 0.05). AAM (≥10 μM) or GA (100 μM) accelerated telomere shortening in HUVECs (p < 0.05). In conclusion, in vitro chronic exposure to AAM or GA at low concentrations induces accelerated senescence. This result suggests that an exposure to AAM might contribute to endothelial aging.

  11. 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

  12. 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.

  13. 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

  14. 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

  15. Modeling Fragile X Syndrome Using Human Pluripotent Stem Cells

    PubMed Central

    Mor-Shaked, Hagar; Eiges, Rachel

    2016-01-01

    Fragile X syndrome (FXS) is the most common heritable form of cognitive impairment. It results from a loss-of-function mutation by a CGG repeat expansion at the 5′ untranslated region of the X-linked fragile X mental retardation 1 (FMR1) gene. Expansion of the CGG repeats beyond 200 copies results in protein deficiency by leading to aberrant methylation of the FMR1 promoter and the switch from active to repressive histone modifications. Additionally, the CGGs become increasingly unstable, resulting in high degree of variation in expansion size between and within tissues of affected individuals. It is still unclear how the FMR1 protein (FMRP) deficiency leads to disease pathology in neurons. Nor do we know the mechanisms by which the CGG expansion results in aberrant DNA methylation, or becomes unstable in somatic cells of patients, at least in part due to the lack of appropriate animal or cellular models. This review summarizes the current contribution of pluripotent stem cells, mutant human embryonic stem cells, and patient-derived induced pluripotent stem cells to disease modeling of FXS for basic and applied research, including the development of new therapeutic approaches. PMID:27690107

  16. Severe combined immunodeficiency mice engrafted with human T cells, B cells, and myeloid cells after transplantation with human fetal bone marrow or liver cells and implanted with human fetal thymus: a model for studying human gene therapy.

    PubMed

    Yurasov, S; Kollmann, T R; Kim, A; Raker, C A; Hachamovitch, M; Wong-Staal, F; Goldstein, H

    1997-03-01

    To develop an in vivo model wherein human hematopoiesis occurs, we transplanted severe combined immunodeficiency (SCID) mice with either human fetal bone marrow (HFBM) or human fetal liver (HFL). After transplantation of SCID mice with cultured HFBM (BM-SCID-hu mice) or HFL cells (Liv-SCID-hu mice), significant engraftment of the mouse bone marrow (BM) and population of the peripheral blood with human leukocytes was detected. Human colony-forming unit-granulocyte macrophage and burst forming unit-erythroid were detected in the BM of the BM-SCID-hu and Liv-SCID-hu mice up to 8 months after transplantation. When the HFBM or HFL cells were transduced with a retroviral vector before transplantation, integrated retroviral sequences were detected in human precursor cells present in the SCID mouse BM and in leukocytes circulating in the peripheral blood (PB) up to 7 months after transplantation. The PB of the BM-SCID-hu mice also became populated with human T cells after implantation with human thymic tissue, which provided a human microenvironment wherein human pre-T cells from the BM could mature. When the HFBM was retrovirally transduced before transplantation, integrated retrovirus was detected in sorted CD4+CD8+ double positive and CD4+ single positive cells from the thymic implant and CD4+ cells from the PB. Taken together, these data indicated that the BM of our BM-SCID-hu and Liv-SCID-hu mice became engrafted with retrovirally transduced human hematopoietic precursors that undergo the normal human hematopoietic program and populate the mouse PB with human cells containing integrated retroviral sequences. In addition to being a model for studying in vivo human hematopoiesis, these mice should also prove to be a useful model for investigating in vivo gene therapy using human stem/precursor cells.

  17. Kidney organoids from human iPS cells contain multiple lineages and model human nephrogenesis.

    PubMed

    Takasato, Minoru; Er, Pei X; Chiu, Han S; Maier, Barbara; Baillie, Gregory J; Ferguson, Charles; Parton, Robert G; Wolvetang, Ernst J; Roost, Matthias S; Chuva de Sousa Lopes, Susana M; Little, Melissa H

    2015-10-22

    The human kidney contains up to 2 million epithelial nephrons responsible for blood filtration. Regenerating the kidney requires the induction of the more than 20 distinct cell types required for excretion and the regulation of pH, and electrolyte and fluid balance. We have previously described the simultaneous induction of progenitors for both collecting duct and nephrons via the directed differentiation of human pluripotent stem cells. Paradoxically, although both are of intermediate mesoderm in origin, collecting duct and nephrons have distinct temporospatial origins. Here we identify the developmental mechanism regulating the preferential induction of collecting duct versus kidney mesenchyme progenitors. Using this knowledge, we have generated kidney organoids that contain nephrons associated with a collecting duct network surrounded by renal interstitium and endothelial cells. Within these organoids, individual nephrons segment into distal and proximal tubules, early loops of Henle, and glomeruli containing podocytes elaborating foot processes and undergoing vascularization. When transcription profiles of kidney organoids were compared to human fetal tissues, they showed highest congruence with first trimester human kidney. Furthermore, the proximal tubules endocytose dextran and differentially apoptose in response to cisplatin, a nephrotoxicant. Such kidney organoids represent powerful models of the human organ for future applications, including nephrotoxicity screening, disease modelling and as a source of cells for therapy.

  18. 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

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

    PubMed

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

    2015-09-01

    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.

  20. Stem cells on the brain: modeling neurodevelopmental and neurodegenerative diseases using human induced pluripotent stem cells.

    PubMed

    Srikanth, Priya; Young-Pearse, Tracy L

    2014-01-01

    Seven years have passed since the initial report of the generation of induced pluripotent stem cells (iPSCs) from adult human somatic cells, and in the intervening time the field of neuroscience has developed numerous disease models using this technology. Here, we review progress in the field and describe both the advantages and potential pitfalls of modeling neurodegenerative and neurodevelopmental diseases using this technology. We include tables with information on neural differentiation protocols and studies that developed human iPSC lines to model neurological diseases. We also discuss how one can: investigate effects of genetic mutations with iPSCs, examine cell fate-specific phenotypes, best determine the specificity of a phenotype, and bring in vivo relevance to this in vitro technique.

  1. 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

  2. Characterization of Human Huntington's Disease Cell Model from Induced Pluripotent Stem Cells

    PubMed Central

    Zhang, Ningzhe; An, Mahru C.; Montoro, Daniel; Ellerby, Lisa M.

    2010-01-01

    Huntington’s disease (HD) is a dominantly inherited neurodegenerative disease caused by a CAG repeat expansion in the first exon of the gene Huntingtin (Htt). A dramatic pathological change in HD is the massive loss of striatal neurons as the disease progresses. A useful advance in HD would be the generation of a human-derived HD model to use for drug screening and understanding mechanisms of HD. We utilized the recently established human iPS cell line derived from HD patient fibroblasts to derive neuronal precursors and human striatal neurons. To achieve this goal, the differentiation of the HD-iPS cells into striatal fate required several steps. First, we generated nestin+/PAX6+/SOX1+/OCT4- neural stem cells (NSCs) from HD-iPS cells using the method of embryoid body formation. HD-NSCs were then subjected to a differentiation condition combining morphogens and neurotrophins to induce striatal lineage commitment. Striatal neuronal precursors/immature neurons stained with β-III tubulin, calbindin and GABA but not DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000) were produced in this step. Finally, maturation and terminal differentiation of the striatal neuronal precursors/immature neurons resulted in striatal neurons expressing markers like DARPP-32. The HD-iPS cells derived striatal neurons and neuronal precursors contain the same CAG expansion as the mutation in the HD patient from whom the iPS cell line was established. Moreover, the HD-NSCs showed enhanced caspase activity upon growth factor deprivation compared to normal NSCs (from iPS or H9 NSCs). Therefore, these differentiated cells may produce a human HD cell model useful in the study of HD mechanisms and drug screening. PMID:21037797

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

    PubMed

    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

    2015-10-01

    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

  4. 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

  5. Modeling hippocampal neurogenesis using human pluripotent stem cells.

    PubMed

    Yu, Diana Xuan; Di Giorgio, Francesco Paolo; Yao, Jun; Marchetto, Maria Carolina; Brennand, Kristen; Wright, Rebecca; Mei, Arianna; McHenry, Lauren; Lisuk, David; Grasmick, Jaeson Michael; Silberman, Pedro; Silberman, Giovanna; Jappelli, Roberto; Gage, Fred H

    2014-03-11

    The availability of human pluripotent stem cells (hPSCs) offers the opportunity to generate lineage-specific cells to investigate mechanisms of human diseases specific to brain regions. Here, we report a differentiation paradigm for hPSCs that enriches for hippocampal dentate gyrus (DG) granule neurons. This differentiation paradigm recapitulates the expression patterns of key developmental genes during hippocampal neurogenesis, exhibits characteristics of neuronal network maturation, and produces PROX1+ neurons that functionally integrate into the DG. Because hippocampal neurogenesis has been implicated in schizophrenia (SCZD), we applied our protocol to SCZD patient-derived human induced pluripotent stem cells (hiPSCs). We found deficits in the generation of DG granule neurons from SCZD hiPSC-derived hippocampal NPCs with lowered levels of NEUROD1, PROX1, and TBR1, reduced neuronal activity, and reduced levels of spontaneous neurotransmitter release. Our approach offers important insights into the neurodevelopmental aspects of SCZD and may be a promising tool for drug screening and personalized medicine.

  6. Use of genome editing tools in human stem cell-based disease modeling and precision medicine.

    PubMed

    Yuda, Wei; Shuang, Li; Gaigai, Liu; Yongxian, Zhang; Qiurong, Ding

    2015-10-01

    Precision medicine emerges as a new approach that takes into account individual variability. The successful conduct of precision medicine requires the use of precise disease models. Human pluripotent stem cells (hPSCs), as well as adult stem cells, can be differentiated into a variety of human somatic cell types that can be used for research and drug screening. The development of genome editing technology over the past few years, especially the CRISPR/Cas system, has made it feasible to precisely and efficiently edit the genetic background. Therefore, disease modeling by using a combination of human stem cells and genome editing technology has offered a new platform to generate " personalized " disease models, which allow the study of the contribution of individual genetic variabilities to disease progression and the development of precise treatments. In this review, recent advances in the use of genome editing in human stem cells and the generation of stem cell models for rare diseases and cancers are discussed.

  7. Human cells and cell membrane molecular models are affected in vitro by chlorpromazine.

    PubMed

    Suwalsky, Mario; Villena, Fernando; Sotomayor, Carlos P; Bolognin, Silvia; Zatta, Paolo

    2008-06-01

    This study presents evidence that chlorpromazine (CPZ) affects human cells and cell membrane molecular models. Human SH-SY5Y neuroblastoma cells incubated with 0.1 mM CPZ suffered a decrease of cell viability. On the other hand, phase contrast microscopy observations of human erythrocytes indicated that they underwent a morphological alteration as 1 microM CPZ changed their discoid normal shape to stomatocytes, and to hemolysis with 1 mM CPZ. X-ray diffraction experiments performed on dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) bilayers, classes of the major phospholipids present in the outer and inner sides of the erythrocyte membrane, respectively showed that CPZ disordered the polar head and acyl chain regions of both DMPC and DMPE, where these interactions were stronger with DMPC bilayers. Fluorescence spectroscopy on DMPC LUV at 18 degrees C confirmed these results. In fact, the assays showed that CPZ induced a significant reduction of their generalized polarization (GP) and anisotropy (r) values, indicative of enhanced disorder at the polar head and acyl chain regions of the DMPC lipid bilayer. PMID:18372093

  8. 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

  9. Induced Pluripotent Stem Cells to Model Human Fibrodysplasia Ossificans Progressiva.

    PubMed

    Cai, Jie; Orlova, Valeria V; Cai, Xiujuan; Eekhoff, Elisabeth M W; Zhang, Keqin; Pei, Duanqing; Pan, Guangjin; Mummery, Christine L; Ten Dijke, Peter

    2015-12-01

    Fibrodysplasia ossificans progressiva (FOP) is a rare disease characterized by progressive ossification of soft tissues, for which there is no effective treatment. Mutations in the bone morphogenetic protein (BMP) type I receptor activin receptor-like kinase 2 (ACVR1/ALK2) are the main cause of FOP. We generated human induced pluripotent stem cells (hiPSCs) from FOP patients with the ALK2 R206H mutation. The mutant ALK2 gene changed differentiation efficiencies of hiPSCs into FOP bone-forming progenitors: endothelial cells (ECs) and pericytes. ECs from FOP hiPSCs showed reduced expression of vascular endothelial growth factor receptor 2 and could transform into mesenchymal cells through endothelial-mesenchymal transition. Increased mineralization of pericytes from FOP hiPSCs could be partly inhibited by the ALK2 kinase inhibitor LDN-212854. Thus, differentiated FOP hiPSCs recapitulate some aspects of the disease phenotype in vitro, and they could be instrumental in further elucidating underlying mechanisms of FOP and development of therapeutic drug candidates. PMID:26626181

  10. Induced Pluripotent Stem Cells to Model Human Fibrodysplasia Ossificans Progressiva

    PubMed Central

    Cai, Jie; Orlova, Valeria V.; Cai, Xiujuan; Eekhoff, Elisabeth M.W.; Zhang, Keqin; Pei, Duanqing; Pan, Guangjin; Mummery, Christine L.; ten Dijke, Peter

    2015-01-01

    Summary Fibrodysplasia ossificans progressiva (FOP) is a rare disease characterized by progressive ossification of soft tissues, for which there is no effective treatment. Mutations in the bone morphogenetic protein (BMP) type I receptor activin receptor-like kinase 2 (ACVR1/ALK2) are the main cause of FOP. We generated human induced pluripotent stem cells (hiPSCs) from FOP patients with the ALK2 R206H mutation. The mutant ALK2 gene changed differentiation efficiencies of hiPSCs into FOP bone-forming progenitors: endothelial cells (ECs) and pericytes. ECs from FOP hiPSCs showed reduced expression of vascular endothelial growth factor receptor 2 and could transform into mesenchymal cells through endothelial-mesenchymal transition. Increased mineralization of pericytes from FOP hiPSCs could be partly inhibited by the ALK2 kinase inhibitor LDN-212854. Thus, differentiated FOP hiPSCs recapitulate some aspects of the disease phenotype in vitro, and they could be instrumental in further elucidating underlying mechanisms of FOP and development of therapeutic drug candidates. PMID:26626181

  11. Induced Pluripotent Stem Cells to Model Human Fibrodysplasia Ossificans Progressiva.

    PubMed

    Cai, Jie; Orlova, Valeria V; Cai, Xiujuan; Eekhoff, Elisabeth M W; Zhang, Keqin; Pei, Duanqing; Pan, Guangjin; Mummery, Christine L; Ten Dijke, Peter

    2015-12-01

    Fibrodysplasia ossificans progressiva (FOP) is a rare disease characterized by progressive ossification of soft tissues, for which there is no effective treatment. Mutations in the bone morphogenetic protein (BMP) type I receptor activin receptor-like kinase 2 (ACVR1/ALK2) are the main cause of FOP. We generated human induced pluripotent stem cells (hiPSCs) from FOP patients with the ALK2 R206H mutation. The mutant ALK2 gene changed differentiation efficiencies of hiPSCs into FOP bone-forming progenitors: endothelial cells (ECs) and pericytes. ECs from FOP hiPSCs showed reduced expression of vascular endothelial growth factor receptor 2 and could transform into mesenchymal cells through endothelial-mesenchymal transition. Increased mineralization of pericytes from FOP hiPSCs could be partly inhibited by the ALK2 kinase inhibitor LDN-212854. Thus, differentiated FOP hiPSCs recapitulate some aspects of the disease phenotype in vitro, and they could be instrumental in further elucidating underlying mechanisms of FOP and development of therapeutic drug candidates.

  12. Human induced pluripotent stem cells in Parkinson's disease: A novel cell source of cell therapy and disease modeling.

    PubMed

    Li, Wen; Chen, Shengdi; Li, Jia-Yi

    2015-11-01

    Human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) are two novel cell sources for studying neurodegenerative diseases. Dopaminergic neurons derived from hiPSCs/hESCs have been implicated to be very useful in Parkinson's disease (PD) research, including cell replacement therapy, disease modeling and drug screening. Recently, great efforts have been made to improve the application of hiPSCs/hESCs in PD research. Considerable advances have been made in recent years, including advanced reprogramming strategies without the use of viruses or using fewer transcriptional factors, optimized methods for generating highly homogeneous neural progenitors with a larger proportion of mature dopaminergic neurons and better survival and integration after transplantation. Here we outline the progress that has been made in these aspects in recent years, particularly during the last year, and also discuss existing issues that need to be addressed.

  13. Alzheimer's disease in a dish: promises and challenges of human stem cell models.

    PubMed

    Young, Jessica E; Goldstein, Lawrence S B

    2012-10-15

    Human pluripotent stem cells can differentiate into disease-relevant cell types, which capture the unique genome of an individual patient and provide insight into pathological mechanisms of human disease. Recently, human stem cell models for Alzheimer's disease (AD), the most common neurodegenerative dementia, have been described. Stem cell-derived neurons from patients with familial and sporadic AD and Down's syndrome recapitulate human disease phenotypes such as amyloid β peptide production, hyperphosphorylation of tau protein and endosomal abnormalities. Treatment of human neurons with small molecules can modulate these phenotypes, demonstrating the utility of this system for drug development and screening. This review will highlight the current AD stem cell models and discuss the remaining challenges and potential future directions of this field.

  14. Asiaticoside enhances normal human skin cell migration, attachment and growth in vitro wound healing model.

    PubMed

    Lee, Jeong-Hyun; Kim, Hye-Lee; Lee, Mi Hee; You, Kyung Eun; Kwon, Byeong-Ju; Seo, Hyok Jin; Park, Jong-Chul

    2012-10-15

    Wound healing proceeds through a complex collaborative process involving many types of cells. Keratinocytes and fibroblasts of epidermal and dermal layers of the skin play prominent roles in this process. Asiaticoside, an active component of Centella asiatica, is known for beneficial effects on keloid and hypertrophic scar. However, the effects of this compound on normal human skin cells are not well known. Using in vitro systems, we observed the effects of asiaticoside on normal human skin cell behaviors related to healing. In a wound closure seeding model, asiaticoside increased migration rates of skin cells. By observing the numbers of cells attached and the area occupied by the cells, we concluded that asiaticoside also enhanced the initial skin cell adhesion. In cell proliferation assays, asiaticoside induced an increase in the number of normal human dermal fibroblasts. In conclusion, asiaticoside promotes skin cell behaviors involved in wound healing; and as a bioactive component of an artificial skin, may have therapeutic value.

  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. Mathematical Modeling of Heterogeneous Electrophysiological Responses in Human β-Cells

    PubMed Central

    Riz, Michela; Braun, Matthias; Pedersen, Morten Gram

    2014-01-01

    Electrical activity plays a pivotal role in glucose-stimulated insulin secretion from pancreatic -cells. Recent findings have shown that the electrophysiological characteristics of human -cells differ from their rodent counterparts. We show that the electrophysiological responses in human -cells to a range of ion channels antagonists are heterogeneous. In some cells, inhibition of small-conductance potassium currents has no effect on action potential firing, while it increases the firing frequency dramatically in other cells. Sodium channel block can sometimes reduce action potential amplitude, sometimes abolish electrical activity, and in some cells even change spiking electrical activity to rapid bursting. We show that, in contrast to L-type -channels, P/Q-type -currents are not necessary for action potential generation, and, surprisingly, a P/Q-type -channel antagonist even accelerates action potential firing. By including SK-channels and dynamics in a previous mathematical model of electrical activity in human -cells, we investigate the heterogeneous and nonintuitive electrophysiological responses to ion channel antagonists, and use our findings to obtain insight in previously published insulin secretion measurements. Using our model we also study paracrine signals, and simulate slow oscillations by adding a glycolytic oscillatory component to the electrophysiological model. The heterogenous electrophysiological responses in human -cells must be taken into account for a deeper understanding of the mechanisms underlying insulin secretion in health and disease, and as shown here, the interdisciplinary combination of experiments and modeling increases our understanding of human -cell physiology. PMID:24391482

  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.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. 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

  5. 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.

  6. Expression kinetics of hepatic progenitor markers in cellular models of human liver development recapitulating hepatocyte and biliary cell fate commitment.

    PubMed

    Chaudhari, Pooja; Tian, Lipeng; Deshmukh, Abhijeet; Jang, Yoon-Young

    2016-09-01

    Due to the limitations of research using human embryos and the lack of a biological model of human liver development, the roles of the various markers associated with liver stem or progenitor cell potential in humans are largely speculative, and based on studies utilizing animal models and certain patient tissues. Human pluripotent stem cell-based in vitro multistage hepatic differentiation systems may serve as good surrogate models for mimicking normal human liver development, pathogenesis and injury/regeneration studies. Here, we describe the implications of various liver stem or progenitor cell markers and their bipotency (i.e. hepatocytic- and biliary-epithelial cell differentiation), based on the pluripotent stem cell-derived model of human liver development. Future studies using the human cellular model(s) of liver and biliary development will provide more human relevant biological and/or pathological roles of distinct markers expressed in heterogeneous liver stem/progenitor cell populations.

  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. 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

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

    PubMed

    Reeb, Ashley N; Ziegler, Andrea; Lin, Reigh-Yi

    2016-03-01

    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

  10. 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.

  11. Modeling Dengue Virus-Hepatic Cell Interactions Using Human Pluripotent Stem Cell-Derived Hepatocyte-like Cells.

    PubMed

    Lang, Jianshe; Vera, Daniel; Cheng, Yichen; Tang, Hengli

    2016-09-13

    The development of dengue antivirals and vaccine has been hampered by the incomplete understanding of molecular mechanisms of dengue virus (DENV) infection and pathology, partly due to the limited suitable cell culture or animal models that can capture the comprehensive cellular changes induced by DENV. In this study, we differentiated human pluripotent stem cells (hPSCs) into hepatocytes, one of the target cells of DENV, to investigate various aspects of DENV-hepatocyte interaction. hPSC-derived hepatocyte-like cells (HLCs) supported persistent and productive DENV infection. The activation of interferon pathways by DENV protected bystander cells from infection and protected the infected cells from massive apoptosis. Furthermore, DENV infection activated the NF-κB pathway, which led to production of proinflammatory cytokines and downregulated many liver-specific genes such as albumin and coagulation factor V. Our study demonstrates the utility of hPSC-derived hepatocytes as an in vitro model for DENV infection and reveals important aspects of DENV-host interactions. PMID:27546535

  12. An in vivo model of human small intestine using pluripotent stem cells

    PubMed Central

    Watson, Carey L; Mahe, Maxime M; Múnera, Jorge; Howell, Jonathan C; Sundaram, Nambirajan; Poling, Holly M; Schweitzer, Jamie I; Vallance, Jefferson E; Mayhew, Christopher N; Sun, Ying; Grabowski, Gregory; Finkbeiner, Stacy R; Spence, Jason R; Shroyer, Noah F; Wells, James M; Helmrath, Michael A

    2015-01-01

    Differentiation of human pluripotent stem cells (hPSCs) into organ-specific subtypes offers an exciting avenue for the study of embryonic development and disease processes, for pharmacologic studies and as a potential resource for therapeutic transplant1,2. To date, limited in vivo models exist for human intestine, all of which are dependent upon primary epithelial cultures or digested tissue from surgical biopsies that include mesenchymal cells transplanted on biodegradable scaffolds3,4. Here, we generated human intestinal organoids (HIOs) produced in vitro from human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs)5,6 that can engraft in vivo. These HIOs form mature human intestinal epithelium with intestinal stem cells contributing to the cryptvillus architecture and a laminated human mesenchyme, both supported by mouse vasculature ingrowth. In vivo transplantation resulted in marked expansion and maturation of the epithelium and mesenchyme, as demonstrated by differentiated intestinal cell lineages (enterocytes, goblet cells, Paneth cells, tuft cells and enteroendocrine cells), presence of functional brush-border enzymes (lactase, sucrase-isomaltase and dipeptidyl peptidase 4) and visible subepithelial and smooth muscle layers when compared with HIOs in vitro. Transplanted intestinal tissues demonstrated digestive functions as shown by permeability and peptide uptake studies. Furthermore, transplanted HIO-derived tissue was responsive to systemic signals from the host mouse following ileocecal resection, suggesting a role for circulating factors in the intestinal adaptive response7–9. This model of the human small intestine may pave the way for studies of intestinal physiology, disease and translational studies. PMID:25326803

  13. Human amniotic fluid stem cells as a model for functional studies of genes involved in human genetic diseases or oncogenesis.

    PubMed

    Rosner, Margit; Dolznig, Helmut; Schipany, Katharina; Mikula, Mario; Brandau, Oliver; Hengstschläger, Markus

    2011-09-01

    functions in AFS cells was established. Since AFS cells are not tumorigenic, gene modulations not only allow to investigate the role of endogenous genes involved in human genetic diseases but also may help to reveal putative oncogenic gene functions in different biological models, both in vitro and in vivo. This concept is discussed and a "proof of principle", already obtained via modulating genes involved in the mammalian target of rapamycin (mTOR) pathway in AFS cells, is presented.

  14. Peritonitis activates transcription of the human prolactin locus in myeloid cells in a humanized transgenic rat model.

    PubMed

    Semprini, Sabrina; McNamara, Anne V; Awais, Raheela; Featherstone, Karen; Harper, Claire V; McNeilly, Judith R; Patist, Amanda; Rossi, Adriano G; Dransfield, Ian; McNeilly, Alan S; Davis, Julian R E; White, Michael R H; Mullins, John J

    2012-06-01

    Prolactin (PRL) is mainly expressed in the pituitary in rodents, whereas in humans, expression is observed in many extrapituitary sites, including lymphocytes. Due to the lack of adequate experimental models, the function of locally produced PRL in the immune system is largely unknown. Using transgenic rats that express luciferase under the control of extensive human PRL regulatory regions, we characterized immune cell responses to thioglycollate (TG)-induced peritonitis. Resident populations of myeloid cells in the peritoneal cavity of untreated rats expressed barely detectable levels of luciferase. In contrast, during TG-induced peritonitis, cell-specific expression in both neutrophils and monocytes/macrophages in peritoneal exudates increased dramatically. Elevated luciferase expression was also detectable in peripheral blood and bone marrow CD11b(+) cells. Ex vivo stimulation of primary myeloid cells showed activation of the human extrapituitary promoter by TNF-α, lipopolysaccharide, or TG. These findings were confirmed in human peripheral blood monocytes, showing that the transgenic rat provided a faithful model for the human gene. Thus, the resolution of an inflammatory response is associated with dramatic activation of the PRL gene promoter in the myeloid lineage.

  15. Large animal induced pluripotent stem cells as pre-clinical models for studying human disease

    PubMed Central

    Plews, Jordan R; Gu, Mingxia; Longaker, Michael T; Wu, Joseph C

    2012-01-01

    Abstract The derivation of human embryonic stem cells and subsequently human induced pluripotent stem cells (iPSCs) has energized regenerative medicine research and enabled seemingly limitless applications. Although small animal models, such as mouse models, have played an important role in the progression of the field, typically, they are poor representations of the human disease phenotype. As an alternative, large animal models should be explored as a potentially better approach for clinical translation of cellular therapies. However, only fragmented information regarding the derivation, characterization and clinical usefulness of pluripotent large animal cells is currently available. Here, we briefly review the latest advances regarding the derivation and use of large animal iPSCs. PMID:22212700

  16. 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

  17. Potential Limitations of the NSG Humanized Mouse as a Model System to Optimize Engineered Human T cell Therapy for Cancer

    PubMed Central

    Alcantar-Orozco, Erik M.; Gornall, Hannah; Baldan, Vania; Hawkins, Robert E.

    2013-01-01

    Abstract The genetic modification of peripheral blood lymphocytes using retroviral vectors to redirect T cells against tumor cells has been recently used as a means to generate large numbers of antigen-specific T cells for adoptive cell therapy protocols. However, commonly used retroviral vector-based genetic modification requires T cells to be driven into cell division; this potent mitogenic stimulus is associated with the development of an effector phenotype that may adversely impact upon the long-term engraftment potential and subsequent antitumor effects of T cells. To investigate whether the cytokines used during culture impact upon the engraftment potential of gene-modified T cells, a humanized model employing T cells engrafted with a MART-1-specific T cell receptor adoptively transferred into NOD/Shi-scid IL-2rγ−/− (NSG) immune-deficient mice bearing established melanoma tumors was used to compare the effects of the common γ chain cytokines IL-2, IL-7, and IL-15 upon gene-modified T cell activity. MART-1-specific T cells cultured in IL-7 and IL-15 demonstrated greater relative in vitro proliferation and viability of T cells compared with the extensively used IL-2. Moreover, the IL-15 culture prolonged the survival of animals bearing melanoma tumors after adoptive transfer. However, the combination of IL-7 and IL-15 produced T cells with improved engraftment potential compared with IL-15 alone; however, a high rate of xenogeneic graft-versus-host disease prevented the identification of a clear improvement in antitumor effect of these T cells. These results clearly demonstrate modulation of gene-modified T cell engraftment in the NSG mouse, which supports the future testing of the combination of IL-7 and IL-15 in adoptive cell therapy protocols; however, this improved engraftment is also associated with the long-term maintenance of xenoreactive T cells, which limits the ultimate usefulness of the NSG mouse model in this situation. PMID:23931270

  18. 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.

  19. Pathway Analysis and Modeling of the Differentiation of Human Embryonic Stem Cells into Hepatocyte-like Cells

    NASA Astrophysics Data System (ADS)

    Daskalaki, Andriani; Jozefczuk, Justyna; Lehrach, Hans; Adjaye, James; Wierling, Christoph

    2011-06-01

    A more detailed understanding of the differentiation of human embryonic and induced pluripotent stem cells into hepatocyte-like cells can help to improve therapies for liver diseases, like steatohepatitis. In this work we used microarray-based expression data to analyze the in vitro differentiation of human embryonic stem cells into hepatocytes. Pathway analysis has been carried out on gene expression data of different stages of the differentiation process from embryonic stem cells into hepatocyte-like cells via definitive endoderm and hepatic endoderm. Based on pathway analysis we identified signaling pathways, like the GPCR signaling pathway as well as FOXA2 regulatory networks. Based on these highly enriched pathways we constructed a model prototype to better understand and study the differentiation of stem cells into hepatocytes.

  20. 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

  1. 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.

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

    PubMed

    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.

  3. 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...

  4. Cell-free transmission of human adenovirus by passive mass transfer in cell culture simulated in a computer model.

    PubMed

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

    2012-09-01

    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.

  5. Alzheimer's disease modeling: ups, downs, and perspectives for human induced pluripotent stem cells.

    PubMed

    Wojda, Urszula; Kuznicki, Jacek

    2013-01-01

    Major breakthroughs are required to win the war against the increasing threat of Alzheimer's disease. Until now, however, despite enormous efforts and funds, effective therapies are lacking, and adequate models for drug validation are still unavailable. In this article, we review the available animal and cellular models of different features of human Alzheimer's disease and critically evaluate their usefulness for understanding the mechanisms of the disease. The majority of the presently used models are based on the amyloid-β and hyperphosphorylated tau hypothesis, which resembles features of familial Alzheimer's disease. Unfortunately, these models offer limited help for understanding the pathomechanisms of the early stages of sporadic Alzheimer's disease. Thus, new models are needed to discover ways to treat or delay the onset of Alzheimer's disease, and we discuss the prospects for such desperately needed models, including human induced pluripotent stem cells and in silico brain models.

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. 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

  11. A Human Cell Line Model for Interferon-α Driven Dendritic Cell Differentiation

    PubMed Central

    Ruben, Jurjen M.; Visser, Lindy L.; Heinhuis, Kimberley M.; O’Toole, Tom; Bontkes, Hetty J.; Westers, Theresia M.; Ossenkoppele, Gert J.; de Gruijl, Tanja D.; van de Loosdrecht, Arjan A.

    2015-01-01

    The CD34+ MUTZ-3 acute myeloid leukemia cell line has been used as a dendritic cell (DC) differentiation model. This cell line can be cultured into Langerhans cell (LC) or interstitial DC-like cells using the same cytokine cocktails used for the differentiation of their primary counterparts. Currently, there is an increasing interest in the study and clinical application of DC generated in the presence of IFNα, as these IFNα-DC produce high levels of inflammatory cytokines and have been suggested to be more potent in their ability to cross-present protein antigens, as compared to the more commonly used IL-4-DC. Here, we report on the generation of IFNα-induced MUTZ-DC. We show that IFNα MUTZ-DC morphologically and phenotypically display characteristic DC features and are functionally equivalent to “classic” IL-4 MUTZ-DC. IFNα MUTZ-DC ingest exogenous antigens and can subsequently cross-present HLA class-I restricted epitopes to specific CD8+ T cells. Importantly, mature IFNα MUTZ-DC express CCR7, migrate in response to CCL21, and are capable of priming naïve antigen-specific CD8+ T cells. In conclusion, we show that the MUTZ-3 cell line offers a viable and sustainable model system to study IFNα driven DC development and functionality. PMID:26252775

  12. 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

  13. Neuro-immune interactions of neural stem cell transplants: from animal disease models to human trials.

    PubMed

    Giusto, Elena; Donegà, Matteo; Cossetti, Chiara; Pluchino, Stefano

    2014-10-01

    Stem cell technology is a promising branch of regenerative medicine that is aimed at developing new approaches for the treatment of severely debilitating human diseases, including those affecting the central nervous system (CNS). Despite the increasing understanding of the mechanisms governing their biology, the application of stem cell therapeutics remains challenging. The initial idea that stem cell transplants work in vivo via the replacement of endogenous cells lost or damaged owing to disease has been challenged by accumulating evidence of their therapeutic plasticity. This new concept covers the remarkable immune regulatory and tissue trophic effects that transplanted stem cells exert at the level of the neural microenvironment to promote tissue healing via combination of immune modulatory and tissue protective actions, while retaining predominantly undifferentiated features. Among a number of promising candidate stem cell sources, neural stem/precursor cells (NPCs) are under extensive investigation with regard to their therapeutic plasticity after transplantation. The significant impact in vivo of experimental NPC therapies in animal models of inflammatory CNS diseases has raised great expectations that these stem cells, or the manipulation of the mechanisms behind their therapeutic impact, could soon be translated to human studies. This review aims to provide an update on the most recent evidence of therapeutically-relevant neuro-immune interactions following NPC transplants in animal models of multiple sclerosis, cerebral stroke and traumas of the spinal cord, and consideration of the forthcoming challenges related to the early translation of some of these exciting experimental outcomes into clinical medicines.

  14. 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.

  15. Application of human induced pluripotent stem cells for modeling and treating neurodegenerative diseases.

    PubMed

    Payne, Natalie L; Sylvain, Aude; O'Brien, Carmel; Herszfeld, Daniella; Sun, Guizhi; Bernard, Claude C A

    2015-01-25

    The advent of human induced pluripotent stem cells (hiPSCs), reprogrammed in vitro from both healthy and disease-state human somatic cells, has triggered an enormous global research effort to realize personalized regenerative medicine for numerous degenerative conditions. hiPSCs have been generated from cells of many tissue types and can be differentiated in vitro to most somatic lineages, not only for the establishment of disease models that can be utilized as novel drug screening platforms and to study the molecular and cellular processes leading to degeneration, but also for the in vivo cell-based repair or modulation of a patient's disease profile. hiPSCs derived from patients with the neurodegenerative diseases amyotrophic lateral sclerosis, Parkinson's disease, Alzheimer's disease and multiple sclerosis have been successfully differentiated in vitro into disease-relevant cell types, including motor neurons, dopaminergic neurons and oligodendrocytes. However, the generation of functional iPSC-derived neural cells that are capable of engraftment in humans and the identification of robust disease phenotypes for modeling neurodegeneration still require several key challenges to be addressed. Here, we discuss these challenges and summarize recent progress toward the application of iPSC technology for these four common neurodegenerative diseases.

  16. Embryonic origins of human vascular smooth muscle cells: implications for in vitro modeling and clinical application.

    PubMed

    Sinha, Sanjay; Iyer, Dharini; Granata, Alessandra

    2014-06-01

    Vascular smooth muscle cells (SMCs) arise from multiple origins during development, raising the possibility that differences in embryological origins between SMCs could contribute to site-specific localization of vascular diseases. In this review, we first examine the developmental pathways and embryological origins of vascular SMCs and then discuss in vitro strategies for deriving SMCs from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We then review in detail the potential for vascular disease modeling using iPSC-derived SMCs and consider the pathological implications of heterogeneous embryonic origins. Finally, we touch upon the role of human ESC-derived SMCs in therapeutic revascularization and the challenges remaining before regenerative medicine using ESC- or iPSC-derived cells comes of age.

  17. 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.

  18. 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

  19. 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

  20. An in vitro triple cell co-culture model with primary cells mimicking the human alveolar epithelial barrier.

    PubMed

    Lehmann, Andrea D; Daum, Nicole; Bur, Michael; Lehr, Claus-Michael; Gehr, Peter; Rothen-Rutishauser, Barbara M

    2011-04-01

    A triple cell co-culture model was recently established by the authors, consisting of either A549 or 16HBE14o- epithelial cells, human blood monocyte-derived macrophages and dendritic cells, which offers the possibility to study the interaction of xenobiotics with those cells. The 16HBE14o- containing co-culture model mimics the airway epithelial barrier, whereas the A549 co-cultures mimic the alveolar type II-like epithelial barrier. The goal of the present work was to establish a new triple cell co-culture model composed of primary alveolar type I-like cells isolated from human lung biopsies (hAEpC) representing a more realistic alveolar epithelial barrier wall, since type I epithelial cells cover >93% of the alveolar surface. Monocultures of A549 and 16HBE14o- were morphologically and functionally compared with the hAEpC using laser scanning microscopy, as well as transmission electron microscopy, and by determining the epithelial integrity. The triple cell co-cultures were characterized using the same methods. It could be shown that the epithelial integrity of hAEpC (mean ± SD, 1180 ± 188 Ω cm(2)) was higher than in A549 (172 ± 59 Ω cm(2)) but similar to 16HBE14o- cells (1469 ± 156 Ω cm(2)). The triple cell co-culture model with hAEpC (1113 ± 30 Ω cm(2)) showed the highest integrity compared to the ones with A549 (93 ± 14 Ω cm(2)) and 16HBE14o- (558 ± 267 Ω cm(2)). The tight junction protein zonula occludens-1 in hAEpC and 16HBE14o- were more regularly expressed but not in A549. The epithelial alveolar model with hAEpC combined with two immune cells (i.e. macrophages and dendritic cells) will offer a novel and more realistic cell co-culture system to study possible cell interactions of inhaled xenobiotics and their toxic potential on the human alveolar type I epithelial wall.

  1. Induced pluripotent stem cells restore function in a human cell loss model of open-angle glaucoma.

    PubMed

    Abu-Hassan, Diala W; Li, Xinbo; Ryan, Eileen I; Acott, Ted S; Kelley, Mary J

    2015-03-01

    Normally, trabecular meshwork (TM) and Schlemm's canal inner wall endothelial cells within the aqueous humor outflow pathway maintain intraocular pressure within a narrow safe range. Elevation in intraocular pressure, because of the loss of homeostatic regulation by these outflow pathway cells, is the primary risk factor for vision loss due to glaucomatous optic neuropathy. A notable feature associated with glaucoma is outflow pathway cell loss. Using controlled cell loss in ex vivo perfused human outflow pathway organ culture, we developed compelling experimental evidence that this level of cell loss compromises intraocular pressure homeostatic function. This function was restored by repopulation of the model with fresh TM cells. We then differentiated induced pluripotent stem cells (iPSCs) and used them to repopulate this cell depletion model. These differentiated cells (TM-like iPSCs) became similar to TM cells in both morphology and expression patterns. When transplanted, they were able to fully restore intraocular pressure homeostatic function. This successful transplantation of TM-like iPSCs establishes the conceptual feasibility of using autologous stem cells to restore intraocular pressure regulatory function in open-angle glaucoma patients, providing a novel alternative treatment option.

  2. Induced pluripotent stem cells restore function in a human cell loss model of open-angle glaucoma.

    PubMed

    Abu-Hassan, Diala W; Li, Xinbo; Ryan, Eileen I; Acott, Ted S; Kelley, Mary J

    2015-03-01

    Normally, trabecular meshwork (TM) and Schlemm's canal inner wall endothelial cells within the aqueous humor outflow pathway maintain intraocular pressure within a narrow safe range. Elevation in intraocular pressure, because of the loss of homeostatic regulation by these outflow pathway cells, is the primary risk factor for vision loss due to glaucomatous optic neuropathy. A notable feature associated with glaucoma is outflow pathway cell loss. Using controlled cell loss in ex vivo perfused human outflow pathway organ culture, we developed compelling experimental evidence that this level of cell loss compromises intraocular pressure homeostatic function. This function was restored by repopulation of the model with fresh TM cells. We then differentiated induced pluripotent stem cells (iPSCs) and used them to repopulate this cell depletion model. These differentiated cells (TM-like iPSCs) became similar to TM cells in both morphology and expression patterns. When transplanted, they were able to fully restore intraocular pressure homeostatic function. This successful transplantation of TM-like iPSCs establishes the conceptual feasibility of using autologous stem cells to restore intraocular pressure regulatory function in open-angle glaucoma patients, providing a novel alternative treatment option. PMID:25377070

  3. 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

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

    PubMed

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

    2016-02-04

    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.

  5. Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models

    PubMed Central

    Liu, Huiping; Patel, Manishkumar R.; Prescher, Jennifer A.; Patsialou, Antonia; Qian, Dalong; Lin, Jiahui; Wen, Susanna; Chang, Ya-Fang; Bachmann, Michael H.; Shimono, Yohei; Dalerba, Piero; Adorno, Maddalena; Lobo, Neethan; Bueno, Janet; Dirbas, Frederick M.; Goswami, Sumanta; Somlo, George; Condeelis, John; Contag, Christopher H.; Gambhir, Sanjiv Sam; Clarke, Michael F.

    2010-01-01

    To examine the role of breast cancer stem cells (BCSCs) in metastasis, we generated human-in-mouse breast cancer orthotopic models using patient tumor specimens, labeled with optical reporter fusion genes. These models recapitulate human cancer features not captured with previous models, including spontaneous metastasis in particular, and provide a useful platform for studies of breast tumor initiation and progression. With noninvasive imaging approaches, as few as 10 cells of stably labeled BCSCs could be tracked in vivo, enabling studies of early tumor growth and spontaneous metastasis. These advances in BCSC imaging revealed that CD44+ cells from both primary tumors and lung metastases are highly enriched for tumor-initiating cells. Our metastatic cancer models, combined with noninvasive imaging techniques, constitute an integrated approach that could be applied to dissect the molecular mechanisms underlying the dissemination of metastatic CSCs (MCSCs) and to explore therapeutic strategies targeting MCSCs in general or to evaluate individual patient tumor cells and predict response to therapy. PMID:20921380

  6. Neovascularization in a mouse model via stem cells derived from human fetal amniotic membranes.

    PubMed

    Kim, Hwi Gon; Choi, Ook Hwan

    2011-03-01

    In this study, we evaluated the effect of culture-expanded mesenchymal stem cells (MSCs), derived from amniotic membranes, on neovascularization and blood flow, in an animal model of limb ischemia in immune-deficient mice. MSCs were cultured from human amniotic membranes by collagenase digestion. Human amniotic mesenchymal stem cells (hAMSCs) were administered intramuscularly at three different sites of the ischemic leg whose femoral vessels were ligated. After 4 weeks of culture, a population of homogeneous mesenchymal cells was isolated from the human amniotic membranes after confluence was reached. We performed three different groups of mice model [controls, hAMSCs, conditioned media from the hAMSCs (hAMSCs-CM)]. The blood flow recovery in the hindlimb ischemia model was significantly higher in the hAMSC-transplanted group than in the control group. Moreover, hAMSCs-CM significantly improved the cutaneous blood flow. The histological examination showed that red fluorescence (CM-DiI)-labeled hAMSCs was detected in the interstitial tissues between the muscle fibers 2 weeks after transplantation. The results of this study showed that hAMSCs may be an attractive, alternative source of progenitor or stem cells for basic research as well as clinical applications.

  7. Methods of Liver Stem Cell Therapy in Rodents as Models of Human Liver Regeneration in Hepatic Failure.

    PubMed

    Hashemi Goradel, Nasser; Darabi, Masoud; Shamsasenjan, Karim; Ejtehadifar, Mostafa; Zahedi, Sarah

    2015-09-01

    Cell therapy is a promising intervention for treating liver diseases and liver failure. Different animal models of human liver cell therapy have been developed in recent years. Rats and mice are the most commonly used liver failure models. In fact, rodent models of hepatic failure have shown significant improvement in liver function after cell infusion. With the advent of stem-cell technologies, it is now possible to re-programme adult somatic cells such as skin or hair-follicle cells from individual patients to stem-like cells and differentiate them into liver cells. Such regenerative stem cells are highly promising in the personalization of cell therapy. The present review article will summarize current approaches to liver stem cell therapy with rodent models. In addition, we discuss common cell tracking techniques and how tracking data help to direct liver cell therapy research in animal models of hepatic failure.

  8. A Werner syndrome stem cell model unveils heterochromatin alterations as a driver of human aging

    PubMed Central

    Zhang, Weiqi; Li, Jingyi; Suzuki, Keiichiro; Qu, Jing; Wang, Ping; Zhou, Junzhi; Liu, Xiaomeng; Ren, Ruotong; Xu, Xiuling; Ocampo, Alejandro; Yuan, Tingting; Yang, Jiping; Li, Ying; Shi, Liang; Guan, Dee; Pan, Huize; Duan, Shunlei; Ding, Zhichao; Li, Mo; Yi, Fei; Bai, Ruijun; Wang, Yayu; Chen, Chang; Yang, Fuquan; Li, Xiaoyu; Wang, Zimei; Aizawa, Emi; Goebl, April; Soligalla, Rupa Devi; Reddy, Pradeep; Esteban, Concepcion Rodriguez; Tang, Fuchou; Liu, Guang-Hui; Belmonte, Juan Carlos Izpisua

    2015-01-01

    Werner syndrome (WS) is a premature aging disorder caused by WRN protein deficiency. Here, we report on the generation of a human WS model in human embryonic stem cells (ESCs). Differentiation of WRN-null ESCs to mesenchymal stem cells (MSCs) recapitulates features of premature cellular aging, a global loss of H3K9me3, and changes in heterochromatin architecture. We show that WRN associates with heterochromatin proteins SUV39H1 and HP1α and nuclear lamina-heterochromatin anchoring protein LAP2β. Targeted knock-in of catalytically inactive SUV39H1 in wild-type MSCs recapitulates accelerated cellular senescence, resembling WRN-deficient MSCs. Moreover, decrease in WRN and heterochromatin marks are detected in MSCs from older individuals. Our observations uncover a role for WRN in maintaining heterochromatin stability and highlight heterochromatin disorganization as a potential determinant of human aging. PMID:25931448

  9. A model of metabolic changes in respiration-deficient human cells.

    PubMed

    Bollmann, F Mathias

    2007-09-01

    Cells lacking aerobic metabolism because of damaged mtDNA accumulate in many postmitotic tissues in the course aging. Although being only a small fraction of cells, they might play a major role in oxidative stress affecting the whole body. However, it remains unclear how such cells, which are under normal circumstances dependent on aerobic metabolism, are able to survive for decades in vivo. Here a new model is presented that proposes a coexistence of anaerobic glycolysis and a partly reversed TCA cycle. Succinate plays a key role in the changed metabolic pathways because it has to be exported by the cell. This hypothesis supports the view that some respiration-deficient cells are able to survive permanently within the body and contribute to human aging.

  10. The therapeutic effects of human adipose-derived stem cells in Alzheimer's disease mouse models.

    PubMed

    Chang, Keun-A; Kim, Hee Jin; Joo, Yuyoung; Ha, Sungji; Suh, Yoo-Hun

    2014-01-01

    Alzheimer's disease (AD) is an irreversible neurodegenerative disease, still lacking proper clinical treatment. Therefore, many researchers have focused on the possibility of therapeutic use of stem cells for AD. Adipose-derived stem cells (ASCs), mesenchymal stem cells (MSCs) isolated from adipose tissue, are well known for their pluripotency and their ability to differentiate into multiple tissue types and have immune modulatory properties similar to those of MSCs from other origins. Because of their biological properties, ASCs can be considered for cell therapy and neuroregeneration. Our recent results clearly showed the therapeutic potential of these cells after transplantation into Tg2576 mice (an AD mouse model). Intravenously or intracerebrally transplanted human ASCs (hASCs) greatly improved the memory impairment and the neuropathology, suggesting that hASCs have a high therapeutic potential for AD.

  11. Urine-derived induced pluripotent stem cells as a modeling tool to study rare human diseases.

    PubMed

    Shi, Liang; Cui, Yazhou; Luan, Jing; Zhou, Xiaoyan; Han, Jinxiang

    2016-08-01

    Rare diseases with a low prevalence are a key public health issue because the causes of those diseases are difficult to determine and those diseases lack a clearly established or curative treatment. Thus, investigating the molecular mechanisms that underlie the pathology of rare diseases and facilitating the development of novel therapies using disease models is crucial. Human induced pluripotent stem cells (iPSCs) are well suited to modeling rare diseases since they have the capacity for self-renewal and pluripotency. In addition, iPSC technology provides a valuable tool to generate patient-specific iPSCs. These cells can be differentiated into cell types that have been affected by a disease. These cells would circumvent ethical concerns and avoid immunological rejection, so they could be used in cell replacement therapy or regenerative medicine. To date, human iPSCs could have been generated from multiple donor sources, such as skin, adipose tissue, and peripheral blood. However, these cells are obtained via invasive procedures. In contrast, several groups of researchers have found that urine may be a better source for producing iPSCs from normal individuals or patients. This review discusses urinary iPSC (UiPSC) as a candidate for modeling rare diseases. Cells obtained from urine have overwhelming advantages compared to other donor sources since they are safely, affordably, and frequently obtained and they are readily obtained from patients. The use of iPSC-based models is also discussed. UiPSCs may prove to be a key means of modeling rare diseases and they may facilitate the treatment of those diseases in the future. PMID:27672542

  12. Urine-derived induced pluripotent stem cells as a modeling tool to study rare human diseases

    PubMed Central

    Shi, Liang; Cui, Yazhou; Luan, Jing; Zhou, Xiaoyan; Han, Jinxiang

    2016-01-01

    Summary Rare diseases with a low prevalence are a key public health issue because the causes of those diseases are difficult to determine and those diseases lack a clearly established or curative treatment. Thus, investigating the molecular mechanisms that underlie the pathology of rare diseases and facilitating the development of novel therapies using disease models is crucial. Human induced pluripotent stem cells (iPSCs) are well suited to modeling rare diseases since they have the capacity for self-renewal and pluripotency. In addition, iPSC technology provides a valuable tool to generate patient-specific iPSCs. These cells can be differentiated into cell types that have been affected by a disease. These cells would circumvent ethical concerns and avoid immunological rejection, so they could be used in cell replacement therapy or regenerative medicine. To date, human iPSCs could have been generated from multiple donor sources, such as skin, adipose tissue, and peripheral blood. However, these cells are obtained via invasive procedures. In contrast, several groups of researchers have found that urine may be a better source for producing iPSCs from normal individuals or patients. This review discusses urinary iPSC (UiPSC) as a candidate for modeling rare diseases. Cells obtained from urine have overwhelming advantages compared to other donor sources since they are safely, affordably, and frequently obtained and they are readily obtained from patients. The use of iPSC-based models is also discussed. UiPSCs may prove to be a key means of modeling rare diseases and they may facilitate the treatment of those diseases in the future. PMID:27672542

  13. 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.

  14. 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

  15. 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.

  16. Human fallopian tube mesenchymal stromal cells enhance bone regeneration in a xenotransplanted model.

    PubMed

    Jazedje, Tatiana; Bueno, Daniela F; Almada, Bruno V P; Caetano, Heloisa; Czeresnia, Carlos E; Perin, Paulo M; Halpern, Silvio; Maluf, Mariangela; Evangelista, Lucila P; Nisenbaum, Marcelo G; Martins, Marília T; Passos-Bueno, Maria R; Zatz, Mayana

    2012-06-01

    We have recently reported that human fallopian tubes, which are discarded during surgical procedures of women submitted to sterilization or hysterectomies, are a rich source of human fallopian tube mesenchymal stromal cells (htMSCs). It has been previously shown that human mesenchymal stromal cells may be useful in enhancing the speed of bone regeneration. This prompted us to investigate whether htMSCs might be useful for the treatment of osteoporosis or other bone diseases, since they present a pronounced capacity for osteogenic differentiation in vitro. Based on this prior knowledge, our aim was to evaluate, in vivo, the osteogenic capacity of htMSCs to regenerate bone through an already described xenotransplantation model: nonimmunosuppressed (NIS) rats with cranial defects. htMSCs were obtained from five 30-50 years old healthy women and characterized by flow cytometry and for their multipotenciality in vitro capacity (osteogenic, chondrogenic and adipogenic differentiations). Two symmetric full-thickness cranial defects on each parietal region of seven NIS rats were performed. The left side (LS) of six animals was covered with CellCeram (Scaffdex)-a bioabsorbable ceramic composite scaffold that contains 60% hydroxyapatite and 40% β-tricalciumphosphate-only, and the right side (RS) with the CellCeram and htMSCs (10(6) cells/scaffold). The animals were euthanized at 30, 60 and 90 days postoperatively and cranial tissue samples were taken for histological analysis. After 90 days we observed neobone formation in both sides. However, in animals euthanized 30 and 60 days after the procedure, a mature bone was observed only on the side with htMSCs. PCR and immunofluorescence analysis confirmed the presence of human DNA and thus that human cells were not rejected, which further supports the imunomodulatory property of htMSCs. In conclusion, htMSCs can be used successfully to enhance bone regeneration in vivo, opening a new field for future treatments of osteoporosis

  17. 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

  18. Aging stem cells. A Werner syndrome stem cell model unveils heterochromatin alterations as a driver of human aging.

    PubMed

    Zhang, Weiqi; Li, Jingyi; Suzuki, Keiichiro; Qu, Jing; Wang, Ping; Zhou, Junzhi; Liu, Xiaomeng; Ren, Ruotong; Xu, Xiuling; Ocampo, Alejandro; Yuan, Tingting; Yang, Jiping; Li, Ying; Shi, Liang; Guan, Dee; Pan, Huize; Duan, Shunlei; Ding, Zhichao; Li, Mo; Yi, Fei; Bai, Ruijun; Wang, Yayu; Chen, Chang; Yang, Fuquan; Li, Xiaoyu; Wang, Zimei; Aizawa, Emi; Goebl, April; Soligalla, Rupa Devi; Reddy, Pradeep; Esteban, Concepcion Rodriguez; Tang, Fuchou; Liu, Guang-Hui; Belmonte, Juan Carlos Izpisua

    2015-06-01

    Werner syndrome (WS) is a premature aging disorder caused by WRN protein deficiency. Here, we report on the generation of a human WS model in human embryonic stem cells (ESCs). Differentiation of WRN-null ESCs to mesenchymal stem cells (MSCs) recapitulates features of premature cellular aging, a global loss of H3K9me3, and changes in heterochromatin architecture. We show that WRN associates with heterochromatin proteins SUV39H1 and HP1α and nuclear lamina-heterochromatin anchoring protein LAP2β. Targeted knock-in of catalytically inactive SUV39H1 in wild-type MSCs recapitulates accelerated cellular senescence, resembling WRN-deficient MSCs. Moreover, decrease in WRN and heterochromatin marks are detected in MSCs from older individuals. Our observations uncover a role for WRN in maintaining heterochromatin stability and highlight heterochromatin disorganization as a potential determinant of human aging.

  19. 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

  20. 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

  1. Model for assessing radiation dose to epithelial cells of the human respiratory tract from radon progeny

    SciTech Connect

    Fisher, D.R.; Hui, T.E.; James, A.C.

    1990-07-01

    A computational model was developed to evaluate radiation doses to sensitive cells from exposure to radon progeny throughout human bronchial epithelium. The model incorporated current information on nasal and oral filtration efficiencies for unattached radon progeny, characteristics of bronchial deposition by diffusive and inertial processes, mucous clearance and possible transfer of radon progeny to the airway epithelium, locations of target nuclei of secretory and basal cells in different regions of the bronchial tree epithelium, and other features. The model is useful for evaluating absorbed doses to various populations of target cell nuclei, the associated microdosimetric probability densities in specific energy, and the likelihood that target nuclei are hit one or more times by alpha-particle tracks. The model was applied to extrapolating lung cancer risks observed in underground miners to the general population exposed to low-level radon progeny in indoor home environments. The effect of increasing exposure rates by one and two orders of magnitude in both environments was modeled to determine the frequency of radiation events in target cell nuclei. The implications of dosimetric modeling for lung cancer risk analysis were also examined. 28 refs., 5 figs., 5 tabs.

  2. 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

  3. 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

  4. A model of human nasal epithelial cells adapted for direct and repeated exposure to airborne pollutants.

    PubMed

    Bardet, Gaëlle; Achard, Sophie; Loret, Thomas; Desauziers, Valérie; Momas, Isabelle; Seta, Nathalie

    2014-08-17

    Airway epithelium lining the nasal cavity plays a pivotal role in respiratory tract defense and protection mechanisms. Air pollution induces alterations linked to airway diseases such as asthma. Only very few in vitro studies to date have succeeded in reproducing physiological conditions relevant to cellular type and chronic atmospheric pollution exposure. We therefore, set up an in vitro model of human Airway Epithelial Cells of Nasal origin (hAECN) close to real human cell functionality, specifically adapted to study the biological effects of exposure to indoor gaseous pollution at the environmental level. hAECN were exposed under air-liquid interface, one, two, or three-times at 24 h intervals for 1 h, to air or formaldehyde (200 μg/m(3)), an indoor air gaseous pollutant. All experiments were ended at day 4, when both cellular viability and cytokine production were assessed. Optimal adherence and confluence of cells were obtained 96 h after cell seeding onto collagen IV-precoated insert. Direct and repeated exposure to formaldehyde did not produce any cellular damage or IL-6 production change, although weak lower IL-8 production was observed only after the third exposure. Our model is significantly better than previous ones due to cell type and the repeated exposure protocol.

  5. Modeling Neurological Disease by Rapid Conversion of Human Urine Cells into Functional Neurons.

    PubMed

    Zhang, Shu-Zhen; Ma, Li-Xiang; Qian, Wen-Jing; Li, Hong-Fu; Wang, Zhong-Feng; Wang, Hong-Xia; Wu, Zhi-Ying

    2016-01-01

    Somatic cells can be directly converted into functional neurons by ectopic expression of defined factors and/or microRNAs. Since the first report of conversion mouse embryonic fibroblasts into functional neurons, the postnatal mouse, and human fibroblasts, astroglia, hepatocytes, and pericyte-derived cells have been converted into functional dopaminergic and motor neurons both in vitro and in vivo. However, it is invasive to get all these materials. In the current study, we provide a noninvasive approach to obtain directly reprogrammed functional neurons by overexpression of the transcription factors Ascl1, Brn2, NeuroD, c-Myc, and Myt1l in human urine cells. These induced neuronal (iN) cells could express multiple neuron-specific proteins and generate action potentials. Moreover, urine cells from Wilson's disease (WD) patient could also be directly converted into neurons. In conclusion, generation of iN cells from nonneural lineages is a feasible and befitting approach for neurological disease modeling. PMID:26770203

  6. In Vitro Modeling of RSV Infection and Cytopathogenesis in Well-Differentiated Human Primary Airway Epithelial Cells (WD-PAECs).

    PubMed

    Broadbent, Lindsay; Villenave, Remi; Guo-Parke, Hong; Douglas, Isobel; Shields, Michael D; Power, Ultan F

    2016-01-01

    The choice of model used to study human respiratory syncytial virus (RSV) infection is extremely important. RSV is a human pathogen that is exquisitely adapted to infection of human hosts. Rodent models, such as mice and cotton rats, are semi-permissive to RSV infection and do not faithfully reproduce hallmarks of RSV disease in humans. Furthermore, immortalized airway-derived cell lines, such as HEp-2, BEAS-2B, and A549 cells, are poorly representative of the complexity of the respiratory epithelium. The development of a well-differentiated primary pediatric airway epithelial cell models (WD-PAECs) allows us to simulate several hallmarks of RSV infection of infant airways. They therefore represent important additions to RSV pathogenesis modeling in human-relevant tissues. The following protocols describe how to culture and differentiate both bronchial and nasal primary pediatric airway epithelial cells and how to use these cultures to study RSV cytopathogenesis. PMID:27464691

  7. A biomimetic physiological model for human adipose tissue by adipocytes and endothelial cell cocultures with spatially controlled distribution.

    PubMed

    Yao, Rui; Du, Yanan; Zhang, Renji; Lin, Feng; Luan, Jie

    2013-08-01

    An in vitro model that recapitulates the characteristics of native human adipose tissue would largely benefit pathology studies and therapy development. In this paper, we fabricated a physiological model composed of both human adipocytes and endothelial cells with spatially controlled distribution that biomimics the structure and composition of human adipose tissue. Detailed studies into the cell-cell interactions between the adipocytes and endothelial cells revealed a mutual-enhanced effect which resembles the in vivo routine. Furthermore, comparisons between planar coculture and model coculture demonstrated improved adipocyte function as well as endothelial cell proliferation under the same conditions. This research provided a reliable model for human adipose tissue development studies and potential obesity-related therapy development.

  8. Expression of estrogenicity genes in a lineage cell culture model of human breast cancer progression.

    PubMed

    Fu, Jiaqi; Weise, Amy M; Falany, Josie L; Falany, Charles N; Thibodeau, Bryan J; Miller, Fred R; Kocarek, Thomas A; Runge-Morris, Melissa

    2010-02-01

    TaqMan Gene Expression assays were used to profile the mRNA expression of estrogen receptor (ERalpha and ERbeta) and estrogen metabolism enzymes including cytosolic sulfotransferases (SULT1E1, SULT1A1, SULT2A1, and SULT2B1), steroid sulfatase (STS), aromatase (CYP19), 17beta-hydroxysteroid dehydrogenases (17betaHSD1 and 2), CYP1B1, and catechol-O-methyltransferase (COMT) in an MCF10A-derived lineage cell culture model for basal-like human breast cancer progression and in ERalpha-positive luminal MCF7 breast cancer cells. Low levels of ERalpha and ERbeta mRNA were present in MCF10A-derived cell lines. SULT1E1 mRNA was more abundant in confluent relative to subconfluent MCF10A cells, a non-tumorigenic proliferative breast disease cell line. SULT1E1 was also expressed in preneoplastic MCF10AT1 and MCF10AT1K.cl2 cells, but was markedly repressed in neoplastic MCF10A-derived cell lines as well as in MCF7 cells. Steroid-metabolizing enzymes SULT1A1 and SULT2B1 were only expressed in MCF7 cells. STS and COMT were widely detected across cell lines. Pro-estrogenic 17betaHSD1 mRNA was most abundant in neoplastic MCF10CA1a and MCF10DCIS.com cells, while 17betaHSD2 mRNA was more prominent in parental MCF10A cells. CYP1B1 mRNA was most abundant in MCF7 cells. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) induced SULT1E1 and CYP19 mRNA but suppressed CYP1B1, STS, COMT, 17betaHSD1, and 17betaHSD2 mRNA in MCF10A lineage cell lines. In MCF7 cells, TSA treatment suppressed ERalpha, CYP1B1, STS, COMT, SULT1A1, and SULT2B1 but induced ERbeta, CYP19 and SULT2A1 mRNA expression. The results indicate that relative to the MCF7 breast cancer cell line, key determinants of breast estrogen metabolism are differentially regulated in the MCF10A-derived lineage model for breast cancer progression.

  9. Genomic editing tools to model human diseases with isogenic pluripotent stem cells.

    PubMed

    Kim, Huen Suk; Bernitz, Jeffrey M; Lee, Dung-Fang; Lemischka, Ihor R

    2014-11-15

    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.

  10. 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

  11. Effects of an antimalarial quinazoline derivative on human erythrocytes and on cell membrane molecular models.

    PubMed

    Rojas-Aguirre, Yareli; Hernández-Luis, Francisco; Mendoza-Martínez, César; Sotomayor, Carlos Patricio; Aguilar, Luis Felipe; Villena, Fernando; Castillo, Ivan; Hernández, David J; Suwalsky, Mario

    2012-03-01

    Plasmodium, the parasite which causes malaria in humans multiplies in the liver and then infects circulating erythrocytes. Thus, the role of the erythrocyte cell membrane in antimalarial drug activity and resistance has key importance. The effects of the antiplasmodial N(6)-(4-methoxybenzyl)quinazoline-2,4,6-triamine (M4), and its inclusion complex (M4/HPβCD) with 2-hydroxypropyl-β-cyclodextrin (HPβCD) on human erythrocytes and on cell membrane molecular models are herein reported. This work evidences that M4/HPβCD interacts with red cells as follows: a) in scanning electron microscopy (SEM) studies on human erythrocytes induced shape changes at a 10μM concentration; b) in isolated unsealed human erythrocyte membranes (IUM) a concentration as low as 1μM induced sharp DPH fluorescence anisotropy decrease whereas increasing concentrations produced a monotonically decrease of DPH fluorescence lifetime at 37°C; c) X-ray diffraction studies showed that 200μM induced a complete structural perturbation of dimyristoylphosphatidylcholine (DMPC) bilayers whereas no significant effects were detected in dimyristoylphosphatidylethanolamine (DMPE) bilayers, classes of lipids present in the outer and inner monolayers of the human erythrocyte membrane, respectively; d) fluorescence spectroscopy data showed that increasing concentrations of the complex interacted with the deep hydrophobic core of DMPC large unilamellar vesicles (LUV) at 18°C. All these experiments are consistent with the insertion of M4/HPβCD in the outer monolayer of the human erythrocyte membrane; thus, it can be considered a promising and novel antimalarial agent. PMID:22155684

  12. Effects of an antimalarial quinazoline derivative on human erythrocytes and on cell membrane molecular models.

    PubMed

    Rojas-Aguirre, Yareli; Hernández-Luis, Francisco; Mendoza-Martínez, César; Sotomayor, Carlos Patricio; Aguilar, Luis Felipe; Villena, Fernando; Castillo, Ivan; Hernández, David J; Suwalsky, Mario

    2012-03-01

    Plasmodium, the parasite which causes malaria in humans multiplies in the liver and then infects circulating erythrocytes. Thus, the role of the erythrocyte cell membrane in antimalarial drug activity and resistance has key importance. The effects of the antiplasmodial N(6)-(4-methoxybenzyl)quinazoline-2,4,6-triamine (M4), and its inclusion complex (M4/HPβCD) with 2-hydroxypropyl-β-cyclodextrin (HPβCD) on human erythrocytes and on cell membrane molecular models are herein reported. This work evidences that M4/HPβCD interacts with red cells as follows: a) in scanning electron microscopy (SEM) studies on human erythrocytes induced shape changes at a 10μM concentration; b) in isolated unsealed human erythrocyte membranes (IUM) a concentration as low as 1μM induced sharp DPH fluorescence anisotropy decrease whereas increasing concentrations produced a monotonically decrease of DPH fluorescence lifetime at 37°C; c) X-ray diffraction studies showed that 200μM induced a complete structural perturbation of dimyristoylphosphatidylcholine (DMPC) bilayers whereas no significant effects were detected in dimyristoylphosphatidylethanolamine (DMPE) bilayers, classes of lipids present in the outer and inner monolayers of the human erythrocyte membrane, respectively; d) fluorescence spectroscopy data showed that increasing concentrations of the complex interacted with the deep hydrophobic core of DMPC large unilamellar vesicles (LUV) at 18°C. All these experiments are consistent with the insertion of M4/HPβCD in the outer monolayer of the human erythrocyte membrane; thus, it can be considered a promising and novel antimalarial agent.

  13. QSAR model for predicting cell viability of human embryonic kidney cells exposed to SiO₂ nanoparticles.

    PubMed

    Manganelli, Serena; Leone, Caterina; Toropov, Andrey A; Toropova, Alla P; Benfenati, Emilio

    2016-02-01

    A predictive model for the viability (%) of cultured human embryonic kidney cells (HEK293) exposed to 20 and 50 nm silica nanoparticles was built using 'optimal descriptors' as mathematical functions of size, concentration and exposure time. The calculation was carried out with CORAL software (http://www.insilico.eu/coral/) on five random splits of combined systems (particle size-particle concentration-cell exposure time) into training, calibration, and validation sets. The R(2) values of the best models were above 0.68. The average statistical quality of the model for the viability (%) of HEK293 exposed to different concentrations of silica nanoparticles measured by MTT assay is satisfactory. PMID:26439516

  14. 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

  15. 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.

  16. Quantifying in vitro growth and metabolism kinetics of human mesenchymal stem cells using a mathematical model.

    PubMed

    Higuera, Gustavo; Schop, Deborah; Janssen, Frank; van Dijkhuizen-Radersma, Riemke; van Boxtel, Ton; van Blitterswijk, C A

    2009-09-01

    Better quantitative understanding of human mesenchymal stem cells (hMSCs) metabolism is needed to identify, understand, and subsequently optimize the processes in expansion of hMSCs in vitro. For this purpose, we analyzed growth of hMSCs in vitro with a mathematical model based on the mass balances for viable cell numbers, glucose, lactate, glutamine, and glutamate. The mathematical modeling had two aims: (1) to estimate kinetic parameters of important metabolites for hMSC monolayer cultures, and (2) to quantitatively assess assumptions on growth of hMSCs. Two cell seeding densities were used to investigate growth and metabolism kinetics of MSCs from three human donors. We analyzed growth up to confluency and used metabolic assumptions described in literature. Results showed a longer initial phase, a slower growth rate, and a higher glucose, lactate, glutamine, and glutamate metabolic rates at the lower cell seeding density. Higher metabolic rates could be induced by a lower contact inhibition effect when seeding at 100 cells/cm2 than when seeding at 1000 cells/cm2. In addition, parameter estimation describing kinetics of hMSCs in culture, depending on the seeding density, showed doubling times in the order of 17-32h, specific glucose consumption in the order of 1.25 x 10(-1) to 3.77 x 10(-1) pmol/cell/h, specific lactate production in the order of 2.48 x 10(-1) to 7.67 x 10(-1)pmol/cell/h, specific glutamine production in the order of 7.04 x 10(-3) to 2.27 pmol/cell/h, and specific glutamate production in the order of 4.87 x 10(-1) to 23.4 pmol/cell/h. Lactate-to-glucose yield ratios confirmed that hMSCs use glucose via anaerobic glycolysis. In addition, glutamine and glutamate metabolic shifts were identified that could be important for understanding growth of hMSCs in vitro. This study showed that the mathematical modeling approach supports quantitative analysis of important mechanisms in proliferation of hMSCs in vitro.

  17. Modeling human cortical development in vitro using induced pluripotent stem cells

    PubMed Central

    Mariani, Jessica; Simonini, Maria Vittoria; Palejev, Dean; Tomasini, Livia; Coppola, Gianfilippo; Szekely, Anna M.; Horvath, Tamas L.; Vaccarino, Flora M.

    2012-01-01

    Human induced pluripotent stem cells (hiPSCs) are emerging as a tool for understanding human brain development at cellular, molecular, and genomic levels. Here we show that hiPSCs grown in suspension in the presence of rostral neuralizing factors can generate 3D structures containing polarized radial glia, intermediate progenitors, and a spectrum of layer-specific cortical neurons reminiscent of their organization in vivo. The hiPSC-derived multilayered structures express a gene expression profile typical of the embryonic telencephalon but not that of other CNS regions. Their transcriptome is highly enriched in transcription factors controlling the specification, growth, and patterning of the dorsal telencephalon and displays highest correlation with that of the early human cerebral cortical wall at 8–10 wk after conception. Thus, hiPSC are capable of enacting a transcriptional program specifying human telencephalic (pallial) development. This model will allow the study of human brain development as well as disorders of the human cerebral cortex. PMID:22761314

  18. Modeling human cortical development in vitro using induced pluripotent stem cells.

    PubMed

    Mariani, Jessica; Simonini, Maria Vittoria; Palejev, Dean; Tomasini, Livia; Coppola, Gianfilippo; Szekely, Anna M; Horvath, Tamas L; Vaccarino, Flora M

    2012-07-31

    Human induced pluripotent stem cells (hiPSCs) are emerging as a tool for understanding human brain development at cellular, molecular, and genomic levels. Here we show that hiPSCs grown in suspension in the presence of rostral neuralizing factors can generate 3D structures containing polarized radial glia, intermediate progenitors, and a spectrum of layer-specific cortical neurons reminiscent of their organization in vivo. The hiPSC-derived multilayered structures express a gene expression profile typical of the embryonic telencephalon but not that of other CNS regions. Their transcriptome is highly enriched in transcription factors controlling the specification, growth, and patterning of the dorsal telencephalon and displays highest correlation with that of the early human cerebral cortical wall at 8-10 wk after conception. Thus, hiPSC are capable of enacting a transcriptional program specifying human telencephalic (pallial) development. This model will allow the study of human brain development as well as disorders of the human cerebral cortex. PMID:22761314

  19. 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

  20. Effect of Mild Heating on Human Lens Epithelial Cells: A Possible Model of Lens Aging

    PubMed Central

    Zhang, Keke; Zhu, Xiangjia; Lu, Yi

    2016-01-01

    This study aims to investigate the effect of mild heating on lens epithelial cells and to explore its possibility as an in vitro model for lens aging. Human lens epithelial cells (LECs) were heated at 50 °C for a cellular lens aging study. Analysis of the head group order of lens membranes was performed using Laurdan labeling. Immunofluorescence was performed to detect changes in α-crystallin expression and its cellular distribution. The chaperone-like activity of α-crystallin was also assessed. After mild heating, α-crystallin in LECs showed a tendency towards accumulation around the nucleus. The membrane head group environment of lens epithelial cells became more fluid with increasing time of exposure to mild heating, as indicated by increased water penetration. Furthermore, the chaperone activity of α-crystallin decreased, and suggests a relatively lower protective effect on other functional proteins in LECs. Thus, compared to the mild heating model based on lens tissue, this cellular model could provide a more convenient and accurate method for studying lens aging in vitro, including changes in membrane head group order in each cell, the real-time observation of crystallin distribution, and the monitoring of functional changes in the chaperone activity of crystallins as a result of aging. PMID:27725687

  1. 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

  2. Sequence-dependent abnormal aggregation of human Tau fragment in an inducible cell model.

    PubMed

    Liu, Xiao-Ling; Hu, Ji-Ying; Hu, Meng-Yun; Zhang, Yi; Hong, Zheng-Yuan; Cheng, Xiao-Qing; Chen, Jie; Pang, Dai-Wen; Liang, Yi

    2015-08-01

    A pathological hallmark of Alzheimer disease (AD) is the accumulation of misfolded hyperphosphorylated microtubule-associated protein Tau within neurons, forming neurofibrillary tangles and leading to synaptic dysfunction and neuronal death. Here we study sequence-dependent abnormal aggregation of human fragment Tau244-372 in an inducible cell model. As evidenced by confocal laser scanning microscopy, Western blot, and immunogold electron microscopy, fibril-forming motifs are essential and sufficient for abnormal aggregation of Tau244-372 in SH-SY5Y neuroblastoma cells induced by Congo red: when its two fibril-forming segments PHF6 and PHF6* are deleted, Tau244-372 does lose its ability to form fibrils in SH-SY5Y cells, and the replacement of PHF6 and PHF6* with an unrelated amyloidogenic sequence IFQINS from human lysozyme does rescue the fibril-forming ability of Tau244-372 in SH-SY5Y cells. By contrast, insertion of a non-fibril forming peptide GGGGGG does not drive the disabled Tau244-372 to misfold in SH-SY5Y cells. Furthermore, as revealed by quantum dots based probes combined with annexin V staining, annexin V-FITC apoptosis detection assay, and immunofluorescence, fibril-forming motifs are essential and sufficient for early apoptosis of living SH-SY5Y cells induced by abnormal aggregation of Tau244-372. Our results suggest that fibril-forming motifs could be the determinants of Tau protein tending to misfold in living cells, thereby inducing neuronal apoptosis and causing the initiation and development of AD.

  3. Isolation and purification of self-renewable human neural stem cells for cell therapy in experimental model of ischemic stroke.

    PubMed

    Azevedo-Pereira, Ricardo L; Daadi, Marcel M

    2013-01-01

    Human embryonic stem cells (hESCs) are pluripotent with a strong self-renewable ability making them a virtually unlimited source of neural cells for structural repair in neurological disorders. Currently, hESCs are one of the most promising cell sources amenable for commercialization of off-shelf cell therapy products. However, along with this strong proliferative capacity of hESCs comes the tumorigenic potential of these cells after transplantation. Thus, the isolation and purification of a homogeneous, population of neural stem cells (hNSCs) are of paramount importance to avoid tumor formation in the host brain. This chapter describes the isolation, neuralization, and long-term perpetuation of hNSCs derived from hESCs through use of specific mitogenic growth factors and the preparation of hNSCs for transplantation in an experimental model of stroke. Additionally, we describe methods to analyze the stroke and size of grafts using magnetic resonance imaging and Osirix software, and neuroanatomical tracing procedures to study axonal remodeling after stroke and cell transplantation.

  4. Genome engineering of isogenic human ES cells to model autism disorders.

    PubMed

    Martinez, Refugio A; Stein, Jason L; Krostag, Anne-Rachel F; Nelson, Angelique M; Marken, John S; Menon, Vilas; May, Ryan C; Yao, Zizhen; Kaykas, Ajamete; Geschwind, Daniel H; Grimley, Joshua S

    2015-05-26

    Isogenic pluripotent stem cells are critical tools for studying human neurological diseases by allowing one to study the effects of a mutation in a fixed genetic background. Of particular interest are the spectrum of autism disorders, some of which are monogenic such as Timothy syndrome (TS); others are multigenic such as the microdeletion and microduplication syndromes of the 16p11.2 chromosomal locus. Here, we report engineered human embryonic stem cell (hESC) lines for modeling these two disorders using locus-specific endonucleases to increase the efficiency of homology-directed repair (HDR). We developed a system to: (1) computationally identify unique transcription activator-like effector nuclease (TALEN) binding sites in the genome using a new software program, TALENSeek, (2) assemble the TALEN genes by combining golden gate cloning with modified constructs from the FLASH protocol, and (3) test the TALEN pairs in an amplification-based HDR assay that is more sensitive than the typical non-homologous end joining assay. We applied these methods to identify, construct, and test TALENs that were used with HDR donors in hESCs to generate an isogenic TS cell line in a scarless manner and to model the 16p11.2 copy number disorder without modifying genomic loci with high sequence similarity.

  5. Establishment and validation of a model for non-luteinized human mural granulosa cell culture.

    PubMed

    Ophir, L; Yung, Y; Maman, E; Rubinstein, N; Yerushalmi, G M; Haas, J; Barzilay, E; Hourvitz, A

    2014-03-25

    Cell culture techniques of human mural granulosa cells (MGCs) serve as a major in vitro tool. However, the use of luteinized MGCs has major limitations due to their luteinized state. Our aim was to establish a standardized protocol for the culture of MGCs as a model for different stages of folliculogenesis. We showed that early-non-luteinized, preovulatory-non-luteinized and luteal-MGCs have distinct gene expression pattern. After 4 days of incubation of luteinized-MGCs, ovulatory genes mRNA's achieve expression levels similar to the early non-luteinized follicles. FSH stimulation for 48 h of these 4 days cultured MGCs showed ovulatory genes mRNA's expression similar to the pre-ovulatory non-luteinized follicles. These FSH-stimulated cells responded to hCG stimulation in a pattern similar to the response of pre-ovulatory follicles. This novel model may provide a standardized research tool for delineation of the molecular processes occurring during the latter stages of follicular development in the human ovary.

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

    PubMed

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

    2016-07-01

    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 hEAG1

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. In vitro Modeling of Ryanodine Receptor 2 Dysfunction Using Human Induced Pluripotent Stem Cells

    PubMed Central

    Fatima, Azra; Xu, Guoxing; Shao, Kaifeng; Papadopoulos, Symeon; Lehmann, Martin; Arnáiz-Cot, Juan J.; Rosa, Angelo O.; Nguemo, Filomain; Matzkies, Matthias; Dittmann, Sven; Stone, Susannah L.; Linke, Matthias; Zechner, Ulrich; Beyer, Vera; Hennies, Hans Christian; Rosenkranz, Stephan; Klauke, Baerbel; Parwani, Abdul S.; Haverkamp, Wilhelm; Pfitzer, Gabriele; Farr, Martin; Cleemann, Lars; Morad, Martin; Milting, Hendrik; Hescheler, Juergen; Šaric, Tomo

    2011-01-01

    Background/Aims: Induced pluripotent stem (iPS) cells generated from accessible adult cells of patients with genetic diseases open unprecedented opportunities for exploring the pathophysiology of human diseases in vitro. Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited cardiac disorder that is caused by mutations in the cardiac ryanodine receptor type 2 gene (RYR2) and is characterized by stress-induced ventricular arrhythmia that can lead to sudden cardiac death in young individuals. The aim of this study was to generate iPS cells from a patient with CPVT1 and determine whether iPS cell-derived cardiomyocytes carrying patient specific RYR2 mutation recapitulate the disease phenotype in vitro. Methods: iPS cells were derived from dermal fibroblasts of healthy donors and a patient with CPVT1 carrying the novel heterozygous autosomal dominant mutation p.F2483I in the RYR2. Functional properties of iPS cell derived-cardiomyocytes were analyzed by using whole-cell current and voltage clamp and calcium imaging techniques. Results: Patch-clamp recordings revealed arrhythmias and delayed afterdepolarizations (DADs) after catecholaminergic stimulation of CPVT1-iPS cell-derived cardiomyocytes. Calcium imaging studies showed that, compared to healthy cardiomyocytes, CPVT1-cardiomyocytes exhibit higher amplitudes and longer durations of spontaneous Ca2+ release events at basal state. In addition, in CPVT1-cardiomyocytes the Ca2+-induced Ca2+-release events continued after repolarization and were abolished by increasing the cytosolic cAMP levels with forskolin. Conclusion: This study demonstrates the suitability of iPS cells in modeling RYR2-related cardiac disorders in vitro and opens new opportunities for investigating the disease mechanism in vitro, developing new drugs, predicting their toxicity, and optimizing current treatment strategies. PMID:22178870

  12. 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

  13. 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

  14. 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.

  15. Transplantation of Human Neuroblastoma Cells, Catecholaminergic and Non-Catecholaminergic: Effects on Rotational Behavoir in Parkinson's Rat Model

    PubMed Central

    Manaster, Jacob S.; Feuerman, Tony; Reynolds, C. Patrick; Markham, Charles H.

    1992-01-01

    Cultured human catecholaminergic and noncatecholaminergic donor cells were used in neural transplantation experiments in a rat model of Parkinson's disease. Using two different human catecholaminergic neuroblastoma cell lines, one control non-catecholaminergic neuroblastoma cell line, and one sham control (tissue culture medium), transplants were made into the striatum using a modified Ungerstedt hemiparkinsonian rat model. Significant decreases in apomorphine-induced rotational behavior were produced by two of three catecholaminergic cell lines. Grafted cells staining positively for tyrosine hydroxylase (TH) and catecholamine fluorescence indicated viable catecholamine activity in the two cell lines which produced reductions in rotational behavior. Catecholamine fluorescence was not detected in either of the two controls. These data suggest a link between catecholamine secretion by transplanted cells and motor improvement using a rat rotational behavior model. PMID:1355366

  16. Mammalian cell cultures as models for Mycobacterium tuberculosis-human immunodeficiency virus (HIV) interaction studies: A review.

    PubMed

    Chingwaru, Walter; Glashoff, Richard H; Vidmar, Jerneja; Kapewangolo, Petrina; Sampson, Samantha L

    2016-09-01

    Mycobacterium tuberculosis and human immunodeficiency virus (HIV) co-infections have remained a major public health concern worldwide, particularly in Southern Africa. Yet our understanding of the molecular interactions between the pathogens has remained poor due to lack of suitable preclinical models for such studies. We reviewed the use, this far, of mammalian cell culture models in HIV-MTB interaction studies. Studies have described the use of primary human cell cultures, including (1) monocyte-derived macrophage (MDM) fractions of peripheral blood mononuclear cell (PBMC), alveolar macrophages (AM), (2) cell lines such as the monocyte-derived macrophage cell line (U937), T lymphocyte cell lines (CEMx174, ESAT-6-specific CD4(+) T-cells) and an alveolar epithelial cell line (A549) and (3) special models such as stem cells, three dimensional (3D) or organoid cell models (including a blood-brain barrier cell model) in HIV-MTB interaction studies. The use of cell cultures from other mammals, including: mouse cell lines [macrophage cell lines RAW 264.7 and J774.2, fibroblast cell lines (NIH 3T3, C3H clones), embryonic fibroblast cell lines and T-lymphoma cell lines (S1A.TB, TIMI.4 and R1.1)]; rat (T cells: Rat2, RGE, XC and HH16, and alveolar cells: NR8383) and primary guinea pigs derived AMs, in HIV-MTB studies is also described. Given the spectrum of the models available, cell cultures offer great potential for host-HIV-MTB interactions studies. PMID:27633294

  17. Effects of AlCl3 on toad skin, human erythrocytes, and model cell membranes.

    PubMed

    Suwalsky, M; Ungerer, B; Villena, F; Norris, B; Cárdenas, H; Zatta, P

    2001-05-15

    Aluminum, a very abundant metal, could play a toxic role in several pathological processes, including neurodegeneration. Although the effects of Al(III) on biological membranes have been extensively described, direct information concerning the molecular basis of its biological activity is rather scanty. To examine aluminum challenges on cell membranes, various concentrations of AlCl3 in aqueous solutions were incubated with human erythrocytes, isolated toad skin, and molecular models of biomembranes. The latter consisted of multilayers of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine, representing phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane. These specimens were studied by scanning electron microscopy, electrophysiological measurements, and x-ray diffraction. The results indicate that Al(III) in the concentration range of 10-100 microM induced the following structural and functional effects: (i) change in the normal discoid shape of human erythrocytes to echinocytes due to the accumulation of Al(III) ions in the outer moiety of the red cell membrane; (ii) perturbation of dimyristoylphosphatidylcholine, and to a lesser extent of dimyristoylphosphatidylethanolamine bilayers, and (iii) decrease in the short-circuit current and in the potential difference of the isolated toad skin, effects that are in accordance with a time-dependent modulation of ion transport in response to changes in the molecular structure of the lipid bilayer. PMID:11470316

  18. 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

  19. Therapeutic potentials of human adipose-derived stem cells on the mouse model of Parkinson's disease.

    PubMed

    Choi, Hee Soon; Kim, Hee Jin; Oh, Jin-Hwan; Park, Hyeong-Geun; Ra, Jeong Chan; Chang, Keun-A; Suh, Yoo-Hun

    2015-10-01

    The treatment of Parkinson's disease (PD) using stem cells has long been the focus of many researchers, but the ideal therapeutic strategy has not yet been developed. The consistency and high reliability of the experimental results confirmed by animal models are considered to be a critical factor in the stability of stem cell transplantation for PD. Therefore, the aim of this study was to investigate the preventive and therapeutic potential of human adipose-derived stem cells (hASC) for PD and was to identify the related factors to this therapeutic effect. The hASC were intravenously injected into the tail vein of a PD mouse model induced by 6-hydroxydopamine. Consequently, the behavioral performances were significantly improved at 3 weeks after the injection of hASC. Additionally, dopaminergic neurons were rescued, the number of structure-modified mitochondria was decreased, and mitochondrial complex I activity was restored in the brains of the hASC-injected PD mouse model. Overall, this study underscores that intravenously transplanted hASC may have therapeutic potential for PD by recovering mitochondrial functions.

  20. 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.

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

    PubMed

    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.

  2. 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

  3. 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

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

    PubMed

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

    2014-02-24

    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 Ωxcm(2)). Overall, this scalable human BBB model may enable a wide range of neuroscience studies.

  5. 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.

  6. Recent advances in animal and human pluripotent stem cell modeling of cardiac laminopathy.

    PubMed

    Lee, Yee-Ki; Jiang, Yu; Ran, Xin-Ru; Lau, Yee-Man; Ng, Kwong-Man; Lai, Wing-Hon Kevin; Siu, Chung-Wah; Tse, Hung-Fat

    2016-01-01

    Laminopathy is a disease closely related to deficiency of the nuclear matrix protein lamin A/C or failure in prelamin A processing, and leads to accumulation of the misfold protein causing progeria. The resultant disrupted lamin function is highly associated with abnormal nuclear architecture, cell senescence, apoptosis, and unstable genome integrity. To date, the effects of loss in nuclear integrity on the susceptible organ, striated muscle, have been commonly associated with muscular dystrophy, dilated cardiac myopathy (DCM), and conduction defeats, but have not been studied intensively. In this review, we aim to summarize recent breakthroughs in an in vivo laminopathy model and in vitro study using patient-specific human induced pluripotent stem cells (iPSCs) that reproduce the pathophysiological phenotype for further drug screening. We describe several in-vivo transgenic mouse models to elucidate the effects of Lmna H222P, N195K mutations, and LMNA knockout on cardiac function, in terms of hemodynamic and electrical signal propagation; certain strategies targeted on stress-related MAPK are mentioned. We will also discuss human iPSC cardiomyocytes serving as a platform to reveal the underlying mechanisms, such as the altered mechanical sensation in electrical coupling of the heart conduction system and ion channel alternation in relation to altered nuclear architecture, and furthermore to enable screening of drugs that can attenuate this cardiac premature aging phenotype by inhibition of prelamin misfolding and oxidative stress, and also enhancement of autophagy protein clearance and cardiac-protective microRNA. PMID:27649756

  7. Recent advances in animal and human pluripotent stem cell modeling of cardiac laminopathy.

    PubMed

    Lee, Yee-Ki; Jiang, Yu; Ran, Xin-Ru; Lau, Yee-Man; Ng, Kwong-Man; Lai, Wing-Hon Kevin; Siu, Chung-Wah; Tse, Hung-Fat

    2016-09-20

    Laminopathy is a disease closely related to deficiency of the nuclear matrix protein lamin A/C or failure in prelamin A processing, and leads to accumulation of the misfold protein causing progeria. The resultant disrupted lamin function is highly associated with abnormal nuclear architecture, cell senescence, apoptosis, and unstable genome integrity. To date, the effects of loss in nuclear integrity on the susceptible organ, striated muscle, have been commonly associated with muscular dystrophy, dilated cardiac myopathy (DCM), and conduction defeats, but have not been studied intensively. In this review, we aim to summarize recent breakthroughs in an in vivo laminopathy model and in vitro study using patient-specific human induced pluripotent stem cells (iPSCs) that reproduce the pathophysiological phenotype for further drug screening. We describe several in-vivo transgenic mouse models to elucidate the effects of Lmna H222P, N195K mutations, and LMNA knockout on cardiac function, in terms of hemodynamic and electrical signal propagation; certain strategies targeted on stress-related MAPK are mentioned. We will also discuss human iPSC cardiomyocytes serving as a platform to reveal the underlying mechanisms, such as the altered mechanical sensation in electrical coupling of the heart conduction system and ion channel alternation in relation to altered nuclear architecture, and furthermore to enable screening of drugs that can attenuate this cardiac premature aging phenotype by inhibition of prelamin misfolding and oxidative stress, and also enhancement of autophagy protein clearance and cardiac-protective microRNA.

  8. Human CD1d knock-in mouse model demonstrates potent antitumor potential of human CD1d-restricted invariant natural killer T cells

    PubMed Central

    Wen, Xiangshu; Rao, Ping; Carreño, Leandro J.; Kim, Seil; Lawrenczyk, Agnieszka; Porcelli, Steven A.; Cresswell, Peter; Yuan, Weiming

    2013-01-01

    Despite a high degree of conservation, subtle but important differences exist between the CD1d antigen presentation pathways of humans and mice. These differences may account for the minimal success of natural killer T (NKT) cell-based antitumor therapies in human clinical trials, which contrast strongly with the powerful antitumor effects in conventional mouse models. To develop an accurate model for in vivo human CD1d (hCD1d) antigen presentation, we have generated a hCD1d knock-in (hCD1d-KI) mouse. In these mice, hCD1d is expressed in a native tissue distribution pattern and supports NKT cell development. Reduced numbers of invariant NKT (iNKT) cells were observed, but at an abundance comparable to that in most normal humans. These iNKT cells predominantly expressed mouse Vβ8, the homolog of human Vβ11, and phenotypically resembled human iNKT cells in their reduced expression of CD4. Importantly, iNKT cells in hCD1d knock-in mice exert a potent antitumor function in a melanoma challenge model. Our results show that replacement of mCD1d by hCD1d can select a population of functional iNKT cells closely resembling human iNKT cells. These hCD1d knock-in mice will allow more accurate in vivo modeling of human iNKT cell responses and will facilitate the preclinical assessment of iNKT cell-targeted antitumor therapies. PMID:23382238

  9. 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.

  10. 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

  11. EAD and DAD mechanisms analyzed by developing a new human ventricular cell model.

    PubMed

    Asakura, K; Cha, C Y; Yamaoka, H; Horikawa, Y; Memida, H; Powell, T; Amano, A; Noma, A

    2014-09-01

    It has long been suggested that the Ca(2+)-mechanisms are largely involved in generating the early afterdepolarization (EAD) as well as the delayed afterdepolarization (DAD). This view was examined in a quantitative manner by applying the lead potential analysis to a new human ventricular cell model. In this ventricular cell model, the tight coupled LCC-RyR model (CaRU) based on local control theory (Hinch et al. 2004) and ion channel models mostly based on human electrophysiological data were included to reproduce realistic Ca(2+) dynamics as well as the membrane excitation. Simultaneously, the Ca(2+) accumulation near the Ca(2+) releasing site was incorporated as observed in real cardiac myocytes. The maximum rate of ventricular repolarization (-1.02 mV/ms) is due to IK1 (-0.55 mV/ms) and the rest is provided nearly equally by INCX (-0.20 mV/ms), INaL (-0.16 mV/ms) and INaT (-0.13 mV/ms). These INaL and INaT components are due to closure of the voltage gate, which remains partially open during the plateau potential. DADs could be evoked by applying high-frequency stimulations supplemented by a partial Na(+)/K(+) pump inhibition, or by a microinjection of Ca(2+). EADs was evoked by retarding the inactivation of INaL. The lead potential (VL) analysis revealed that IK1 and IKr played the primary role to reverse the AP repolarization to depolarizing limb of EAD. ICaL and INCX amplified EAD, while the remaining currents partially antagonized dVL/dt. The maximum rate of rise of EAD was attributable to the rapid activation of both ICaL (45.5%) and INCX (54.5%). PMID:25192800

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

    PubMed

    Keefe, Douglas H

    2015-05-01

    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.

  13. 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

  14. 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.

  15. Is this a brain which I see before me? Modeling human neural development with pluripotent stem cells.

    PubMed

    Suzuki, Ikuo K; Vanderhaeghen, Pierre

    2015-09-15

    The human brain is arguably the most complex structure among living organisms. However, the specific mechanisms leading to this complexity remain incompletely understood, primarily because of the poor experimental accessibility of the human embryonic brain. Over recent years, technologies based on pluripotent stem cells (PSCs) have been developed to generate neural cells of various types. While the translational potential of PSC technologies for disease modeling and/or cell replacement therapies is usually put forward as a rationale for their utility, they are also opening novel windows for direct observation and experimentation of the basic mechanisms of human brain development. PSC-based studies have revealed that a number of cardinal features of neural ontogenesis are remarkably conserved in human models, which can be studied in a reductionist fashion. They have also revealed species-specific features, which constitute attractive lines of investigation to elucidate the mechanisms underlying the development of the human brain, and its link with evolution.

  16. 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

  17. 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.

  18. 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

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

    PubMed

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

    2015-07-01

    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.

  20. 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.

  1. 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

  2. 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.

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

    PubMed Central

    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

  4. Toad skin extract cinobufatini inhibits migration of human breast carcinoma MDA-MB-231 cells into a model stromal tissue.

    PubMed

    Nakata, Munehiro; Mori, Shuya; Kamoshida, Yo; Kawaguchi, Shota; Fujita-Yamaguchi, Yoko; Gao, Bo; Tang, Wei

    2015-08-01

    Toad skin extract cinobufatini study has been focused on anticancer activity, especially apoptosis-inducing activity by bufosteroids. The present study examined effect of the toad skin extract on cancer cell migration into model stromal tissues. Human breast carcinoma cell line MDA-MB-231 was incubated in the presence or absence of toad skin extract on a surface of reconstituted type I collagen gel as a model stromal tissue allowing the cells to migrate into the gel. Frozen sections were microscopically observed after azan staining. Data showed a decrease of cell number in a microscopic field and shortening of cell migration into the model stromal tissue in a dose dependent manner. This suggests that toad skin extract may possess migration-preventing activity in addition to cell toxicity such as apoptosis-inducing activity. The multifaceted effects including apoptosis-inducing and cancer cell migration-preventing activities would improve usefulness of toad skin extract cinobufatini as an anticancer medicine.

  5. 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

  6. Generation of an ICF syndrome model by efficient genome editing of human induced pluripotent stem cells using the CRISPR system.

    PubMed

    Horii, Takuro; Tamura, Daiki; Morita, Sumiyo; Kimura, Mika; Hatada, Izuho

    2013-09-30

    Genome manipulation of human induced pluripotent stem (iPS) cells is essential to achieve their full potential as tools for regenerative medicine. To date, however, gene targeting in human pluripotent stem cells (hPSCs) has proven to be extremely difficult. Recently, an efficient genome manipulation technology using the RNA-guided DNase Cas9, the clustered regularly interspaced short palindromic repeats (CRISPR) system, has been developed. Here we report the efficient generation of an iPS cell model for immunodeficiency, centromeric region instability, facial anomalies syndrome (ICF) syndrome using the CRISPR system. We obtained iPS cells with mutations in both alleles of DNA methyltransferase 3B (DNMT3B) in 63% of transfected clones. Our data suggest that the CRISPR system is highly efficient and useful for genome engineering of human iPS cells.

  7. Cardiac disease modeling using induced pluripotent stem cell-derived human cardiomyocytes

    PubMed Central

    Dell’Era, Patrizia; Benzoni, Patrizia; Crescini, Elisabetta; Valle, Matteo; Xia, Er; Consiglio, Antonella; Memo, Maurizio

    2015-01-01

    Causative mutations and variants associated with cardiac diseases have been found in genes encoding cardiac ion channels, accessory proteins, cytoskeletal components, junctional proteins, and signaling molecules. In most cases the functional evaluation of the genetic alteration has been carried out by expressing the mutated proteins in in-vitro heterologous systems. While these studies have provided a wealth of functional details that have greatly enhanced the understanding of the pathological mechanisms, it has always been clear that heterologous expression of the mutant protein bears the intrinsic limitation of the lack of a proper intracellular environment and the lack of pathological remodeling. The results obtained from the application of the next generation sequencing technique to patients suffering from cardiac diseases have identified several loci, mostly in non-coding DNA regions, which still await functional analysis. The isolation and culture of human embryonic stem cells has initially provided a constant source of cells from which cardiomyocytes (CMs) can be obtained by differentiation. Furthermore, the possibility to reprogram cellular fate to a pluripotent state, has opened this process to the study of genetic diseases. Thus induced pluripotent stem cells (iPSCs) represent a completely new cellular model that overcomes the limitations of heterologous studies. Importantly, due to the possibility to keep spontaneously beating CMs in culture for several months, during which they show a certain degree of maturation/aging, this approach will also provide a system in which to address the effect of long-term expression of the mutated proteins or any other DNA mutation, in terms of electrophysiological remodeling. Moreover, since iPSC preserve the entire patients’ genetic context, the system will help the physicians in identifying the most appropriate pharmacological intervention to correct the functional alteration. This article summarizes the current

  8. 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

  9. 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

  10. Yeast models of mutations in the mitochondrial ATP6 gene found in human cancer cells.

    PubMed

    Niedzwiecka, Katarzyna; Kabala, Anna Magdalena; Lasserre, Jean-Paul; Tribouillard-Tanvier, Déborah; Golik, Pawel; Dautant, Alain; di Rago, Jean-Paul; Kucharczyk, Roza

    2016-07-01

    Since the discovery of somatic mtDNA mutations in tumor cells, multiple studies have focused on establishing a causal relationship between those changes and alterations in energy metabolism, a hallmark of cancer cells. Yet the consequences of these mutations on mitochondrial function remain largely unknown. In this study, Saccharomyces cerevisiae has been used as a model to investigate the functional consequences of four cancer-associated missense mutations (8914C>A, 8932C>T, 8953A>G, 9131T>C) found in the mitochondrial MT-ATP6 gene. This gene encodes the a-subunit of F1FO-ATP synthase, which catalyzes the last steps of ATP production in mitochondria. Although the four studied mutations affected well-conserved residues of the a-subunit, only one of them (8932C>T) had a significant impact on mitochondrial function, due to a less efficient incorporation of the a-subunit into ATP synthase. Our findings indicate that these ATP6 genetic variants found in human tumors are neutral mitochondrial genome substitutions with a limited, if any, impact on the energetic function of mitochondria.

  11. 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

  12. 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

  13. Better science with human cell-based organ and tissue models.

    PubMed

    Heinonen, Tuula

    2015-03-01

    At present, animal-based models are the major test systems for assessing the tolerability and safety of chemical substances for regulatory purposes, and also for pivotal efficacy testing in pharmaceutical development. In spite of the high genetic similarity between many laboratory animals and humans, animal models are very poor predictors of human health effects and pathophysiological processes. Thus, models and testing strategies that are more relevant to human biology, are needed for these purposes. The best predictability is achieved with human organotypic models that mimic the microenvironment of human tissues. During their development, such models have to be characterised at the structural, genetic and functional levels, and compared to the respective human tissues. Their predictivity should be confirmed by using known reference chemicals with corresponding human data. The use of these methods in safety assessment and biomedical research, combined with the knowledge gained of the underlying biological processes on gene and protein expression, as well as on cellular signalling, will ultimately lead to better human science and animal welfare.

  14. Interplay between human microglia and neural stem/progenitor cells in an allogeneic co-culture model.

    PubMed

    Liu, Jia; Hjorth, Erik; Zhu, Mingqin; Calzarossa, Cinzia; Samuelsson, Eva-Britt; Schultzberg, Marianne; Åkesson, Elisabet

    2013-11-01

    Experimental neural cell therapies, including donor neural stem/progenitor cells (NPCs) have been reported to offer beneficial effects on the recovery after an injury and to counteract inflammatory and degenerative processes in the central nervous system (CNS). The interplay between donor neural cells and the host CNS still to a large degree remains unclear, in particular in human allogeneic conditions. Here, we focused our studies on the interaction of human NPCs and microglia utilizing a co-culture model. In co-cultures, both NPCs and microglia showed increased survival and proliferation compared with mono-cultures. In the presence of microglia, a larger subpopulation of NPCs expressed the progenitor cell marker nestin, whereas a smaller group of NPCs expressed the neural markers polysialylated neural cell adhesion molecule, A2B5 and glial fibrillary acidic protein compared with NPC mono-cultures. Microglia thus hindered differentiation of NPCs. The presence of human NPCs increased microglial phagocytosis of latex beads. Furthermore, we observed that the expression of CD200 molecules on NPCs and the CD200 receptor protein on microglia was enhanced in co-cultures, whereas the release of transforming growth factor-β was increased suggesting anti-inflammatory features of the co-cultures. To conclude, the interplay between human allogeneic NPCs and microglia, significantly affected their respective proliferation and phenotype. Neural cell therapy including human donor NPCs may in addition to offering cell replacement, modulate host microglial phenotypes and functions to benefit neuroprotection and repair.

  15. Syringotoxin pore formation and inactivation in human red blood cell and model bilayer lipid membranes.

    PubMed

    Szabó, Zsófia; Gróf, Pál; Schagina, Ludmila V; Gurnev, Philip A; Takemoto, Jon Y; Mátyus, Edit; Blaskó, Katalin

    2002-12-23

    The effect of syringotoxin (ST), a member of the cyclic lipodepsipeptides family (CLPs) produced by Pseudomonas syringae pv. syringae on the membrane permeability of human red blood cells (RBCs) and model bilayer lipid membranes (BLMs) was studied and compared to that of two recently investigated CLPs, syringomycin E (SRE) and syringopeptin 22A (SP22A) [Biochim. Biophys. Acta 1466 (2000) 79 and Bioelectrochemistry 52 (2000) 161]. The permeability-increasing effect of ST on RBCs was the least among the three CLPs. A time-dependent ST pore inactivation was observed on RBCs at 20 and 37 degrees C but not at 8 degrees C. From the kinetic model worked out parameters as permeability coefficient of RBC membrane for 86Rb(+) and pores mean lifetime were calculated. A shorter pores mean lifetime was calculated at 37 degrees C then at 20 degrees C, which gave us an explanation for the unusual slower rate of tracer efflux measured at 37 degrees C then that at 20 degrees C. The results obtained on BLM showed that the pore inactivation was due to a decrease in the number of pores but not to a change of their dwell time or conductance.

  16. Is there a role for human pluripotent stem cells in modelling interstitial cells of Cajal and gut motility disorders?

    PubMed

    Meng, Wenbo; Zhou, Jerry; Elliott, Ross; Murphy, Patricia; Ho, Vincent; O'Connor, Michael

    2015-01-01

    Gastrointestinal motility disorders affect millions of people worldwide, resulting in significant morbidity and mortality. Current treatments for these disorders are inadequate and often provide little to no relief for patients. As a result, gastrointestinal motility disorders produce substantial long-term social and economic burdens in both developed and developing countries. These limited treatment options arise largely from our relatively poor understanding of the molecular etiology for the majority of gastrointestinal motility disorders. In turn, this is due to our limited access to normal or diseased human gut tissue for use in research. In particular while the interstitial cells of Cajal (ICC) are known to be important for gastrointestinal motility, little is known of how these cells function or how they are involved in disease initiation and progression. The advent of human pluripotent stem cell technology offers an opportunity to generate large amounts of human tissue for both research and clinical applications. The application of this technology to gastrointestinal motility disorders is currently only in its infancy and as yet no studies have described ICC production from human pluripotent cells. By considering the present understanding of the anatomical, cellular and molecular basis of gut motility with particular emphasis on ICC, this review provides a clear framework for the application of human pluripotent stem cell technology to answer fundamental questions of ICC involvement in gut motility. PMID:25391378

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. Chimeric antigen receptor T cells secreting anti-PD-L1 antibodies more effectively regress renal cell carcinoma in a humanized mouse model

    PubMed Central

    Suarez, Eloah Rabello; Chang, De-Kuan; Sun, Jiusong; Sui, Jianhua; Freeman, Gordon J.; Signoretti, Sabina; Zhu, Quan; Marasco, Wayne A.

    2016-01-01

    Advances in the treatment of metastatic clear cell renal cell carcinoma (ccRCC) have led to improved progression-free survival of many patients; however the therapies are toxic, rarely achieve durable long-term complete responses and are not curative. Herein we used a single bicistronic lentiviral vector to develop a new combination immunotherapy that consists of human anti-carbonic anhydrase IX (CAIX)-targeted chimeric antigen receptor (CAR) T cells engineered to secrete human anti-programmed death ligand 1 (PD-L1) antibodies at the tumor site. The local antibody delivery led to marked immune checkpoint blockade. Tumor growth diminished 5 times and tumor weight reduced 50–80% when compared with the anti-CAIX CAR T cells alone in a humanized mice model of ccRCC. The expression of PD-L1 and Ki67 in the tumors decreased and an increase in granzyme B levels was found in CAR T cells. The anti-PD-L1 IgG1 isotype, which is capable of mediating ADCC, was also able to recruit human NK cells to the tumor site in vivo. These armed second-generation CAR T cells empowered to secrete human anti-PD-L1 antibodies in the ccRCC milieu to combat T cell exhaustion is an innovation in this field that should provide renewed potential for CAR T cell immunotherapy of solid tumors where limited efficacy is currently seen. PMID:27145284

  2. 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.

  3. 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

  4. 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

    2016-08-17

    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.

  5. 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

  6. Human decellularized adipose tissue scaffold as a model for breast cancer cell growth and drug treatments.

    PubMed

    Dunne, Lina W; Huang, Zhao; Meng, Weixu; Fan, Xuejun; Zhang, Ningyan; Zhang, Qixu; An, Zhiqiang

    2014-06-01

    Human adipose tissue extracellular matrix, derived through decellularization processing, has been shown to provide a biomimetic microenvironment for adipose tissue regeneration. This study reports the use of human adipose tissue-derived extracellular matrix (hDAM) scaffolds as a three-dimensional cell culturing system for the investigation of breast cancer growth and drug treatments. The hDAM scaffolds have similar extracellular matrix composition to the microenvironment of breast tissues. Breast cancer cells were cultured in hDAM scaffolds, and cell proliferation, migration, morphology, and drug responses were investigated. The growth profiles of multiple breast cancer cell lines cultured in hDAM scaffolds differed from the growth of those cultured on two-dimensional surfaces and more closely resembled the growth of xenografts. hDAM-cultured breast cancer cells also differed from those cultured on two-dimensional surfaces in terms of cell morphology, migration, expression of adhesion molecules, and sensitivity to drug treatment. Our results demonstrated that the hDAM system provides breast cancer cells with a biomimetic microenvironment in vitro that more closely mimics the in vivo microenvironment than existing two-dimensional and Matrigel three-dimensional cultures do, and thus can provide vital information for the characterization of cancer cells and screening of cancer therapeutics.

  7. 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

  8. 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

  9. 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

  10. 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...

  11. Multiple Intravenous Administrations of Human Umbilical Cord Blood Cells Benefit in a Mouse Model of ALS

    PubMed Central

    Garbuzova-Davis, Svitlana; Rodrigues, Maria C. O.; Mirtyl, Santhia; Turner, Shanna; Mitha, Shazia; Sodhi, Jasmine; Suthakaran, Subatha; Eve, David J.; Sanberg, Cyndy D.; Kuzmin-Nichols, Nicole; Sanberg, Paul R.

    2012-01-01

    Background A promising therapeutic strategy for amyotrophic lateral sclerosis (ALS) is the use of cell-based therapies that can protect motor neurons and thereby retard disease progression. We recently showed that a single large dose (25×106 cells) of mononuclear cells from human umbilical cord blood (MNC hUCB) administered intravenously to pre-symptomatic G93A SOD1 mice is optimal in delaying disease progression and increasing lifespan. However, this single high cell dose is impractical for clinical use. The aim of the present pre-clinical translation study was therefore to evaluate the effects of multiple low dose systemic injections of MNC hUCB cell into G93A SOD1 mice at different disease stages. Methodology/Principal Findings Mice received weekly intravenous injections of MNC hUCB or media. Symptomatic mice received 106 or 2.5×106 cells from 13 weeks of age. A third, pre-symptomatic, group received 106 cells from 9 weeks of age. Control groups were media-injected G93A and mice carrying the normal hSOD1 gene. Motor function tests and various assays determined cell effects. Administered cell distribution, motor neuron counts, and glial cell densities were analyzed in mouse spinal cords. Results showed that mice receiving 106 cells pre-symptomatically or 2.5×106 cells symptomatically significantly delayed functional deterioration, increased lifespan and had higher motor neuron counts than media mice. Astrocytes and microglia were significantly reduced in all cell-treated groups. Conclusions/Significance These results demonstrate that multiple injections of MNC hUCB cells, even beginning at the symptomatic disease stage, could benefit disease outcomes by protecting motor neurons from inflammatory effectors. This multiple cell infusion approach may promote future clinical studies. PMID:22319620

  12. 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

  13. 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.

  14. 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.

  15. Studies Using an in Vitro Model Show Evidence of Involvement of Epithelial-Mesenchymal Transition of Human Endometrial Epithelial Cells in Human Embryo Implantation*

    PubMed Central

    Uchida, Hiroshi; Maruyama, Tetsuo; Nishikawa-Uchida, Sayaka; Oda, Hideyuki; Miyazaki, Kaoru; Yamasaki, Akiko; Yoshimura, Yasunori

    2012-01-01

    Human embryo implantation is a critical multistep process consisting of embryo apposition/adhesion, followed by penetration and invasion. Through embryo penetration, the endometrial epithelial cell barrier is disrupted and remodeled by an unknown mechanism. We have previously developed an in vitro model for human embryo implantation employing the human choriocarcinoma cell line JAR and the human endometrial adenocarcinoma cell line Ishikawa. Using this model we have shown that stimulation with ovarian steroid hormones (17β-estradiol and progesterone, E2P4) and suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, enhances the attachment and adhesion of JAR spheroids to Ishikawa. In the present study we showed that the attachment and adhesion of JAR spheroids and treatment with E2P4 or SAHA individually induce the epithelial-mesenchymal transition (EMT) in Ishikawa cells. This was evident by up-regulation of N-cadherin and vimentin, a mesenchymal cell marker, and concomitant down-regulation of E-cadherin in Ishikawa cells. Stimulation with E2P4 or SAHA accelerated Ishikawa cell motility, increased JAR spheroid outgrowth, and enhanced the unique redistribution of N-cadherin, which was most prominent in proximity to the adhered spheroids. Moreover, an N-cadherin functional blocking antibody attenuated all events but not JAR spheroid adhesion. These results collectively provide evidence suggesting that E2P4- and implanting embryo-induced EMT of endometrial epithelial cells may play a pivotal role in the subsequent processes of human embryo implantation with functional control of N-cadherin. PMID:22174415

  16. 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

  17. 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

  18. 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.

  19. 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

  20. Retinoic acid and hydrocortisone strengthen the barrier function of human RPMI 2650 cells, a model for nasal epithelial permeability.

    PubMed

    Kürti, Levente; Veszelka, Szilvia; Bocsik, Alexandra; Ozsvári, Béla; Puskás, László G; Kittel, Agnes; Szabó-Révész, Piroska; Deli, Mária A

    2013-05-01

    The nasal pathway represents an alternative route for non-invasive systemic administration of drugs. The main advantages of nasal drug delivery are the rapid onset of action, the avoidance of the first-pass metabolism in the liver and the easy applicability. In vitro cell culture systems offer an opportunity to model biological barriers. Our aim was to develop and characterize an in vitro model based on confluent layers of the human RPMI 2650 cell line. Retinoic acid, hydrocortisone and cyclic adenosine monophosphate, which influence cell attachment, growth and differentiation have been investigated on the barrier formation and function of the nasal epithelial cell layers. Real-time cell microelectronic sensing, a novel label-free technique was used for dynamic monitoring of cell growth and barrier properties of RPMI 2650 cells. Treatments enhanced the formation of adherens and tight intercellular junctions visualized by electron microscopy, the presence and localization of junctional proteins ZO-1 and β-catenin demonstrated by fluorescent immunohistochemistry, and the barrier function of nasal epithelial cell layers. The transepithelial resistance of the RPMI 2650 cell model reached 50 to 200 Ω × cm(2), the permeability coefficient for 4.4 kDa FITC-dextran was 9.3 to 17 × 10(-6) cm/s, in agreement with values measured on nasal mucosa from in vivo and ex vivo experiments. Based on these results human RPMI 2650 cells seem to be a suitable nasal epithelial model to test different pharmaceutical excipients and various novel formulations, such as nanoparticles for toxicity and permeability.

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

    PubMed Central

    Chudasama, Vaishali L.; Ovacik, Meric A.; Abernethy, Darrell R.

    2015-01-01

    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

  2. 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.

  3. Survivin as a potential mediator to support autoreactive cell survival in myasthenia gravis: a human and animal model study.

    PubMed

    Kusner, Linda L; Ciesielski, Michael J; Marx, Alexander; Kaminski, Henry J; Fenstermaker, Robert A

    2014-01-01

    The mechanisms that underlie the development and maintenance of autoimmunity in myasthenia gravis are poorly understood. In this investigation, we evaluate the role of survivin, a member of the inhibitor of apoptosis protein family, in humans and in two animal models. We identified survivin expression in cells with B lymphocyte and plasma cells markers, and in the thymuses of patients with myasthenia gravis. A portion of survivin-expressing cells specifically bound a peptide derived from the alpha subunit of acetylcholine receptor indicating that they recognize the peptide. Thymuses of patients with myasthenia gravis had large numbers of survivin-positive cells with fewer cells in the thymuses of corticosteroid-treated patients. Application of a survivin vaccination strategy in mouse and rat models of myasthenia gravis demonstrated improved motor assessment, a reduction in acetylcholine receptor specific autoantibodies, and a retention of acetylcholine receptor at the neuromuscular junction, associated with marked reduction of survivin-expressing circulating CD20+ cells. These data strongly suggest that survivin expression in cells with lymphocyte and plasma cell markers occurs in patients with myasthenia gravis and in two animal models of myasthenia gravis. Survivin expression may be part of a mechanism that inhibits the apoptosis of autoreactive B cells in myasthenia gravis and other autoimmune disorders.

  4. Human pluripotent stem cells: Prospects and challenges as a source of cardiomyocytes for in vitro modeling and cell-based cardiac repair.

    PubMed

    Hartman, Matthew E; Dai, Dao-Fu; Laflamme, Michael A

    2016-01-15

    Human pluripotent stem cells (PSCs) represent an attractive source of cardiomyocytes with potential applications including disease modeling, drug discovery and safety screening, and novel cell-based cardiac therapies. Insights from embryology have contributed to the development of efficient, reliable methods capable of generating large quantities of human PSC-cardiomyocytes with cardiac purities ranging up to 90%. However, for human PSCs to meet their full potential, the field must identify methods to generate cardiomyocyte populations that are uniform in subtype (e.g. homogeneous ventricular cardiomyocytes) and have more mature structural and functional properties. For in vivo applications, cardiomyocyte production must be highly scalable and clinical grade, and we will need to overcome challenges including graft cell death, immune rejection, arrhythmogenesis, and tumorigenic potential. Here we discuss the types of human PSCs, commonly used methods to guide their differentiation into cardiomyocytes, the phenotype of the resultant cardiomyocytes, and the remaining obstacles to their successful translation.

  5. Boron neutron capture therapy (BNCT) selectively destroys human clear cell sarcoma in mouse model.

    PubMed

    Fujimoto, Takuya; Andoh, Tooru; Sudo, Tamotsu; Fujita, Ikuo; Moritake, Hiroshi; Sugimoto, Tohru; Sakuma, Toshiko; Akisue, Toshihiro; Kawabata, Shinji; Kirihata, Mitsunori; Suzuki, Minoru; Sakurai, Yoshinori; Ono, Koji; Fukumori, Yoshinobu; Kurosaka, Masahiro; Ichikawa, Hideki

    2013-03-01

    Clear cell sarcoma of tendons and aponeuroses (CCS) is a rare malignant tumor with no effective treatment. This study demonstrates the efficacy of BNCT with the use of human CCS-bearing nude mice. Groups A and C were administered saline, and groups B and D were injected with p-borono-L-phenylalanine-fructose complex. Groups C and D were then irradiated with thermal neutrons. The tumors in only group D disappeared, demonstrating that BNCT is a potentially new option for the treatment of human CCS.

  6. Human Atrial Cell Models to Analyse Haemodialysis-Related Effects on Cardiac Electrophysiology: Work in Progress

    PubMed Central

    2014-01-01

    During haemodialysis (HD) sessions, patients undergo alterations in the extracellular environment, mostly concerning plasma electrolyte concentrations, pH, and volume, together with a modification of sympathovagal balance. All these changes affect cardiac electrophysiology, possibly leading to an increased arrhythmic risk. Computational modeling may help to investigate the impact of HD-related changes on atrial electrophysiology. However, many different human atrial action potential (AP) models are currently available, all validated only with the standard electrolyte concentrations used in experiments. Therefore, they may respond in different ways to the same environmental changes. After an overview on how the computational approach has been used in the past to investigate the effect of HD therapy on cardiac electrophysiology, the aim of this work has been to assess the current state of the art in human atrial AP models, with respect to the HD context. All the published human atrial AP models have been considered and tested for electrolytes, volume changes, and different acetylcholine concentrations. Most of them proved to be reliable for single modifications, but all of them showed some drawbacks. Therefore, there is room for a new human atrial AP model, hopefully able to physiologically reproduce all the HD-related effects. At the moment, work is still in progress in this specific field. PMID:25587348

  7. Use of a SCID mouse/human lymphoma model to evaluate cytokine-induced killer cells with potent antitumor cell activity

    PubMed Central

    1991-01-01

    C.B-17 severe combined immune deficient (SCID) mice, which lack functional B and T lymphocytes, allow xenografts and, therefore, can be used to study the biology of human malignancies. Two different human B cell lymphoma cell lines, SU-DHL-4 and OCI-Ly8, which both harbor the t(14;18) chromosomal translocation, were injected into C.B-17 SCID mice. Mice injected intravenously or intraperitoneally developed tumors and died in a dose-dependent manner. The presence of tumor cells in various murine tissues could be demonstrated by a clonogenic tumor assay, staining of frozen sections with a monoclonal antibody (mAb) against a human B cell antigen (CD19), and with the polymerase chain reaction technique. A protocol using cytotoxic effector cells was developed and used to selectively deplete the tumor cells from bone marrow. These cells were developed by growing peripheral blood mononuclear cells in the presence of interferon gamma (IFN-gamma), anti- CD3 mAb, and interleukin 2 (IL-2). The timing of IFN-gamma treatment was critical and optimal if IFN-gamma was added before IL-2 treatment. The cells that were stimulated by IFN-gamma, followed by IL-2, could be expanded by treatment with a mAb directed against CD3. These cells could be further activated by IL-1, but not by tumor necrosis factor alpha. With this protocol, a tumor cell kill of 3 logs was obtained as measured by a clonogenic assay. Interestingly, despite their high cytotoxic activity against lymphoma cells, these cells had little toxicity against a subset of normal human hematopoietic precursor cells (granulocyte/macrophage colony-forming units). These cells were further tested by treating murine bone marrow contaminated with the human lymphoma cell line SU-DHL-4, and injecting these cells into SCID mice to assay for tumor growth in vivo. The animals injected with bone marrow contaminated with SU-DHL-4 cells had enhanced survival if the bone marrow was treated with the cytokine-induced killer cells before

  8. Cytotoxic Capacity of IL-15-Stimulated Cytokine-Induced Killer Cells Against Human Acute Myeloid Leukemia and Rhabdomyosarcoma in Humanized Preclinical Mouse Models

    PubMed Central

    Rettinger, Eva; Meyer, Vida; Kreyenberg, Hermann; Volk, Andreas; Kuçi, Selim; Willasch, Andre; Koscielniak, Ewa; Fulda, Simone; Wels, Winfried S.; Boenig, Halvard; Klingebiel, Thomas; Bader, Peter

    2012-01-01

    Allogeneic stem cell transplantation (allo-SCT) has become an important treatment modality for patients with high-risk acute myeloid leukemia (AML) and is also under investigation for soft tissue sarcomas. The therapeutic success is still limited by minimal residual disease (MRD) status ultimately leading to patients’ relapse. Adoptive donor lymphocyte infusions based on MRD status using IL-15-expanded cytokine-induced killer (CIK) cells may prevent relapse without causing graft-versus-host-disease (GvHD). To generate preclinical data we developed mouse models to study anti-leukemic- and anti-tumor-potential of CIK cells in vivo. Immunodeficient mice (NOD/SCID/IL-2Rγc−, NSG) were injected intravenously with human leukemic cell lines THP-1, SH-2 and with human rhabdomyosarcoma (RMS) cell lines RH41 and RH30 at minimal doses required for leukemia or tumor engraftment. Mice transplanted with THP-1 or RH41 cells were randomly assigned for analysis of CIK cell treatment. Organs of mice were analyzed by flow cytometry as well as quantitative polymerase chain reaction for engraftment of malignant cells and CIK cells. Potential of CIK cells to induce GvHD was determined by histological analysis. Tissues of the highest degree of THP-1 cell expansion included bone marrow followed by liver, lung, spleen, peripheral blood (PB), and brain. RH30 and RH41 engraftment mainly took place in liver and lung, but was also detectable in spleen and PB. In spite of delayed CIK cell expansion compared with malignant cells, CIK cells injected at equal amounts were sufficient for significant reduction of RH41 cells, whereas against fast-expanding THP-1 cells 250 times more CIK than THP-1 cells were needed to achieve comparable results. Our preclinical in vivo mouse models showed a reliable 100% engraftment of malignant cells which is essential for analysis of anti-cancer therapy. Furthermore our data demonstrated that IL-15-activated CIK cells have potent cytotoxic capacity against AML

  9. 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

  10. 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.

  11. 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

  12. 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

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

    PubMed

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

    2015-01-28

    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.

  14. Urine-derived induced pluripotent stem cells as a modeling tool to study rare human diseases

    PubMed Central

    Shi, Liang; Cui, Yazhou; Luan, Jing; Zhou, Xiaoyan; Han, Jinxiang

    2016-01-01

    Summary Rare diseases with a low prevalence are a key public health issue because the causes of those diseases are difficult to determine and those diseases lack a clearly established or curative treatment. Thus, investigating the molecular mechanisms that underlie the pathology of rare diseases and facilitating the development of novel therapies using disease models is crucial. Human induced pluripotent stem cells (iPSCs) are well suited to modeling rare diseases since they have the capacity for self-renewal and pluripotency. In addition, iPSC technology provides a valuable tool to generate patient-specific iPSCs. These cells can be differentiated into cell types that have been affected by a disease. These cells would circumvent ethical concerns and avoid immunological rejection, so they could be used in cell replacement therapy or regenerative medicine. To date, human iPSCs could have been generated from multiple donor sources, such as skin, adipose tissue, and peripheral blood. However, these cells are obtained via invasive procedures. In contrast, several groups of researchers have found that urine may be a better source for producing iPSCs from normal individuals or patients. This review discusses urinary iPSC (UiPSC) as a candidate for modeling rare diseases. Cells obtained from urine have overwhelming advantages compared to other donor sources since they are safely, affordably, and frequently obtained and they are readily obtained from patients. The use of iPSC-based models is also discussed. UiPSCs may prove to be a key means of modeling rare diseases and they may facilitate the treatment of those diseases in the future. PMID:27672542

  15. Cross-talk between human neural stem/progenitor cells and peripheral blood mononuclear cells in an allogeneic co-culture model.

    PubMed

    Zhang, Hongxia; Shao, Bei; Zhuge, Qichuan; Wang, Peng; Zheng, Chengcai; Huang, Weilong; Yang, Chenqi; Wang, Brian; Su, Dong-Ming; Jin, Kunlin

    2015-01-01

    Transplantation of human neural stem/progenitor cells (hNSCs) as a regenerative cell replacement therapy holds great promise. However, the underlying mechanisms remain unclear. We, here, focused on the interaction between hNSCs and allogeneic peripheral blood mononuclear cells (PBMCs) in a co-culture model. We found that hNSCs significantly decrease the CD3+ and CD8+ T cells, reduce the gamma delta T cells and increase the regulatory T cells, along with reduced pro-inflammatory cytokines and increased anti-inflammatory cytokines after co-culture. We also found that PBMCs, in turn, significantly promote the proliferation and differentiation of hNSCs. Our data suggest that hNSCs cross-talk with immune cells.

  16. 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.

  17. Human cells and cell membrane molecular models are affected in vitro by the nonsteroidal anti-inflammatory drug ibuprofen.

    PubMed

    Manrique-Moreno, Marcela; Villena, Fernando; Sotomayor, Carlos P; Edwards, Ana M; Muñoz, Marcelo A; Garidel, Patrick; Suwalsky, Mario

    2011-11-01

    This report presents evidence that ibuprofen interacts with red cell membranes as follows: a) in scanning electron microscopy (SEM) studies on human erythrocytes induced shape changes at a concentration as low as 10μM; b) in isolated unsealed human erythrocyte membranes (IUM) induced mild increase in the water content or in their molecular dynamics at the hydrophobic-hydrophilic interphase, while a corresponding ordering decrease at the deep phospholipids acyl chain level; c) at physiological temperature (37°C), 300μM ibuprofen induced a significant increase in the generalized polarization (GP) of dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles (LUV), an indication that ibuprofen molecules locate in the head polar group region of DMPC; d) X-ray diffraction studies showed that ibuprofen concentrations≥300μM induced increasing structural perturbation to DMPC bilayers; e) differential scanning calorimetry (DSC) data showed that ibuprofen was able to alter the cooperativity of DMPC phase transition in a concentration-dependent manner, to destabilize the gel phase and that ibuprofen did not significantly perturb the organization of the lipid hydrocarbon chains. Additionally, the effect on the viability of both human promyelocytic leukemia HL-60 and human cervical carcinoma HeLa cells was studied. PMID:21798236

  18. Influenza A Viruses Grow in Human Pancreatic Cells and Cause Pancreatitis and Diabetes in an Animal Model

    PubMed Central

    Mercalli, Alessia; Pizzuto, Matteo S.; Romero-Tejeda, Aurora; Kasloff, Samantha; De Battisti, Cristian; Bonfante, Francesco; Patrono, Livia V.; Vicenzi, Elisa; Zappulli, Valentina; Lampasona, Vito; Stefani, Annalisa; Doglioni, Claudio; Terregino, Calogero; Cattoli, Giovanni; Piemonti, Lorenzo

    2013-01-01

    Influenza A viruses commonly cause pancreatitis in naturally and experimentally infected animals. In this study, we report the results of in vivo investigations carried out to establish whether influenza virus infection could cause metabolic disorders linked to pancreatic infection. In addition, in vitro tests in human pancreatic islets and in human pancreatic cell lines were performed to evaluate viral growth and cell damage. Infection of an avian model with two low-pathogenicity avian influenza isolates caused pancreatic damage resulting in hyperlipasemia in over 50% of subjects, which evolved into hyperglycemia and subsequently diabetes. Histopathology of the pancreas showed signs of an acute infection resulting in severe fibrosis and disruption of the structure of the organ. Influenza virus nucleoprotein was detected by immunohistochemistry (IHC) in the acinar tissue. Human seasonal H1N1 and H3N2 viruses and avian H7N1 and H7N3 influenza virus isolates were able to infect a selection of human pancreatic cell lines. Human viruses were also shown to be able to infect human pancreatic islets. In situ hybridization assays indicated that viral nucleoprotein could be detected in beta cells. The cytokine activation profile indicated a significant increase of MIG/CXCL9, IP-10/CXCL10, RANTES/CCL5, MIP1b/CCL4, Groa/CXCL1, interleukin 8 (IL-8)/CXCL8, tumor necrosis factor alpha (TNF-α), and IL-6. Our findings indicate that influenza virus infection may play a role as a causative agent of pancreatitis and diabetes in humans and other mammals. PMID:23097451

  19. 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

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

    PubMed

    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

  1. 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

  2. Structural effects in vitro of the anti-inflammatory drug diclofenac on human erythrocytes and molecular models of cell membranes.

    PubMed

    Suwalsky, Mario; Manrique, Marcela; Villena, Fernando; Sotomayor, Carlos P

    2009-04-01

    Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), has been widely investigated in terms of its pharmacological action, but less is known about its effects on cell membranes and particularly on those of human erythrocytes. In the present work, the structural effects on the human erythrocyte membrane and molecular models have been investigated and reported. This report presents the following evidence that diclofenac interacts with red cell membranes: a) X-ray diffraction and fluorescence spectroscopy of phospholipid bilayers showed that diclofenac interacted with a class of lipids found in the outer moiety of the erythrocyte membrane; b) in isolated unsealed human erythrocyte membranes (IUM) the drug induced a disordering effect on the acyl chains of the membrane lipid bilayer; c) in scanning electron microscopy (SEM) studies on human erythrocytes it was observed that the drug induced changes different from the normal biconcave morphology of most red blood cells. This is the first time in which structural effects of diclofenac on the human erythrocyte membrane have been described. PMID:19157682

  3. 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.

  4. Three Dimensional Human Neuro-Spheroid Model of Alzheimer’s Disease Based on Differentiated Induced Pluripotent Stem Cells

    PubMed Central

    Lee, Han-Kyu; Velazquez Sanchez, Clara; Chen, Mei; Morin, Peter J.; Wells, John M.; Hanlon, Eugene B.

    2016-01-01

    The testing of candidate drugs to slow progression of Alzheimer’s disease (AD) requires clinical trials that are lengthy and expensive. Efforts to model the biochemical milieu of the AD brain may be greatly facilitated by combining two cutting edge technologies to generate three-dimensional (3D) human neuro-spheroid from induced pluripotent stem cells (iPSC) derived from AD subjects. We created iPSC from blood cells of five AD patients and differentiated them into 3D human neuronal culture. We characterized neuronal markers of our 3D neurons by immunocytochemical staining to validate the differentiation status. To block the generation of pathologic amyloid β peptides (Aβ), the 3D-differentiated AD neurons were treated with inhibitors targeting β-secretase (BACE1) and γ-secretases. As predicted, both BACE1 and γ-secretase inhibitors dramatically decreased Aβ generation in iPSC-derived neural cells derived from all five AD patients, under standard two-dimensional (2D) differentiation conditions. However, BACE1 and γ-secretase inhibitors showed less potency in decreasing Aβ levels in neural cells differentiated under 3D culture conditions. Interestingly, in a single subject AD1, we found that BACE1 inhibitor treatment was not able to significantly reduce Aβ42 levels. To investigate underlying molecular mechanisms, we performed proteomic analysis of 3D AD human neuronal cultures including AD1. Proteomic analysis revealed specific reduction of several proteins that might contribute to a poor inhibition of BACE1 in subject AD1. To our knowledge, this is the first iPSC-differentiated 3D neuro-spheroid model derived from AD patients’ blood. Our results demonstrate that our 3D human neuro-spheroid model can be a physiologically relevant and valid model for testing efficacy of AD drug. PMID:27684569

  5. 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.

  6. Modeling anhydrobiosis: activation of the mitogen-activated protein kinase ERK by dehydration in both human cells and nematodes.

    PubMed

    Huang, Zebo; Banton, Matthew C; Tunnacliffe, Alan

    2010-12-01

    Anhydrobiosis ("life without water") is the state of suspended animation that certain organisms, including some nematodes, tardigrades, and bdelloid rotifers, enter during desiccation. Extreme water loss imposes considerable stress on biomolecules, cells, and tissues, and must require specific sensing and response mechanisms for survival. However, these mechanisms are poorly understood, in part owing to the lack of amenable model systems. We have, therefore, begun to develop mammalian cell lines as tools for investigating the eukaryotic response to desiccation, and have an additional long-term goal of generating a desiccation-tolerant mammalian cell. Here, we investigate the role of the mitogen-activated protein kinases (MAPKs) in controlling gene expression in response to evaporative water loss. We report that the ERK MAPK pathway inhibitor U0126 can almost completely block induction of desiccation early response genes in a human cell line, suggesting a role for the ERK signal transduction pathway in the stress response. Accordingly, ERK is activated by phosphorylation during desiccation of human cells. Importantly, nematodes also activate ERK on drying, showing that the mammalian cell model behaves similarly to invertebrates experiencing similar stress conditions. We further reveal that, in response to desiccation, human cells can rapidly initiate complex stress signaling networks involving all three MAPK pathways, with transient activation of ERK and sustained activation of JNK and p38. These results are consistent with a role for MAPK pathways in anhydrobiotic adaptation and suggest that non-anhydrobiotes are able to sense and, at least to some extent, respond appropriately to evaporative water loss.

  7. 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

  8. 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.

  9. Human tonsil-derived mesenchymal stromal cells enhanced myelopoiesis in a mouse model of allogeneic bone marrow transplantation

    PubMed Central

    Ryu, Jung-Hwa; Park, Minhwa; Kim, Bo-Kyung; Kim, Yu-Hee; Woo, So-Youn; Ryu, Kyung-Ha

    2016-01-01

    Mesenchymal stromal cells (MSCs) have therapeutic potential for repairing tissue damage and are involved in immune regulation. MSCs are predominantly isolated from bone marrow (BM), adipose tissue or placental tissue. Further to these well-known sources, the isolation of MSCs from human tonsils was previously reported. The aim of the present study was to investigate a potential role for tonsil-derived MSCs (T-MSCs) in BM reconstitution and application towards supplementing hematopoiesis in a mouse model of BM transplantation (BMT). Eight-week-old BALB/c female mice received 80 mg/kg busulfan (Bu)/200 mg/kg cyclophosphamide (Cy) conditioning chemotherapy for BM ablation. Subsequently, human T-MSCs were injected into the Bu/Cy-treated mice with or without BM cells (BMCs) obtained from allogeneic C57BL/6 male mice. After 3 weeks, peripheral blood and BM was collected for analysis. The red blood cell count in the group that received BMCs had almost returned to normal, whereas mononuclear cell counts and BM cellularity were most improved in the T-MSCs + BMCs group. These results indicate that the T-MSCs enhanced myelopoiesis in the allogeneic BMT mouse model, as evidenced by the restoration of BM with hematopoietic cells, as well as increased myeloid colony formation in vitro. Therefore, T-MSCs may provide a source of MSCs to facilitate myelopoiesis and megakaryocytosis following BMT. PMID:27511380

  10. Borrelia burgdorferi Induces TLR2-Mediated Migration of Activated Dendritic Cells in an Ex Vivo Human Skin Model

    PubMed Central

    Wagemakers, Alex; van ‘t Veer, Cornelis; Oei, Anneke; van der Pot, Wouter J.; Ahmed, Kalam; van der Poll, Tom; Geijtenbeek, Teunis B. H.; Hovius, Joppe W. R.

    2016-01-01

    Borrelia burgdorferi is transmitted into the skin of the host where it encounters and interacts with two dendritic cell (DC) subsets; Langerhans cells (LCs) and dermal DCs (DDCs). These cells recognize pathogens via pattern recognition receptors, mature and migrate out of the skin into draining lymph nodes, where they orchestrate adaptive immune responses. In order to investigate the response of skin DCs during the early immunopathogenesis of Lyme borreliosis, we injected B. burgdorferi intradermally into full-thickness human skin and studied the migration of DCs out of the skin, the activation profile and phenotype of migrated cells. We found a significant increase in the migration of LCs and DDCs in response to B. burgdorferi. Notably, migration was prevented by blocking TLR2. DCs migrated from skin inoculated with higher numbers of spirochetes expressed significantly higher levels of CD83 and produced pro-inflammatory cytokines. No difference was observed in the expression of HLA-DR, CD86, CD38, or CCR7. To conclude, we have established an ex vivo human skin model to study DC-B. burgdorferi interactions. Using this model, we have demonstrated that B. burgdorferi-induced DC migration is mediated by TLR2. Our findings underscore the utility of this model as a valuable tool to study immunity to spirochetal infections. PMID:27695100

  11. 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.

  12. 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.

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

    PubMed Central

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

    2015-01-01

    Occupational and environmental exposure to arsenic (III) and chromium VI (Cr(VI)) have been confirmed to cause lung cancer. Mechanisms of these metals-induced carcinogenesis are still under investigation. Selection of cell lines to be used is essential for the mechanistic 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

  14. 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

  15. Modeling ALS using motor neurons derived from human induced pluripotent stem cells

    PubMed Central

    Sances, S; Bruijn, LI; Chandran, S; Eggan, K; Ho, R; Klim, J; Livesey, MR; Lowry, E; Macklis, JD; Rushton, D; Sadegh, C; Sareen, D; Wichterle, H; Zhang, SC; Svendsen, CN

    2016-01-01

    Directing the differentiation of induced pluripotent stem cells into motor neurons has allowed investigators to develop novel models of ALS. However, techniques vary between laboratories and the cells do not appear to mature into fully functional adult motor neurons. Here we discuss common developmental principles of both lower and upper motor neuron development that have led to specific derivation techniques. We then suggest how these motor neurons may be matured further either through direct expression or administration of specific factors or co-culture approaches with other tissues. Ultimately, through a greater understanding of motor neuron biology, it will be possible to establish more reliable models of ALS. These in turn will have a greater chance of validating new drugs that may be effective for the disease. PMID:27021939

  16. 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

  17. Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes as a Model for Heart Development and Congenital Heart Disease.

    PubMed

    Doyle, Michelle J; Lohr, Jamie L; Chapman, Christopher S; Koyano-Nakagawa, Naoko; Garry, Mary G; Garry, Daniel J

    2015-10-01

    Congenital heart disease (CHD) remains a significant health problem, with a growing population of survivors with chronic disease. Despite intense efforts to understand the genetic basis of CHD in humans, the etiology of most CHD is unknown. Furthermore, new models of CHD are required to better understand the development of CHD and to explore novel therapies for this patient population. In this review, we highlight the role that human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes can serve to enhance our understanding of the development, pathophysiology and potential therapeutic targets for CHD. We highlight the use of hiPSC-derived cardiomyocytes to model gene regulatory interactions, cell-cell interactions and tissue interactions contributing to CHD. We further emphasize the importance of using hiPSC-derived cardiomyocytes as personalized research models. The use of hiPSCs presents an unprecedented opportunity to generate disease-specific cellular models, investigate the underlying molecular mechanisms of disease and uncover new therapeutic targets for CHD.

  18. Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes as a Model for Heart Development and Congenital Heart Disease

    PubMed Central

    Doyle, Michelle J.; Lohr, Jamie L.; Chapman, Christopher S.; Nakagawa-Koyano, Naoko; Garry, Mary G.; Garry, Daniel J.

    2015-01-01

    Congenital heart disease (CHD) remains a significant health problem, with a growing population of survivors with chronic disease. Despite intense efforts to understand the genetic basis of CHD in humans, the etiology of most CHD is unknown. Furthermore, new models of CHD are required to better understand the development of CHD and to explore novel therapies for this patient population. In this review, we highlight the role that human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes can serve to enhance our understanding of the development, pathophysiology and potential therapeutic targets in CHD. We highlight the use of hiPSC-derived cardiomyocytes to model gene regulatory interactions, cell-cell interactions and tissue interactions contributing to CHD. We further emphasize the importance of using hiPSC-derived cardiomyocytes as personalized research models. The use of hiPSCs presents an unprecedented opportunity to generate disease-specific cellular models, investigate the underlying molecular mechanisms of disease and uncover new therapeutic targets for CHD. PMID:26085192

  19. Modeling Human Bone Marrow Failure Syndromes Using Pluripotent Stem Cells and Genome Engineering.

    PubMed

    Jung, Moonjung; Dunbar, Cynthia E; Winkler, Thomas

    2015-12-01

    The combination of epigenetic reprogramming with advanced genome editing technologies opened a new avenue to study disease mechanisms, particularly of disorders with depleted target tissue. Bone marrow failure syndromes (BMFS) typically present with a marked reduction of peripheral blood cells due to a destroyed or dysfunctional bone marrow compartment. Somatic and germline mutations have been etiologically linked to many cases of BMFS. However, without the ability to study primary patient material, the exact pathogenesis for many entities remained fragmentary. Capturing the pathological genotype in induced pluripotent stem cells (iPSCs) allows studying potential developmental defects leading to a particular phenotype. The lack of hematopoietic stem and progenitor cells in these patients can also be overcome by differentiating patient-derived iPSCs into hematopoietic lineages. With fast growing genome editing techniques, such as CRISPR/Cas9, correction of disease-causing mutations in iPSCs or introduction of mutations in cells from healthy individuals enable comparative studies that may identify other genetic or epigenetic events contributing to a specific disease phenotype. In this review, we present recent progresses in disease modeling of inherited and acquired BMFS using reprogramming and genome editing techniques. We also discuss the challenges and potential shortcomings of iPSC-based models for hematological diseases.

  20. 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.

  1. BMS-708163 and Nilotinib restore synaptic dysfunction in human embryonic stem cell-derived Alzheimer’s disease models

    PubMed Central

    Nishioka, Hisae; Tooi, Norie; Isobe, Takehisa; Nakatsuji, Norio; Aiba, Kazuhiro

    2016-01-01

    Alzheimer’s disease (AD) is the most common form of dementia. Cellular AD models derived from human pluripotent stem cells are promising tools in AD research. We recently developed human embryonic stem cell-derived AD models which overexpress mutant Presenilin1 genes, and which exhibit AD phenotypes, including synaptic dysfunction. In this study, we found that our AD models showed reduced levels of RAB3A and SV2B proteins in the pre-synapses, which is a possible cause of electrophysiological abnormalities. Through the screening of chemical compounds using our AD models, we have identified Aβ peptide inhibitors which decrease the concentration of Aβ in culture supernatant. Among these, BMS-708163 and Nilotinib were found to improve the expression levels of RAB3A and SV2B proteins and to recover the electrophysiological function in our AD models. These results suggest that the AD models we developed are promising materials for the discovery of AD drugs that target the expression of pre-synaptic proteins and synaptic function. PMID:27641902

  2. BMS-708163 and Nilotinib restore synaptic dysfunction in human embryonic stem cell-derived Alzheimer’s disease models

    PubMed Central

    Nishioka, Hisae; Tooi, Norie; Isobe, Takehisa; Nakatsuji, Norio; Aiba, Kazuhiro

    2016-01-01

    Alzheimer’s disease (AD) is the most common form of dementia. Cellular AD models derived from human pluripotent stem cells are promising tools in AD research. We recently developed human embryonic stem cell-derived AD models which overexpress mutant Presenilin1 genes, and which exhibit AD phenotypes, including synaptic dysfunction. In this study, we found that our AD models showed reduced levels of RAB3A and SV2B proteins in the pre-synapses, which is a possible cause of electrophysiological abnormalities. Through the screening of chemical compounds using our AD models, we have identified Aβ peptide inhibitors which decrease the concentration of Aβ in culture supernatant. Among these, BMS-708163 and Nilotinib were found to improve the expression levels of RAB3A and SV2B proteins and to recover the electrophysiological function in our AD models. These results suggest that the AD models we developed are promising materials for the discovery of AD drugs that target the expression of pre-synaptic proteins and synaptic function. PMID:27641902

  3. BMS-708163 and Nilotinib restore synaptic dysfunction in human embryonic stem cell-derived Alzheimer's disease models.

    PubMed

    Nishioka, Hisae; Tooi, Norie; Isobe, Takehisa; Nakatsuji, Norio; Aiba, Kazuhiro

    2016-01-01

    Alzheimer's disease (AD) is the most common form of dementia. Cellular AD models derived from human pluripotent stem cells are promising tools in AD research. We recently developed human embryonic stem cell-derived AD models which overexpress mutant Presenilin1 genes, and which exhibit AD phenotypes, including synaptic dysfunction. In this study, we found that our AD models showed reduced levels of RAB3A and SV2B proteins in the pre-synapses, which is a possible cause of electrophysiological abnormalities. Through the screening of chemical compounds using our AD models, we have identified Aβ peptide inhibitors which decrease the concentration of Aβ in culture supernatant. Among these, BMS-708163 and Nilotinib were found to improve the expression levels of RAB3A and SV2B proteins and to recover the electrophysiological function in our AD models. These results suggest that the AD models we developed are promising materials for the discovery of AD drugs that target the expression of pre-synaptic proteins and synaptic function. PMID:27641902

  4. Effects of the nonsteroidal anti-inflammatory drug naproxen on human erythrocytes and on cell membrane molecular models.

    PubMed

    Manrique-Moreno, Marcela; Suwalsky, Mario; Villena, Fernando; Garidel, Patrick

    2010-03-01

    Naproxen, a nonsteroidal anti-inflammatory drug (NSAID), has been widely investigated in terms of its pharmacological action, but less is known about its effects on cell membranes and particularly those of human erythrocytes. In the present work, the structural effects on the human erythrocyte membrane and molecular models have been investigated. The latter consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), classes of lipids found in the outer and inner moieties of the erythrocyte and most cell membranes, respectively. This report presents evidence that naproxen interacts with red cell membranes as follows: a) in scanning electron microscopy (SEM) studies on human erythrocytes it has been observed that the drug induced shape changes, forming echinocytes at a concentration as low as 10microM; b) X-ray diffraction showed that naproxen strongly interacted with DMPC multilayers; in contrast, no perturbing effects on DMPE multilayers were detected; c) differential scanning calorimetry (DSC) data showed a decrease in the melting temperature (T(m)) of DMPC liposomes, which was attributed to a destabilization of the gel phase, effect that was less pronounced for DMPE. These experimental results were observed at concentrations lower than those reported for plasma after therapeutic administration. This is the first time in which the structural effects of naproxen on the human erythrocyte membrane have been described. PMID:20083338

  5. Human olfactory bulb neural stem cells expressing hNGF restore cognitive deficit in Alzheimer's disease rat model.

    PubMed

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

    2015-01-01

    In this study, we aim to demonstrate the fate of allogenic adult human olfactory bulb neural stem/progenitor cells (OBNSC/NPCs) transplanted into the rat hippocampus treated with ibotenic acid (IBO), a neurotoxicant specific to hippocampal cholinergic neurons that are lost in Alzheimer's disease. We assessed their possible ability to survive, integrate, proliferate, and differentiate into different neuronal and glial elements: we also evaluate their possible therapeutic potential, and the mechanism(s) relevant to neuroprotection following their engraftment into the CNS milieu. OBNSC/NPCs were isolated from adult human olfactory bulb patients, genetically engineered to express GFP and human nerve growth factor (hNGF) by lentivirus-mediated infection, and stereotaxically transplanted into the hippocampus of IBO-treated animals and controls. Stereological analysis of engrafted OBNSCs eight weeks post transplantation revealed a 1.89 fold increase with respect to the initial cell population, indicating a marked ability for survival and proliferation. In addition, 54.71 ± 11.38%, 30.18 ± 6.00%, and 15.09 ± 5.38% of engrafted OBNSCs were identified by morphological criteria suggestive of mature neurons, oligodendrocytes and astrocytes respectively. Taken together, this work demonstrated that human OBNSCs expressing NGF ameliorate the cognitive deficiencies associated with IBO-induced lesions in AD model rats, and the improvement can probably be attributed primarily to neuronal and glial cell replacement as well as the trophic influence exerted by the secreted NGF.

  6. 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

  7. Animal models relevant to human prostate carcinogenesis underlining the critical implication of prostatic stem/progenitor cells

    PubMed Central

    Mimeault, Murielle; Batra, Surinder K.

    2012-01-01

    Recent development of animal models relevant to human prostate cancer (PC) etiopathogenesis has provided important information on the specific functions provided by key gene products altered during disease initiation and progression to locally invasive, metastatic and hormone-refractory stages. Especially, the characterization of transgenic mouse models has indicated that the inactivation of distinct tumor suppressor proteins such as phosphatase tensin homolog deleted on chromosome 10 (PTEN), Nkx3.1, p27KIP1 and p53 and retinoblastoma (pRb) may cooperate for the malignant transformation of prostatic stem/progenitor cells into PC stem/progenitor cells and tumor development and metastases. Moreover, the sustained activation of diverse oncogenic signaling elements, including epidermal growth factor receptor (EGFR), sonic hedgehog, Wnt/β-catenin, c-Myc, Akt and nuclear factor-kappaB (NF-κB) also may contribute to the acquisition of more aggressive and hormone-refractory phenotypes by PC stem/progenitor cells and their progenies during disease progression. Importantly, it has also been shown that an enrichment of PC stem/progenitor cells expressing stem cell-like markers may occur after androgen deprivation therapy and docetaxel treatment in the transgenic mouse models of PC suggesting the critical implication of these immature PC cells in treatment resistance, tumor re-growth and disease recurrence. Of clinical interest, the molecular targeting of distinct gene products altered in PC cells by using different dietary compounds has also been shown to counteract PC initiation and progression in animal models supporting their potential use as chemopreventive or chemotherapeutic agents for eradicating the total tumor cell mass, improving current anti-hormonal and chemotherapies and preventing disease relapse. PMID:21396984

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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

  14. In vitro modeling of host-parasite interactions: the 'subgingival' biofilm challenge of primary human epithelial cells

    PubMed Central

    2009-01-01

    Background Microbial biofilms are known to cause an increasing number of chronic inflammatory and infectious conditions. A classical example is chronic periodontal disease, a condition initiated by the subgingival dental plaque biofilm on gingival epithelial tissues. We describe here a new model that permits the examination of interactions between the bacterial biofilm and host cells in general. We use primary human gingival epithelial cells (HGEC) and an in vitro grown biofilm, comprising nine frequently studied and representative subgingival plaque bacteria. Results We describe the growth of a mature 'subgingival' in vitro biofilm, its composition during development, its ability to adapt to aerobic conditions and how we expose in vitro a HGEC monolayer to this biofilm. Challenging the host derived HGEC with the biofilm invoked apoptosis in the epithelial cells, triggered release of pro-inflammatory cytokines and in parallel induced rapid degradation of the cytokines by biofilm-generated enzymes. Conclusion We developed an experimental in vitro model to study processes taking place in the gingival crevice during the initiation of inflammation. The new model takes into account that the microbial challenge derives from a biofilm community and not from planktonically cultured bacterial strains. It will facilitate easily the introduction of additional host cells such as neutrophils for future biofilm:host cell challenge studies. Our methodology may generate particular interest, as it should be widely applicable to other biofilm-related chronic inflammatory diseases. PMID:20043840

  15. Mouse models of human non-small-cell lung cancer: raising the bar.

    PubMed

    Kim, C F B; Jackson, E L; Kirsch, D G; Grimm, J; Shaw, A T; Lane, K; Kissil, J; Olive, K P; Sweet-Cordero, A; Weissleder, R; Jacks, T

    2005-01-01

    Lung cancer is a devastating disease that presents a challenge to basic research to provide new steps toward therapeutic advances. The cell-type-specific responses to oncogenic mutations that initiate and regulate lung cancer remain poorly defined. A better understanding of the relevant signaling pathways and mechanisms that control therapeutic outcome could also provide new insight. Improved conditional mouse models are now available as tools to improve the understanding of the cellular and molecular origins of adenocarcinoma. These models have already proven their utility in proof-of-principle experiments with new technologies including genomics and imaging. Integrated thinking to apply technological advances while using the appropriate mouse model is likely to facilitate discoveries that will significantly improve lung cancer detection and intervention.

  16. 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

  17. 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

  18. Asymmetric dimethylarginine exacerbates Aβ-induced toxicity and oxidative stress in human cell and Caenorhabditis elegans models of Alzheimer disease.

    PubMed

    Luo, Yunfeng; Yue, Wenhui; Quan, Xin; Wang, Yue; Zhao, Baolu; Lu, Zhongbing

    2015-02-01

    Growing evidence suggests a strong association between cardiovascular risk factors and incidence of Alzheimer disease (AD). Asymmetric dimethylarginine (ADMA), the endogenous nitric oxide synthase inhibitor, has been identified as an independent cardiovascular risk factor and is also increased in plasma of patients with AD. However, whether ADMA is involved in the pathogenesis of AD is unknown. In this study, we found that ADMA content was increased in a transgenic Caenorhabditis elegans β-amyloid (Aβ) overexpression model, strain CL2006, and in human SH-SY5Y cells overexpressing the Swedish mutant form of human Aβ precursor protein (APPsw). Moreover, ADMA treatment exacerbated Aβ-induced paralysis and oxidative stress in CL2006 worms and further elevated oxidative stress and Aβ secretion in APPsw cells. Knockdown of type 1 protein arginine N-methyltransferase to reduce ADMA production failed to show a protective effect against Aβ toxicity, but resulted in more paralysis in CL2006 worms as well as increased oxidative stress and Aβ secretion in APPsw cells. However, overexpression of dimethylarginine dimethylaminohydrolase 1 (DDAH1) to promote ADMA degradation significantly attenuated oxidative stress and Aβ secretion in APPsw cells. Collectively, our data support the hypothesis that elevated ADMA contributes to the pathogenesis of AD. Our findings suggest that strategies to increase DDAH1 activity in neuronal cells may be a novel approach to attenuating AD development.

  19. Innate immune mediator profiles and their regulation in a novel polarized immortalized epithelial cell model derived from human endocervix.

    PubMed

    Buckner, Lyndsey R; Schust, Danny J; Ding, Jian; Nagamatsu, Takeshi; Beatty, Wandy; Chang, Theresa L; Greene, Sheila J; Lewis, Maria E; Ruiz, Bernardo; Holman, Stacey L; Spagnuolo, Rae Ann; Pyles, Richard B; Quayle, Alison J

    2011-12-01

    The endocervix in the female reproductive tract (FRT) is susceptible to sexually transmitted pathogens such as Chlamydia trachomatis and Neisseria gonorrhoeae. Endocervical epithelial cells in vivo make innate immune mediators that likely aid in the protection from these pathogens. In vitro studies to investigate the innate epithelial cell immune response to endocervical pathogens have been hindered by the paucity of human endocervix-derived epithelial cell lines that display the differentiation proteins and functional characteristics of their site of origin. We have established an immortalized epithelial cell line (A2EN) derived from an endocervical tissue explant that can be polarized to exhibit distinct apical and basolateral membrane domains. Polarized A2EN cells secrete mucus at their apical surface, and express MUC5B, a mucin specific to the endocervix. Polarized A2EN cells also express hormone receptors that respond appropriately to female steroid hormones. Polarized A2EN cells can be stimulated with the toll-like receptor 3 agonist, polyI:C, to express anti-microbial peptides (AMPs) as well as pro-inflammatory cytokines and chemokines. Cytokines and chemokines are also differentially secreted depending on the hormone milieu in which the cells are exposed. We conclude that polarized A2EN cells maintain distinctive phenotypic and functional characteristics of the epithelial cells found in the endocervix and, hence, could provide a useful, new in vitro model system for investigations on the role of endogenous and exogenous factors that regulate endocervical epithelial cell immunity including studies on sexually transmitted infections and topical microbicides.

  20. A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells.

    PubMed

    Marchetto, Maria C N; Carromeu, Cassiano; Acab, Allan; Yu, Diana; Yeo, Gene W; Mu, Yangling; Chen, Gong; Gage, Fred H; Muotri, Alysson R

    2010-11-12

    Autism spectrum disorders (ASD) are complex neurodevelopmental diseases in which different combinations of genetic mutations may contribute to the phenotype. Using Rett syndrome (RTT) as an ASD genetic model, we developed a culture system using induced pluripotent stem cells (iPSCs) from RTT patients' fibroblasts. RTT patients' iPSCs are able to undergo X-inactivation and generate functional neurons. Neurons derived from RTT-iPSCs had fewer synapses, reduced spine density, smaller soma size, altered calcium signaling and electrophysiological defects when compared to controls. Our data uncovered early alterations in developing human RTT neurons. Finally, we used RTT neurons to test the effects of drugs in rescuing synaptic defects. Our data provide evidence of an unexplored developmental window, before disease onset, in RTT syndrome where potential therapies could be successfully employed. Our model recapitulates early stages of a human neurodevelopmental disease and represents a promising cellular tool for drug screening, diagnosis and personalized treatment. PMID:21074045

  1. A logistic model for the detection of circulating tumour cells in human metastatic colorectal cancer

    PubMed Central

    Barbazán, Jorge; Vieito, María; Abalo, Alicia; Alonso-Alconada, Lorena; Muinelo-Romay, Laura; Alonso-Nocelo, Marta; León, Luís; Candamio, Sonia; Gallardo, Elena; Anido, Urbano; Doll, Andreas; los Ángeles Casares, María; Gómez-Tato, Antonio; Abal, Miguel; López-López, Rafael

    2012-01-01

    The accuracy in the diagnosis of metastatic colorectal cancer (mCRC) represents one of the challenges in the clinical management of patients. The detection of circulating tumour cells (CTC) is becoming a promising alternative to current detection techniques, as it focuses on one of the players of the metastatic disease and it should provide with more specific and sensitive detection rates. Here, we describe an improved method of detection of CTC from mCRC patients by combining immune-enrichment, optimal purification of RNA from very low cell numbers, and the selection of accurate PCR probes. As a result, we obtained a logistic model that combines GAPDH and VIL1 normalized to CD45 rendering powerful results in the detection of CTC from mCRC patients (AUROC value 0.8599). We further demonstrated the utility of this model at the clinical setting, as a reliable prognosis tool to determine progression-free survival in mCRC patients. Overall, we developed a strategy that ameliorates the specificity and sensitivity in the detection of CTC, resulting in a robust and promising logistic model for the clinical management of metastatic colorectal cancer patients. PMID:22304365

  2. Computational Model of Steroidogenesis in Human H295R Cells to Predict Biochemical Response to Endocrine-Active Chemicals: Model Development for Metyrapone

    PubMed Central

    Terasaki, Natsuko; Yamazaki, Makoto; Conolly, Rory B.

    2009-01-01

    Background: An in vitro steroidogenesis assay using the human adrenocortical carcinoma cell line H295R is being evaluated as a possible screening assay to detect and assess the impact of endocrine-active chemicals (EACs) capable of altering steroid biosynthesis. Data interpretation and their quantitative use in human and ecological risk assessments can be enhanced with mechanistic computational models to help define mechanisms of action and improve understanding of intracellular concentrationresponse behavior. Objectives: The goal of this study was to develop a mechanistic computational model of the metabolic network of adrenal steroidogenesis to estimate the synthesis and secretion of adrenal steroids in human H295R cells and their biochemical response to steroidogenesis-disrupting EAC. Methods: We developed a deterministic model that describes the biosynthetic pathways for the conversion of cholesterol to adrenal steroids and the kinetics for enzyme inhibition by metryrapone (MET), a model EAC. Using a nonlinear parameter estimation method, the model was fitted to the measurements from an in vitro steroidogenesis assay using H295R cells. Results: Model-predicted steroid concentrations in cells and culture medium corresponded well to the time-course measurements from control and MET-exposed cells. A sensitivity analysis indicated the parameter uncertainties and identified transport and metabolic processes that most influenced the concentrations of primary adrenal steroids, aldosterone and cortisol. Conclusions: Our study demonstrates the feasibility of using a computational model of steroidogenesis to estimate steroid concentrations in vitro. This capability could be useful to help define mechanisms of action for poorly characterized chemicals and mixtures in support of predictive hazard and risk assessments with EACs. PMID:20123619

  3. 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

  4. Human mesenchymal stem cells attenuate early damage in a ventilated pig model of acute lung injury.

    PubMed

    Moodley, Yuben; Sturm, Marian; Shaw, Kathryn; Shimbori, Chiko; Tan, Dino B A; Kolb, Martin; Graham, Ruth

    2016-07-01

    Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a major cause of global morbidity and mortality. Mesenchymal stem cells (MSC) have shown promise in treating inflammatory lung conditions. We hypothesised that human MSC (hMSC) can improve ALI/ARDS through their anti-inflammatory actions. We subjected pigs (n=6) to intravenous oleic acid (OA) injury, ventilation and hMSC infusion, while the controls (n=5) had intravenous OA, ventilation and an infusion vehicle control. hMSC were infused 1h after the administration of OA. The animals were monitored for additional 4h. Nuclear translocation of nuclear factor-light chain enhancer of activated B cells (NF-κB), a transcription factor that mediates several inflammatory pathways was reduced in hMSC treated pigs compared to controls (p=0.04). There was no significant difference in lung injury, assessed by histological scoring in hMSC treated pigs versus controls (p=0.063). There was no difference in neutrophil counts between hMSC-treated pigs and controls. Within 4h, there was no difference in the levels of IL-10 and IL-8 pre- and post-treatment with hMSC. In addition, there was no difference in hemodynamics, lung mechanics or arterial blood gases between hMSC treated animals and controls. Subsequent studies are required to determine if the observed decrease in inflammatory transcription factors will translate into improvement in inflammation and in physiological parameters over the long term.

  5. 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

  6. Factors regulating the bovine, caprine, rat and human ovarian aromatase promoters in a bovine granulosa cell model.

    PubMed

    Sahmi, Fatiha; Nicola, Edmir S; Zamberlam, Gustavo O; Gonçalves, Paulo D B; Vanselow, Jens; Price, Christopher A

    2014-05-01

    The ovarian promoter of the primate and rodent genes encoding cytochrome P450 aromatase (CYP19A1) are robustly responsive to forskolin in luteinized cell models, whereas the ruminant ovarian promoter is minimally active. We explored this discrepancy by investigating the activity of the bovine ovarian promoter in two bovine granulosa cell models, luteinizing and non-luteinizing cells in vitro. In non-luteinizing cells, both FSH and IGF1 increased abundance of transcripts derived from the ovarian promoter. Comparison of the activity of promoters of several species in response to transcription factors (forskolin, NR5A2, FOXL2) in luteinizing cells demonstrated that a rat ovarian promoter-luciferase reporter was regulated mainly by forskolin (18-fold increase over basal expression) and addition of NR5A2 or FOXL2 had no further effect. Activity of a human promoter was significantly increased by NR5A2 plus forskolin (153-fold) compared with forskolin alone (71-fold over basal); addition of FOXL2 did not significantly increase promoter activity. Forskolin alone provoked minor activation of caprine and bovine promoter reporters (3-fold over basal), and addition of NR5A2 increased activity (7- to 11-fold). When forskolin, NR5A2 and FOXL2 treatments were combined, the activity of the caprine and bovine promoters increased to 20- and 34-fold, respectively. These data suggest that a major reason why CYP19A1 is not expressed in luteinized cells (and the corpus luteum) of ruminants may be the stimulatory effect of FOXL2, which does not appear to be the case in the human and rat. PMID:24556528

  7. 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.

  8. Satellite cells in human skeletal muscle plasticity.

    PubMed

    Snijders, Tim; Nederveen, Joshua P; McKay, Bryon R; Joanisse, Sophie; Verdijk, Lex B; van Loon, Luc J C; Parise, Gianni

    2015-01-01

    Skeletal muscle satellite cells are considered to play a crucial role in muscle fiber maintenance, repair and remodeling. Our knowledge of the role of satellite cells in muscle fiber adaptation has traditionally relied on in vitro cell and in vivo animal models. Over the past decade, a genuine effort has been made to translate these results to humans under physiological conditions. Findings from in vivo human studies suggest that satellite cells play a key role in skeletal muscle fiber repair/remodeling in response to exercise. Mounting evidence indicates that aging has a profound impact on the regulation of satellite cells in human skeletal muscle. Yet, the precise role of satellite cells in the development of muscle fiber atrophy with age remains unresolved. This review seeks to integrate recent results from in vivo human studies on satellite cell function in muscle fiber repair/remodeling in the wider context of satellite cell biology whose literature is largely based on animal and cell models.

  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. In vitro kinetics of amiodarone and its major metabolite in two human liver cell models after acute and repeated treatments.

    PubMed

    Pomponio, Giuliana; Savary, Camille C; Parmentier, Céline; Bois, Frederic; Guillouzo, André; Romanelli, Luca; Richert, Lysiane; Di Consiglio, Emma; Testai, Emanuela

    2015-12-25

    The limited value of in vitro toxicity data for the in vivo extrapolation has been often attributed to the lack of kinetic data. Here the in vitro kinetics of amiodarone (AMI) and its mono-N-desethyl (MDEA) metabolite was determined and modelled in primary human hepatocytes (PHH) and HepaRG cells, after single and repeated administration of clinically relevant concentrations. AMI bioavailability was influenced by adsorption to the plastic and the presence of protein in the medium (e.g. 10% serum protein reduced the uptake by half in HepaRG cells). The cell uptake was quick (within 3h), AMI metabolism was efficient and a dynamic equilibrium was reached in about a week after multiple dosing. In HepaRG cells the metabolic clearance was higher than in PHH and increased over time, as well as CYP3A4. The interindividual variability in MDEA production in PHHs was not proportional to the differences in CYP3A4 activities, suggesting the involvement of other CYPs and/or AMI-related CYP inhibition. After repeated treatment AMI showed a slight potential for bioaccumulation, whereas much higher intracellular MDEA levels accumulated over time, especially in the HepaRG cells, associated with occurrence of phospholipidosis. The knowledge of in vitro biokinetics is important to transform an actual in vitro concentration-effect into an in vivo dose-effect relationship by using appropriate modelling, thus improving the in vitro-to-in vivo extrapolation.

  11. A model for persistent infection with Epstein-Barr virus: the stealth virus of human B cells.

    PubMed

    Thorley-Lawson, D A; Babcock, G J

    1999-01-01

    Most adult humans are infected benignly and for life with the herpesvirus Epstein-Barr virus. EBV has been a focus of research because of its status as a candidate tumor virus for a number of lymphomas and carcinomas. In vitro EBV has the ability to establish a latent infection in proliferating B lymphoblasts. This is the only system available for studying human herpesvirus latency in culture and has been extremely useful for elucidating how EBV promotes cellular growth. However, to understand how EBV survives in the healthy host and what goes awry, leading to disease, it is essential to know how EBV establishes and maintains a persistent infection in vivo. Early studies on the mechanism of EBV persistence produced inconclusive and often contradictory results because the techniques available were crude and insensitive. Recent advances in PCR technology and the application of sophisticated cell fractionation techniques have now provided new insights into the behavior of the virus. Most dramatically it has been shown that EBV in vivo does not establish latency in a proliferating lymphoblast, but in a resting memory B cell. The contrasting behaviors of being able to establish a latent infection in proliferating B blasts and resting memory B cells can be resolved in terms of a model where EBV performs its complete life cycle in B lymphocytes. The virus achieves this not by disrupting normal B cell biology but by using it.

  12. Optogenetics enables functional analysis of human embryonic stem cell-derived grafts in a Parkinson's disease model.

    PubMed

    Steinbeck, Julius A; Choi, Se Joon; Mrejeru, Ana; Ganat, Yosif; Deisseroth, Karl; Sulzer, David; Mosharov, Eugene V; Studer, Lorenz

    2015-02-01

    Recent studies have shown evidence of behavioral recovery after transplantation of human pluripotent stem cell (PSC)-derived neural cells in animal models of neurological disease. However, little is known about the mechanisms underlying graft function. Here we use optogenetics to modulate in real time electrophysiological and neurochemical properties of mesencephalic dopaminergic (mesDA) neurons derived from human embryonic stem cells (hESCs). In mice that had recovered from lesion-induced Parkinsonian motor deficits, light-induced selective silencing of graft activity rapidly and reversibly re-introduced the motor deficits. The re-introduction of motor deficits was prevented by the dopamine agonist apomorphine. These results suggest that functionality depends on graft neuronal activity and dopamine release. Combining optogenetics, slice electrophysiology and pharmacological approaches, we further show that mesDA-rich grafts modulate host glutamatergic synaptic transmission onto striatal medium spiny neurons in a manner reminiscent of endogenous mesDA neurons. Thus, application of optogenetics in cell therapy can link transplantation, animal behavior and postmortem analysis to enable the identification of mechanisms that drive recovery.

  13. Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model.

    PubMed

    Plaza Reyes, Alvaro; Petrus-Reurer, Sandra; Antonsson, Liselotte; Stenfelt, Sonya; Bartuma, Hammurabi; Panula, Sarita; Mader, Theresa; Douagi, Iyadh; André, Helder; Hovatta, Outi; Lanner, Fredrik; Kvanta, Anders

    2016-01-12

    Human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells could replace lost tissue in geographic atrophy (GA) but efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model. PMID:26724907

  14. Human Mesenchymal Stem Cells Genetically Engineered to Overexpress Brain-derived Neurotrophic Factor Improve Outcomes in Huntington's Disease Mouse Models

    PubMed Central

    Pollock, Kari; Dahlenburg, Heather; Nelson, Haley; Fink, Kyle D; Cary, Whitney; Hendrix, Kyle; Annett, Geralyn; Torrest, Audrey; Deng, Peter; Gutierrez, Joshua; Nacey, Catherine; Pepper, Karen; Kalomoiris, Stefanos; D Anderson, Johnathon; McGee, Jeannine; Gruenloh, William; Fury, Brian; Bauer, Gerhard; Duffy, Alexandria; Tempkin, Theresa; Wheelock, Vicki; Nolta, Jan A

    2016-01-01

    Huntington's disease (HD) is a fatal degenerative autosomal dominant neuropsychiatric disease that causes neuronal death and is characterized by progressive striatal and then widespread brain atrophy. Brain-derived neurotrophic factor (BDNF) is a lead candidate for the treatment of HD, as it has been shown to prevent cell death and to stimulate the growth and migration of new neurons in the brain in transgenic mouse models. BDNF levels are reduced in HD postmortem human brain. Previous studies have shown efficacy of mesenchymal stem/stromal cells (MSC)/BDNF using murine MSCs, and the present study used human MSCs to advance the therapeutic potential of the MSC/BDNF platform for clinical application. Double-blinded studies were performed to examine the effects of intrastriatally transplanted human MSC/BDNF on disease progression in two strains of immune-suppressed HD transgenic mice: YAC128 and R6/2. MSC/BDNF treatment decreased striatal atrophy in YAC128 mice. MSC/BDNF treatment also significantly reduced anxiety as measured in the open-field assay. Both MSC and MSC/BDNF treatments induced a significant increase in neurogenesis-like activity in R6/2 mice. MSC/BDNF treatment also increased the mean lifespan of the R6/2 mice. Our genetically modified MSC/BDNF cells set a precedent for stem cell-based neurotherapeutics and could potentially be modified for other neurodegenerative disorders such as amyotrophic lateral sclerosis, Alzheimer's disease, and some forms of Parkinson's disease. These cells provide a platform delivery system for future studies involving corrective gene-editing strategies. PMID:26765769

  15. Human Mesenchymal Stem Cells Genetically Engineered to Overexpress Brain-derived Neurotrophic Factor Improve Outcomes in Huntington's Disease Mouse Models.

    PubMed

    Pollock, Kari; Dahlenburg, Heather; Nelson, Haley; Fink, Kyle D; Cary, Whitney; Hendrix, Kyle; Annett, Geralyn; Torrest, Audrey; Deng, Peter; Gutierrez, Joshua; Nacey, Catherine; Pepper, Karen; Kalomoiris, Stefanos; D Anderson, Johnathon; McGee, Jeannine; Gruenloh, William; Fury, Brian; Bauer, Gerhard; Duffy, Alexandria; Tempkin, Theresa; Wheelock, Vicki; Nolta, Jan A

    2016-05-01

    Huntington's disease (HD) is a fatal degenerative autosomal dominant neuropsychiatric disease that causes neuronal death and is characterized by progressive striatal and then widespread brain atrophy. Brain-derived neurotrophic factor (BDNF) is a lead candidate for the treatment of HD, as it has been shown to prevent cell death and to stimulate the growth and migration of new neurons in the brain in transgenic mouse models. BDNF levels are reduced in HD postmortem human brain. Previous studies have shown efficacy of mesenchymal stem/stromal cells (MSC)/BDNF using murine MSCs, and the present study used human MSCs to advance the therapeutic potential of the MSC/BDNF platform for clinical application. Double-blinded studies were performed to examine the effects of intrastriatally transplanted human MSC/BDNF on disease progression in two strains of immune-suppressed HD transgenic mice: YAC128 and R6/2. MSC/BDNF treatment decreased striatal atrophy in YAC128 mice. MSC/BDNF treatment also significantly reduced anxiety as measured in the open-field assay. Both MSC and MSC/BDNF treatments induced a significant increase in neurogenesis-like activity in R6/2 mice. MSC/BDNF treatment also increased the mean lifespan of the R6/2 mice. Our genetically modified MSC/BDNF cells set a precedent for stem cell-based neurotherapeutics and could potentially be modified for other neurodegenerative disorders such as amyotrophic lateral sclerosis, Alzheimer's disease, and some forms of Parkinson's disease. These cells provide a platform delivery system for future studies involving corrective gene-editing strategies. PMID:26765769

  16. Remyelination Is Correlated with Regulatory T Cell Induction Following Human Embryoid Body-Derived Neural Precursor Cell Transplantation in a Viral Model of Multiple Sclerosis.

    PubMed

    Plaisted, Warren C; Zavala, Angel; Hingco, Edna; Tran, Ha; Coleman, Ronald; Lane, Thomas E; Loring, Jeanne F; Walsh, Craig M

    2016-01-01

    We have recently described sustained clinical recovery associated with dampened neuroinflammation and remyelination following transplantation of neural precursor cells (NPCs) derived from human embryonic stem cells (hESCs) in a viral model of the human demyelinating disease multiple sclerosis. The hNPCs used in that study were derived by a novel direct differentiation method (direct differentiation, DD-NPCs) that resulted in a unique gene expression pattern when compared to hNPCs derived by conventional methods. Since the therapeutic potential of human NPCs may differ greatly depending on the method of derivation and culture, we wanted to determine whether NPCs differentiated using conventional methods would be similarly effective in improving clinical outcome under neuroinflammatory demyelinating conditions. For the current study, we utilized hNPCs differentiated from a human induced pluripotent cell line via an embryoid body intermediate stage (EB-NPCs). Intraspinal transplantation of EB-NPCs into mice infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV) resulted in decreased accumulation of CD4+ T cells in the central nervous system that was concomitant with reduced demyelination at the site of injection. Dampened neuroinflammation and remyelination was correlated with a transient increase in CD4+FOXP3+ regulatory T cells (Tregs) concentrated within the peripheral lymphatics. However, compared to our earlier study, pathological improvements were modest and did not result in significant clinical recovery. We conclude that the genetic signature of NPCs is critical to their effectiveness in this model of viral-induced neurologic disease. These comparisons will be useful for understanding what factors are critical for the sustained clinical improvement. PMID:27310015

  17. Remyelination Is Correlated with Regulatory T Cell Induction Following Human Embryoid Body-Derived Neural Precursor Cell Transplantation in a Viral Model of Multiple Sclerosis

    PubMed Central

    Plaisted, Warren C.; Zavala, Angel; Hingco, Edna; Tran, Ha; Coleman, Ronald; Lane, Thomas E.; Loring, Jeanne F.; Walsh, Craig M.

    2016-01-01

    We have recently described sustained clinical recovery associated with dampened neuroinflammation and remyelination following transplantation of neural precursor cells (NPCs) derived from human embryonic stem cells (hESCs) in a viral model of the human demyelinating disease multiple sclerosis. The hNPCs used in that study were derived by a novel direct differentiation method (direct differentiation, DD-NPCs) that resulted in a unique gene expression pattern when compared to hNPCs derived by conventional methods. Since the therapeutic potential of human NPCs may differ greatly depending on the method of derivation and culture, we wanted to determine whether NPCs differentiated using conventional methods would be similarly effective in improving clinical outcome under neuroinflammatory demyelinating conditions. For the current study, we utilized hNPCs differentiated from a human induced pluripotent cell line via an embryoid body intermediate stage (EB-NPCs). Intraspinal transplantation of EB-NPCs into mice infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV) resulted in decreased accumulation of CD4+ T cells in the central nervous system that was concomitant with reduced demyelination at the site of injection. Dampened neuroinflammation and remyelination was correlated with a transient increase in CD4+FOXP3+ regulatory T cells (Tregs) concentrated within the peripheral lymphatics. However, compared to our earlier study, pathological improvements were modest and did not result in significant clinical recovery. We conclude that the genetic signature of NPCs is critical to their effectiveness in this model of viral-induced neurologic disease. These comparisons will be useful for understanding what factors are critical for the sustained clinical improvement. PMID:27310015

  18. A 3-dimensional human embryonic stem cell (hESC)-derived model to detect developmental neurotoxicity of nanoparticles.

    PubMed

    Hoelting, Lisa; Scheinhardt, Benjamin; Bondarenko, Olesja; Schildknecht, Stefan; Kapitza, Marion; Tanavde, Vivek; Tan, Betty; Lee, Qian Yi; Mecking, Stefan; Leist, Marcel; Kadereit, Suzanne

    2013-04-01

    Nanoparticles (NPs) have been shown to accumulate in organs, cross the blood-brain barrier and placenta, and have the potential to elicit developmental neurotoxicity (DNT). Here, we developed a human embryonic stem cell (hESC)-derived 3-dimensional (3-D) in vitro model that allows for testing of potential developmental neurotoxicants. Early central nervous system PAX6(+) precursor cells were generated from hESCs and differentiated further within 3-D structures. The 3-D model was characterized for neural marker expression revealing robust differentiation toward neuronal precursor cells, and gene expression profiling suggested a predominantly forebrain-like development. Altered neural gene expression due to exposure to non-cytotoxic concentrations of the known developmental neurotoxicant, methylmercury, indicated that the 3-D model could detect DNT. To test for specific toxicity of NPs, chemically inert polyethylene NPs (PE-NPs) were chosen. They penetrated deep into the 3-D structures and impacted gene expression at non-cytotoxic concentrations. NOTCH pathway genes such as HES5 and NOTCH1 were reduced in expression, as well as downstream neuronal precursor genes such as NEUROD1 and ASCL1. FOXG1, a patterning marker, was also reduced. As loss of function of these genes results in severe nervous system impairments in mice, our data suggest that the 3-D hESC-derived model could be used to test for Nano-DNT.

  19. 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

  20. Qualitative modeling identifies IL-11 as a novel regulator in maintaining self-renewal in human pluripotent stem cells

    PubMed Central

    Peterson, Hedi; Abu Dawud, Raed; Garg, Abhishek; Wang, Ying; Vilo, Jaak; Xenarios, Ioannis; Adjaye, James

    2013-01-01

    Pluripotency in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is regulated by three transcription factors—OCT3/4, SOX2, and NANOG. To fully exploit the therapeutic potential of these cells it is essential to have a good mechanistic understanding of the maintenance of self-renewal and pluripotency. In this study, we demonstrate a powerful systems biology approach in which we first expand literature-based network encompassing the core regulators of pluripotency by assessing the behavior of genes targeted by perturbation experiments. We focused our attention on highly regulated genes encoding cell surface and secreted proteins as these can be more easily manipulated by the use of inhibitors or recombinant proteins. Qualitative modeling based on combining boolean networks and in silico perturbation experiments were employed to identify novel pluripotency-regulating genes. We validated Interleukin-11 (IL-11) and demonstrate that this cytokine is a novel pluripotency-associated factor capable of supporting self-renewal in the absence of exogenously added bFGF in culture. To date, the various protocols for hESCs maintenance require supplementation with bFGF to activate the Activin/Nodal branch of the TGFβ signaling pathway. Additional evidence supporting our findings is that IL-11 belongs to the same protein family as LIF, which is known to be necessary for maintaining pluripotency in mouse but not in human ESCs. These cytokines operate through the same gp130 receptor which interacts with Janus kinases. Our finding might explain why mESCs are in a more naïve cell state compared to hESCs and how to convert primed hESCs back to the naïve state. Taken together, our integrative modeling approach has identified novel genes as putative candidates to be incorporated into the expansion of the current gene regulatory network responsible for inducing and maintaining pluripotency. PMID:24194720

  1. Humanized Mouse Model of Skin Inflammation Is Characterized by Disturbed Keratinocyte Differentiation and Influx of IL-17A Producing T Cells

    PubMed Central

    de Oliveira, Vivian L.; Keijsers, Romy R. M. C.; van de Kerkhof, Peter C. M.; Seyger, Marieke M. B.; Fasse, Esther; Svensson, Lars; Latta, Markus; Norsgaard, Hanne; Labuda, Tord; Hupkens, Pieter; van Erp, Piet E. J.; Joosten, Irma; Koenen, Hans J. P. M.

    2012-01-01

    Humanized mouse models offer a challenging possibility to study human cell function in vivo. In the huPBL-SCID-huSkin allograft model human skin is transplanted onto immunodeficient mice and allowed to heal. Thereafter allogeneic human peripheral blood mononuclear cells are infused intra peritoneally to induce T cell mediated inflammation and microvessel destruction of the human skin. This model has great potential for in vivo study of human immune cells in (skin) inflammatory processes and for preclinical screening of systemically administered immunomodulating agents. Here we studied the inflammatory skin response of human keratinocytes and human T cells and the concomitant systemic human T cell response. As new findings in the inflamed human skin of the huPBL-SCID-huSkin model we here identified: 1. Parameters of dermal pathology that enable precise quantification of the local skin inflammatory response exemplified by acanthosis, increased expression of human β-defensin-2, Elafin, K16, Ki67 and reduced expression of K10 by microscopy and immunohistochemistry. 2. Induction of human cytokines and chemokines using quantitative real-time PCR. 3. Influx of inflammation associated IL-17A-producing human CD4+ and CD8+ T cells as well as immunoregulatory CD4+Foxp3+ cells using immunohistochemistry and -fluorescence, suggesting that active immune regulation is taking place locally in the inflamed skin. 4. Systemic responses that revealed activated and proliferating human CD4+ and CD8+ T cells that acquired homing marker expression of CD62L and CLA. Finally, we demonstrated the value of the newly identified parameters by showing significant changes upon systemic treatment with the T cell inhibitory agents cyclosporine-A and rapamycin. In summary, here we equipped the huPBL-SCID-huSkin humanized mouse model with relevant tools not only to quantify the inflammatory dermal response, but also to monitor the peripheral immune status. This combined approach will gain our

  2. Long-Term Expression of Human Adenosine Deaminase in Vascular Smooth Muscle Cells of Rats: A Model for Gene Therapy

    NASA Astrophysics Data System (ADS)

    Lynch, Carmel M.; Clowes, Monika M.; Osborne, William R. A.; Clowes, Alexander W.; Dusty Miller, A.

    1992-02-01

    Gene transfer into vascular smooth muscle cells in animals was examined by using recombinant retroviral vectors containing an Escherichia coli β-galactosidase gene or a human adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) gene. Direct gene transfer by infusion of virus into rat carotid arteries was not observed. However, gene transfer by infection of smooth muscle cells in culture and seeding of the transduced cells onto arteries that had been denuded of endothelial cells was successful. Potentially therapeutic levels of human adenosine deaminase activity were detected over 6 months of observation, indicating the utility of vascular smooth muscle cells for gene therapy in humans.

  3. Intracoronary Delivery of Human Mesenchymal/Stromal Stem Cells: Insights from Coronary Microcirculation Invasive Assessment in a Swine Model

    PubMed Central

    Fiarresga, António; Mata, Márcia F.; Cavaco-Gonçalves, Sandra; Selas, Mafalda; Simões, Irina N.; Oliveira, Eunice; Carrapiço, Belmira; Cardim, Nuno; Cabral, Joaquim M. S.; Ferreira, Rui Cruz; da Silva, Cláudia L.

    2015-01-01

    Background Mesenchymal stem/stromal cells have unique properties favorable to their use in clinical practice and have been studied for cardiac repair. However, these cells are larger than coronary microvessels and there is controversy about the risk of embolization and microinfarctions, which could jeopardize the safety and efficacy of intracoronary route for their delivery. The index of microcirculatory resistance (IMR) is an invasive method for quantitatively assessing the coronary microcirculation status. Objectives To examine heart microcirculation after intracoronary injection of mesenchymal stem/stromal cells with the index of microcirculatory resistance. Methods Healthy swine were randomized to receive by intracoronary route either 30x106 MSC or the same solution with no cells (1% human albumin/PBS) (placebo). Blinded operators took coronary pressure and flow measurements, prior to intracoronary infusion and at 5 and 30 minutes post-delivery. Coronary flow reserve (CFR) and the IMR were compared between groups. Results CFR and IMR were done with a variance within the 3 transit time measurements of 6% at rest and 11% at maximal hyperemia. After intracoronary infusion there were no significant differences in CFR. The IMR was significantly higher in MSC-injected animals (at 30 minutes, 14.2U vs. 8.8U, p = 0.02) and intragroup analysis showed a significant increase of 112% from baseline to 30 minutes after cell infusion, although no electrocardiographic changes or clinical deterioration were noted. Conclusion Overall, this study provides definitive evidence of microcirculatory disruption upon intracoronary administration of mesenchymal stem/stromal cells, in a large animal model closely resembling human cardiac physiology, function and anatomy. PMID:26479722

  4. A Comparative Study of Early Afterdepolarization-Mediated Fibrillation in Two Mathematical Models for Human Ventricular Cells

    PubMed Central

    Zimik, Soling; Vandersickel, Nele; Nayak, Alok Ranjan; Panfilov, Alexander V.; Pandit, Rahul

    2015-01-01

    Early afterdepolarizations (EADs), which are abnormal oscillations of the membrane potential at the plateau phase of an action potential, are implicated in the development of cardiac arrhythmias like Torsade de Pointes. We carry out extensive numerical simulations of the TP06 and ORd mathematical models for human ventricular cells with EADs. We investigate the different regimes in both these models, namely, the parameter regimes where they exhibit (1) a normal action potential (AP) with no EADs, (2) an AP with EADs, and (3) an AP with EADs that does not go back to the resting potential. We also study the dependence of EADs on the rate of at which we pace a cell, with the specific goal of elucidating EADs that are induced by slow or fast rate pacing. In our simulations in two- and three-dimensional domains, in the presence of EADs, we find the following wave types: (A) waves driven by the fast sodium current and the L-type calcium current (Na-Ca-mediated waves); (B) waves driven only by the L-type calcium current (Ca-mediated waves); (C) phase waves, which are pseudo-travelling waves. Furthermore, we compare the wave patterns of the various wave-types (Na-Ca-mediated, Ca-mediated, and phase waves) in both these models. We find that the two models produce qualitatively similar results in terms of exhibiting Na-Ca-mediated wave patterns that are more chaotic than those for the Ca-mediated and phase waves. However, there are quantitative differences in the wave patterns of each wave type. The Na-Ca-mediated waves in the ORd model show short-lived spirals but the TP06 model does not. The TP06 model supports more Ca-mediated spirals than those in the ORd model, and the TP06 model exhibits more phase-wave patterns than does the ORd model. PMID:26125185

  5. A Comparative Study of Early Afterdepolarization-Mediated Fibrillation in Two Mathematical Models for Human Ventricular Cells.

    PubMed

    Zimik, Soling; Vandersickel, Nele; Nayak, Alok Ranjan; Panfilov, Alexander V; Pandit, Rahul

    2015-01-01

    Early afterdepolarizations (EADs), which are abnormal oscillations of the membrane potential at the plateau phase of an action potential, are implicated in the development of cardiac arrhythmias like Torsade de Pointes. We carry out extensive numerical simulations of the TP06 and ORd mathematical models for human ventricular cells with EADs. We investigate the different regimes in both these models, namely, the parameter regimes where they exhibit (1) a normal action potential (AP) with no EADs, (2) an AP with EADs, and (3) an AP with EADs that does not go back to the resting potential. We also study the dependence of EADs on the rate of at which we pace a cell, with the specific goal of elucidating EADs that are induced by slow or fast rate pacing. In our simulations in two- and three-dimensional domains, in the presence of EADs, we find the following wave types: (A) waves driven by the fast sodium current and the L-type calcium current (Na-Ca-mediated waves); (B) waves driven only by the L-type calcium current (Ca-mediated waves); (C) phase waves, which are pseudo-travelling waves. Furthermore, we compare the wave patterns of the various wave-types (Na-Ca-mediated, Ca-mediated, and phase waves) in both these models. We find that the two models produce qualitatively similar results in terms of exhibiting Na-Ca-mediated wave patterns that are more chaotic than those for the Ca-mediated and phase waves. However, there are quantitative differences in the wave patterns of each wave type. The Na-Ca-mediated waves in the ORd model show short-lived spirals but the TP06 model does not. The TP06 model supports more Ca-mediated spirals than those in the ORd model, and the TP06 model exhibits more phase-wave patterns than does the ORd model. PMID:26125185

  6. A Human Espophageal Epithelial Cell Model for Study of Radiation Induced Cancer and DNA Damage Repair

    NASA Technical Reports Server (NTRS)

    Huff, Janice L.; Patel, Zarana S.; Hada, Megumi; Cucinotta, Francis A.

    2008-01-01

    For cancer risk assessment in astronauts and for countermeasure development, it is essential to understand the molecular mechanisms of radiation carcinogenesis and how these mechanisms are influenced by exposure to the types of radiation found in space. We are developing an in vitro model system for the study of radiation-induced initiation and progression of esophageal carcinoma, a type of cancer found to have a significant enhancement in incidence in the survivors of the atomic bomb detonations in Japan. Here we present the results of our preliminary characterization of both normal and hTERT immortalized esophageal epithelial cells grown in 2-dimensional culture. We analyzed DNA repair capacity by measuring the kinetics of formation and loss of - H2AX foci following radiation exposure. Additionally, we analyzed induction of chromosomal aberrations using 3-color fluorescence in situ hybridization (FISH). Data were generated using both low LET (gamma rays) and high LET ions (1000 MeV/nucleon iron).

  7. Infusion of Human Bone Marrow-Derived Mesenchymal Stem Cells Alleviates Autoimmune Nephritis in a Lupus Model by Suppressing Follicular Helper T-Cell Development.

    PubMed

    Jang, Eunkyeong; Jeong, Mini; Kim, Sukhyung; Jang, Kiseok; Kang, Bo-Kyeong; Lee, Dong Yun; Bae, Sang-Cheol; Kim, Kyung Suk; Youn, Jeehee

    2016-01-01

    Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies to components of the cell nucleus. These autoantibodies are predominantly produced with the help of follicular helper T (Tfh) cells and form immune complexes that trigger widespread inflammatory damage, including nephritis. In recent studies, mesenchymal stem cells (MSCs) elicited diverse, even opposing, effects in experimental and clinical SLE. Here we investigated the effect of human bone marrow-derived MSCs (hBM-MSCs) in a murine model of SLE, the F1 hybrid between New Zealand Black and New Zealand White strains (NZB/W). We found that infusion of female NZB/W mice with hBM-MSCs attenuated glomerulonephritis; it also decreased levels of autoantibodies and the incidence of proteinuria and improved survival. These effects coincided with a decrease in Tfh cells and downstream components. Infiltration of long-lived plasma cells into the inflamed kidney was also reduced in the hBM-MSC-treated mice. Importantly, hBM-MSCs directly suppressed the in vitro differentiation of naive CD4(+) T cells toward Tfh cells in a contact-dependent manner. These results suggest that MSCs attenuate lupus nephritis by suppressing the development of Tfh cells and the subsequent activation of humoral immune components. They thus reveal a novel mechanism by which MSCs regulate humoral autoimmune diseases such as SLE.

  8. LDLR Expression and Localization Are Altered in Mouse and Human Cell Culture Models of Alzheimer's Disease

    PubMed Central

    Abisambra, Jose F.; Fiorelli, Tina; Padmanabhan, Jaya; Neame, Peter; Wefes, Inge; Potter, Huntington

    2010-01-01

    Background Alzheimer's disease (AD) is a chronic neurodegenerative disorder and the most common form of dementia. The major molecular risk factor for late-onset AD is expression of the ε-4 allele of apolipoprotein E (apoE), the major cholesterol transporter in the brain. The low-density lipoprotein receptor (LDLR) has the highest affinity for apoE and plays an important role in brain cholesterol metabolism. Methodology/Principal Findings Using RT-PCR and western blotting techniques we found that over-expression of APP caused increases in both LDLR mRNA and protein levels in APP transfected H4 neuroglioma cells compared to H4 controls. Furthermore, immunohistochemical experiments showed aberrant localization of LDLR in H4-APP neuroglioma cells, Aβ-treated primary neurons, and in the PSAPP transgenic mouse model of AD. Finally, immunofluorescent staining of LDLR and of γ- and α-tubulin showed a change in LDLR localization preferentially away from the plasma membrane that was paralleled by and likely the result of a disruption of the microtubule-organizing center and associated microtubule network. Conclusions/Significance These data suggest that increased APP expression and Aβ exposure alters microtubule function, leading to reduced transport of LDLR to the plasma membrane. Consequent deleterious effects on apoE uptake and function will have implications for AD pathogenesis and/or progression. PMID:20049331

  9. A human esophageal epithelial cell model for study of radiation induced cancer and DNA repair

    NASA Astrophysics Data System (ADS)

    Huff, Janice; Patel, Zarana; Hada, Megumi; Cucinotta, Francis A.

    For cancer risk assessment in astronauts and for countermeasure development, it is essential to understand the molecular mechanisms of radiation carcinogenesis and how these mechanisms are influenced by exposure to the types of radiation found in space. We are developing an in vitro model system for the study of radiation-induced initiation and progression of esophageal carcinoma. Development of squamous cell carcinoma of the esophagus is associated with radiation exposure, as revealed by the significant enhanced in incidence rates for this type of cancer in the survivors of the atomic bomb detonations in Japan. It is also associated with poor nutritional status and micronutrient deficiencies, which are also important issues for long duration spaceflight. The possible synergies between nutritional issues and radiation exposure are unknown. Here we present the results of preliminary characterization of both normal and hTERT-immortalized esophageal epithelial cells grown in 2-dimensional culture. We analyzed DNA repair capacity by measuring the kinetics of formation and loss of gamma-H2AX foci following radiation exposure. Additionally, we analyzed induction of chromosomal aberrations using 3-color fluorescence in situ hybridization (FISH). Data were generated using both low LET (gamma rays) and high LET ions (1000 MeV/nucleon iron.

  10. Measuring and modeling of binary mixture effects of pharmaceuticals and nickel on cell viability/cytotoxicity in the human hepatoma derived cell line HepG2

    SciTech Connect

    Rudzok, S.; Schlink, U.; Herbarth, O.; Bauer, M.

    2010-05-01

    The interaction of drugs and non-therapeutic xenobiotics constitutes a central role in human health risk assessment. Still, available data are rare. Two different models have been established to predict mixture toxicity from single dose data, namely, the concentration addition (CA) and independent action (IA) model. However, chemicals can also act synergistic or antagonistic or in dose level deviation, or in a dose ratio dependent deviation. In the present study we used the MIXTOX model (EU project ENV4-CT97-0507), which incorporates these algorithms, to assess effects of the binary mixtures in the human hepatoma cell line HepG2. These cells possess a liver-like enzyme pattern and a variety of xenobiotic-metabolizing enzymes (phases I and II). We tested binary mixtures of the metal nickel, the anti-inflammatory drug diclofenac, and the antibiotic agent irgasan and compared the experimental data to the mathematical models. Cell viability was determined by three different methods the MTT-, AlamarBlue (registered) and NRU assay. The compounds were tested separately and in combinations. We could show that the metal nickel is the dominant component in the mixture, affecting an antagonism at low-dose levels and a synergism at high-dose levels in combination with diclofenac or irgasan, when using the NRU and the AlamarBlue assay. The dose-response surface of irgasan and diclofenac indicated a concentration addition. The experimental data could be described by the algorithms with a regression of up to 90%, revealing the HepG2 cell line and the MIXTOX model as valuable tool for risk assessment of binary mixtures for cytotoxic endpoints. However the model failed to predict a specific mode of action, the CYP1A1 enzyme activity.

  11. Human induced pluripotent stem cells can reach complete terminal maturation: in vivo and in vitro evidence in the erythropoietic differentiation model

    PubMed Central

    Kobari, Ladan; Yates, Frank; Oudrhiri, Noufissa; Francina, Alain; Kiger, Laurent; Mazurier, Christelle; Rouzbeh, Shaghayegh; El-Nemer, Wassim; Hebert, Nicolas; Giarratana, Marie-Catherine; François, Sabine; Chapel, Alain; Lapillonne, Hélène; Luton, Dominique; Bennaceur-Griscelli, Annelise; Douay, Luc

    2012-01-01

    Background Human induced pluripotent stem cells offer perspectives for cell therapy and research models for diseases. We applied this approach to the normal and pathological erythroid differentiation model by establishing induced pluripotent stem cells from normal and homozygous sickle cell disease donors. Design and Methods We addressed the question as to whether these cells can reach complete erythroid terminal maturation notably with a complete switch from fetal to adult hemoglobin. Sickle cell disease induced pluripotent stem cells were differentiated in vitro into red blood cells and characterized for their terminal maturation in terms of hemoglobin content, oxygen transport capacity, deformability, sickling and adherence. Nucleated erythroblast populations generated from normal and pathological induced pluripotent stem cells were then injected into non-obese diabetic severe combined immunodeficiency mice to follow the in vivo hemoglobin maturation. Results We observed that in vitro erythroid differentiation results in predominance of fetal hemoglobin which rescues the functionality of red blood cells in the pathological model of sickle cell disease. We observed, in vivo, the switch from fetal to adult hemoglobin after infusion of nucleated erythroid precursors derived from either normal or pathological induced pluripotent stem cells into mice. Conclusions These results demonstrate that human induced pluripotent stem cells: i) can achieve complete terminal erythroid maturation, in vitro in terms of nucleus expulsion and in vivo in terms of hemoglobin maturation; and ii) open the way to generation of functionally corrected red blood cells from sickle cell disease induced pluripotent stem cells, without any genetic modification or drug treatment. PMID:22733021

  12. A human dendritic cell-based in vitro model to assess Mycobacterium tuberculosis SO2 vaccine immunogenicity.

    PubMed

    Etna, Marilena P; Giacomini, Elena; Severa, Martina; Pardini, Manuela; Aguilo, Nacho; Martin, Carlos; Coccia, Eliana M

    2014-01-01

    Among the tuberculosis (TB) vaccine candidates, SO2 is the prototype of the first live-attenuated vaccine that recently entered into clinical trials. To investigate the capacity of SO2 to stimulate an appropriate immune response in vitro within a human immunological context, a comparative analysis of the effects promoted by SO2, the current Bacille Calmette-Guerin (BCG) vaccine and Mycobacterium tuberculosis (Mtb) was conducted in human primary dendritic cells (DC), which are critical modulators of vaccine-induced immunity. In particular, we found that SO2 promotes the expression of maturation markers similarly to BCG but at a lower extent than Mtb. Moreover, SO2-infected DC released higher levels of interleukin (IL)-23 than BCG-infected cells, which account for the expansion of interferon (IFN)-γ-producing T cells in an IL-12-independent manner. In the autologous mixed leukocyte reaction setting, the expansion of IL-17-producing T cells was also observed in response to SO2 infection. Interestingly, apoptosis and autophagic flux, events required for the antigen presentation within MHC class II complex, were not affected in DC infected with SO2, conversely to what observed upon Mtb stimulation. Collectively, our results indicate that SO2 represents a promising TB vaccine candidate, which displays an attenuated phenotype and promotes in DC a stronger capacity to stimulate the Th response than BCG vaccine. Interestingly, the data obtained by using the human DC-based experimental setting mirrored the results derived from studies in animal models, suggesting that this system could be used for an efficient and rapid down-selection of new TB vaccine candidates, contributing to achieve the "3Rs" objective.

  13. Human erythrocytes and molecular models of cell membranes are affected in vitro by Balbisia peduncularis (Amancay) extracts.

    PubMed

    Suwalsky, Mario; Oyarce, Karina; Avello, Marcia; Villena, F; Sotomayor, Carlos P

    2009-05-15

    Balbisia peduncularis, also known as "Amancay", is a plant of the Ledocarpaceae family that can be found in the Atacama Desert in northern Chile. Infusions of the plant have long being used in traditional herbal medicine. Its chemical composition indicates the presence of flavonoids, which have antioxidant properties. Aqueous extracts from its stems were prepared to induce their interaction with human erythrocytes and their membrane models in order to elucidate whether this rare and unstudied plant produced perturbations to cell membranes. Scanning electron microscopy (SEM) of intact human red blood cells showed that the extract changed the normal erythrocytes morphology as a function of its concentration, first inducing echinocytes, and then stomatocytes and spherocytes. According to the bilayer couple hypothesis, the shape changes indicated that the flavonoids were first located in the outer monolayer of the erythrocyte membrane, and at the highest assayed concentration in both monolayers. The results obtained by fluorescence spectroscopy measurements of isolated unsealed human erythrocytes (IUM), of unilamellar vesicles (LUV) of dimyristoylphosphatidylcholine (DMPC), and by X-ray diffraction of DMPC and dimyristoylphosphatidylethanolamine (DMPE) multilayers, confirmed this conclusion. In fact, they showed that the plant aqueous extract molecules were located in both the hydrophilic polar head and in the hydrophobic acyl chain regions of the lipid bilayers. As a consequence, perturbations of the phospholipid bilayer packing arrangement were produced. PMID:19146840

  14. Wound Dressing Model of Human Umbilical Cord Mesenchymal Stem Cells-Alginates Complex Promotes Skin Wound Healing by Paracrine Signaling

    PubMed Central

    Yang, Huachao; Tang, Zhenrui; Long, Gang; Huang, Wen

    2016-01-01

    Purpose. To probe growth characteristics of human umbilical cord mesenchymal stem cells (hUCMSCs) cultured with alginate gel scaffolds, and to explore feasibility of wound dressing model of hUCMSCs-alginates compound. Methods. hUCMSCs were isolated, cultured, and identified in vitro. Then cells were cultivated in 100 mM calcium alginate gel, and the capacity of proliferation and migration and the expression of vascular endothelial growth factors (VEGF) were investigated regularly. Wound dressing model of hUCMSCs-alginate gel mix was transplanted into Balb/c mice skin defects. Wound healing rate and immunohistochemistry were examined. Results. hUCMSCs grew well but with little migration ability in the alginate gel. Compared with control group, a significantly larger cell number and more VEGF expression were shown in the gel group after culturing for 3–6 days (P < 0.05). In addition, a faster skin wound healing rate with more neovascularization was observed in the hUCMSCs-alginate gel group than in control groups at 15th day after surgery (P < 0.05). Conclusion. hUCMSCs can proliferate well and express massive VEGF in calcium alginate gel porous scaffolds. Wound dressing model of hUCMSCs-alginate gel mix can promote wound healing through paracrine signaling. PMID:26880953

  15. Role of CEACAM1, ECM, and mesenchymal stem cells in an orthotopic model of human breast cancer.

    PubMed

    Samineni, Sridhar; Glackin, Carlotta; Shively, John E

    2011-01-01

    Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is a morphogen in an in vitro model for lumen formation and plays a similar role in breast epithelial cells implanted in humanized mammary fat pads in NOD-SCID mice. Although extra cellular matrix alone is sufficient to stimulate lumen formation in CEACAM1 transfected MCF-7 cells grown in 3D culture, there is an additional requirement for stromal or mesenchymal cells (MSCs) for these cells to form xenografts with glandular structures in an orthotopic site. We demonstrate that optimal in vitro conditions include both Matrigel and MSCs and that the inclusion of collagen I inhibits xenograft differentiation. Additionally, there is no need to remove the nascent murine mammary gland. The previously observed difference in gland development between the long and short cytoplasmic domain isoforms of CEACAM1 is no longer observed in pregnant NOD/SCID mice suggesting that stimulation of the mammary fat pad by pregnancy critically affects xenograft differentiation.

  16. Antiepileptic and neuroprotective effects of human umbilical cord blood mononuclear cells in a pilocarpine-induced epilepsy model.

    PubMed

    Costa-Ferro, Zaquer Suzana Munhoz; de Borba Cunha, Fernanda; de Freitas Souza, Bruno Solano; Leal, Marcos Maurício Tosta; da Silva, Adelson Alves; de Bellis Kühn, Telma Ingrid Borges; Forte, Andresa; Sekiya, Eliseo Joji; Soares, Milena Botelho Pereira; Dos Santos, Ricardo Ribeiro

    2014-03-01

    Status epilepticus (SE) is a condition of persistent seizure that leads to brain damage and, frequently, to the establishment of chronic epilepsy. Cord blood is an important source of adult stem cells for the treatment of neurological disorders. The present study aimed to evaluate the effects of human umbilical cord blood mononuclear cells (HUCBC) transplanted into rats after induction of SE by the administration of lithium and pilocarpine chloride. Transplantation of HUCBC into epileptic rats protected against neuronal loss in the hippocampal subfields CA1, CA3 and in the hilus of the dentate gyrus, up to 300 days after SE induction. Moreover, transplanted rats had reduced frequency and duration of spontaneous recurrent seizures (SRS) 15, 120 and 300 days after the SE. Our study shows that HUCBC provide prominent antiepileptic and neuroprotective effects in the experimental model of epilepsy and reinforces that early interventions can protect the brain against the establishment of epilepsy.

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

    PubMed

    Zeng, Huawei; Jackson, Matthew I; Cheng, Wen-Hsing; Combs, Gerald F

    2011-11-01

    Determining the effect of selenium (Se) chemical form on uptake, transport, and glutathione peroxidase activity in human intestinal cells is critical to assess Se bioavailability at nutritional doses. In this study, we found that two sources of L-selenomethionine (SeMet) and Se-enriched yeast each increased intracellular Se content more effectively than selenite or methylselenocysteine (SeMSC) in the human intestinal Caco-2 cell model. Interestingly, SeMSC, SeMet, and digested Se-enriched yeast were transported at comparable efficacy from the apical to basolateral sides, each being about 3-fold that of selenite. In addition, these forms of Se, whether before or after traversing from apical side to basolateral side, did not change the potential to support glutathione peroxidase (GPx) activity. Although selenoprotein P has been postulated to be a key Se transport protein, its intracellular expression did not differ when selenite, SeMSC, SeMet, or digested Se-enriched yeast was added to serum-contained media. Taken together, our data show, for the first time, that the chemical form of Se at nutritional doses can affect the absorptive (apical to basolateral side) efficacy and retention of Se by intestinal cells; but that, these effects are not directly correlated to the potential to support GPx activity.

  18. A novel human model of the neurodegenerative disease GM1 gangliosidosis using induced pluripotent stem cells demonstrates inflammasome activation.

    PubMed

    Son, Mi-Young; Kwak, Jae Eun; Seol, Binna; Lee, Da Yong; Jeon, Hyejin; Cho, Yee Sook

    2015-09-01

    GM1 gangliosidosis (GM1) is an inherited neurodegenerative disorder caused by mutations in the lysosomal β-galactosidase (β-gal) gene. Insufficient β-gal activity leads to abnormal accumulation of GM1 gangliosides in tissues, particularly in the central nervous system, resulting in progressive neurodegeneration. Here, we report an in vitro human GM1 model, based on induced pluripotent stem cell (iPSC) technology. Neural progenitor cells differentiated from GM1 patient-derived iPSCs (GM1-NPCs) recapitulated the biochemical and molecular phenotypes of GM1, including defective β-gal activity and increased lysosomes. Importantly, the characterization of GM1-NPCs established that GM1 is significantly associated with the activation of inflammasomes, which play a critical role in the pathogenesis of various neurodegenerative diseases. Specific inflammasome inhibitors potently alleviated the disease-related phenotypes of GM1-NPCs in vitro and in vivo. Our data demonstrate that GM1-NPCs are a valuable in vitro human GM1 model and suggest that inflammasome activation is a novel target pathway for GM1 drug development.

  19. The Food-Associated Ribotoxin Deoxynivalenol Modulates Inducible NO Synthase in Human Intestinal Cell Model.

    PubMed

    Graziani, Fabien; Pujol, Ange; Nicoletti, Cendrine; Pinton, Philippe; Armand, Loriane; Di Pasquale, Eric; Oswald, Isabelle P; Perrier, Josette; Maresca, Marc

    2015-06-01

    The intestinal epithelium possesses active immune functions including the production of proinflammatory cytokines and antimicrobial molecules such as nitric oxide (NO). As observed with immune cells, the production of NO by the intestinal epithelium is mainly due to the expression of the inducible NO synthase (iNOS or NOS2). Epithelial immune functions could be affected by many factors including pathogenic microorganisms and food-associated toxins (bacterial and fungal). Among the various mycotoxins, deoxynivalenol (DON) is known to alter the systemic and intestinal immunity. However, little is known about the effect of DON on the production of NO by the intestinal epithelium. We studied the impact of DON on the intestinal expression of iNOS using the Caco-2 cell model. In line with its proinflammatory activity, we observed that DON dose-dependently up-regulates the expression of iNOS mRNA. Surprisingly, DON failed to increase the expression of iNOS protein. When testing the effects of DON on cytokine-mediated induction of iNOS, we found that very low concentrations of DON (ie, 1 µM) decrease the amount of iNOS protein but not of iNOS mRNA. We demonstrated that DON's effect on iNOS protein relies on its ability to activate signal pathways and to increase iNOS ubiquitinylation and degradation through the proteasome pathway. Taken together, our results demonstrate that although DON causes intestinal inflammation, it suppresses the ability of the gut epithelium to express iNOS and to produce NO, potentially explaining the increased susceptibility of animals to intestinal infection following exposure to low doses of DON.

  20. Transplantation of human embryonic stem cell-derived retinal tissue in two primate models of retinal degeneration

    PubMed Central

    Shirai, Hiroshi; Mandai, Michiko; Matsushita, Keizo; Kuwahara, Atsushi; Yonemura, Shigenobu; Nakano, Tokushige; Assawachananont, Juthaporn; Kimura, Toru; Saito, Koichi; Terasaki, Hiroko; Eiraku, Mototsugu; Sasai, Yoshiki; Takahashi, Masayo

    2016-01-01

    Retinal transplantation therapy for retinitis pigmentosa is increasingly of interest due to accumulating evidence of transplantation efficacy from animal studies and development of techniques for the differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells into retinal tissues or cells. In this study, we aimed to assess the potential clinical utility of hESC-derived retinal tissues (hESC-retina) using newly developed primate models of retinal degeneration to obtain preparatory information regarding the potential clinical utility of these hESC-retinas in transplantation therapy. hESC-retinas were first transplanted subretinally into nude rats with or without retinal degeneration to confirm their competency as a graft to mature to form highly specified outer segment structure and to integrate after transplantation. Two focal selective photoreceptor degeneration models were then developed in monkeys by subretinal injection of cobalt chloride or 577-nm optically pumped semiconductor laser photocoagulation. The utility of the developed models and a practicality of visual acuity test developed for monkeys were evaluated. Finally, feasibility of hESC-retina transplantation was assessed in the developed monkey models under practical surgical procedure and postoperational examinations. Grafted hESC-retina was observed differentiating into a range of retinal cell types, including rod and cone photoreceptors that developed structured outer nuclear layers after transplantation. Further, immunohistochemical analyses suggested the formation of host–graft synaptic connections. The findings of this study demonstrate the clinical feasibility of hESC-retina transplantation and provide the practical tools for the optimization of transplantation strategies for future clinical applications. PMID:26699487

  1. Improved Neurological Outcome by Intramuscular Injection of Human Amniotic Fluid Derived Stem Cells in a Muscle Denervation Model

    PubMed Central

    Su, Hong-Lin; Sheu, Meei-Ling; Lu, Zong-Han; Chiang, Chien-Yi; Yang, Dar-Yu; Sheehan, Jason; Pan, Hung-Chuan

    2015-01-01

    Purpose The skeletal muscle develops various degrees of atrophy and metabolic dysfunction following nerve injury. Neurotrophic factors are essential for muscle regeneration. Human amniotic fluid derived stem cells (AFS) have the potential to secrete various neurotrophic factors necessary for nerve regeneration. In the present study, we assess the outcome of neurological function by intramuscular injection of AFS in a muscle denervation and nerve anastomosis model. Materials and Methods Seventy two Sprague-Dawley rats weighing 200–250 gm were enrolled in this study. Muscle denervation model was conducted by transverse resection of a sciatic nerve with the proximal end sutured into the gluteal muscle. The nerve anastomosis model was performed by transverse resection of the sciatic nerve followed by four stitches reconnection. These animals were allocated to three groups: control, electrical muscle stimulation, and AFS groups. Results NT-3 (Neurotrophin 3), BDNF (Brain derived neurotrophic factor), CNTF (Ciliary neurotrophic factor), and GDNF (Glia cell line derived neurotrophic factor) were highly expressed in AFS cells and supernatant of culture medium. Intra-muscular injection of AFS exerted significant expression of several neurotrophic factors over the distal end of nerve and denervated muscle. AFS caused high expression of Bcl-2 in denervated muscle with a reciprocal decrease of Bad and Bax. AFS preserved the muscle morphology with high expression of desmin and acetylcholine receptors. Up to two months, AFS produced significant improvement in electrophysiological study and neurological functions such as SFI (sciatic nerve function index) and Catwalk gait analysis. There was also significant preservation of the number of anterior horn cells and increased nerve myelination as well as muscle morphology. Conclusion Intramuscular injection of AFS can protect muscle apoptosis and likely does so through the secretion of various neurotrophic factors. This protection

  2. Transplantation of GABAergic cells derived from bioreactor-expanded human neural precursor cells restores motor and cognitive behavioral deficits in a rodent model of Huntington's disease.

    PubMed

    McLeod, Marcus C; Kobayashi, Nao R; Sen, Arindom; Baghbaderani, Behnam A; Sadi, Damaso; Ulalia, Ruperto; Behie, Leo A; Mendez, Ivar

    2013-01-01

    Huntington's disease (HD) is a neurodegenerative disorder that is characterized by progressive dementia, choreiform involuntary movements, and emotional deterioration. Neuropathological features include the progressive degeneration of striatal γ-aminobutyric acid (GABA) neurons. New therapeutic approaches, such as the transplantation of human neural precursor cells (hNPCs) to replace damaged or degenerated cells, are currently being investigated. The aim of this study was to investigate the potential for utilizing telencephalic hNPCs expanded in suspension bioreactors for cell restorative therapy in a rodent model of HD. hNPCs were expanded in a hydrodynamically controlled and homogeneous environment under serum-free conditions. In vitro analysis revealed that the bioreactor-expanded telencephalic (BET)-hNPCs could be differentiated into a highly enriched population of GABAergic neurons. Behavioral assessments of unilateral striatal quinolinic acid-lesioned rodents revealed a significant improvement in motor and memory deficits following transplantation with GABAergic cells differentiated from BET-hNPCs. Immunohistochemical analysis revealed that transplanted BET-hNPCs retained a GABAergic neuronal phenotype without aberrant transdifferentiation or tumor formation, indicating that BET-hNPCs are a safe source of cells for transplantation. This preclinical study has important implications as the transplantation of GABAergic cells derived from predifferentiated BET-hNPCs may be a safe and feasible cell replacement strategy to promote behavioral recovery in HD.

  3. Therapeutic effect of transplanted human Wharton's jelly stem cell-derived oligodendrocyte progenitor cells (hWJ-MSC-derived OPCs) in an animal model of multiple sclerosis.

    PubMed

    Mikaeili Agah, Elmira; Parivar, Kazem; Joghataei, Mohammad Taghi

    2014-04-01

    Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS). A potential new therapeutic approach for MS is cell transplantation which may promote remyelination. We transplanted human Wharton's jelly stem cell-derived oligodendrocyte progenitor cells (hWJ-MSC-derived OPCs) into the brain ventricles of mice induced with experimental autoimmune encephalomyelitis (EAE), the animal model of MS. We studied the effect of the transplanted OPCs on the functional and pathological manifestations of the disease. Transplanted hWJ-MSC-derived OPCs significantly reduced the clinical signs of EAE. Histological examinations showed that remyelination was significantly increased after transplantation. These results suggest that hWJ-MSC-derived OPCs promote the regeneration of myelin sheaths in the brain.

  4. Establishment and Characterization of a Human Small Cell Osteosarcoma Cancer Stem Cell Line: A New Possible In Vitro Model for Discovering Small Cell Osteosarcoma Biology

    PubMed Central

    Zonefrati, Roberto; Mavilia, Carmelo; Franchi, Alessandro; Capanna, Rodolfo

    2016-01-01

    Osteosarcoma (OSA) is the most common primary malignant bone tumor, usually arising in the long bones of children and young adults. There are different subtypes of OSA, among which we find the conventional OS (also called medullary or central osteosarcoma) which has a high grade of malignancy and an incidence of 80%. There are different subtypes of high grade OS like chondroblastic, fibroblastic, osteoblastic, telangiectatic, and the small cell osteosarcoma (SCO). In this study, for the first time, we have isolated, established, and characterized a cell line of cancer stem cells (CSCs) from a human SCO. First of all, we have established a primary finite cell line of SCO, from which we have isolated the CSCs by the sphere formation assay. We have proved their in vitro mesenchymal and embryonic stem phenotype. Additionally, we have showed their neoplastic phenotype, since the original tumor bulk is a high grade osteosarcoma. This research demonstrates the existence of CSCs also in human primary SCO and highlights the establishment of this particular stabilized cancer stem cell line. This will represent a first step into the study of the biology of these cells to discover new molecular targets molecules for new incisive therapeutic strategies against this highly aggressive OSA.

  5. Establishment and Characterization of a Human Small Cell Osteosarcoma Cancer Stem Cell Line: A New Possible In Vitro Model for Discovering Small Cell Osteosarcoma Biology.

    PubMed

    Palmini, Gaia; Zonefrati, Roberto; Romagnoli, Cecilia; Aldinucci, Alessandra; Mavilia, Carmelo; Leoncini, Gigliola; Franchi, Alessandro; Capanna, Rodolfo; Brandi, Maria Luisa

    2016-01-01

    Osteosarcoma (OSA) is the most common primary malignant bone tumor, usually arising in the long bones of children and young adults. There are different subtypes of OSA, among which we find the conventional OS (also called medullary or central osteosarcoma) which has a high grade of malignancy and an incidence of 80%. There are different subtypes of high grade OS like chondroblastic, fibroblastic, osteoblastic, telangiectatic, and the small cell osteosarcoma (SCO). In this study, for the first time, we have isolated, established, and characterized a cell line of cancer stem cells (CSCs) from a human SCO. First of all, we have established a primary finite cell line of SCO, from which we have isolated the CSCs by the sphere formation assay. We have proved their in vitro mesenchymal and embryonic stem phenotype. Additionally, we have showed their neoplastic phenotype, since the original tumor bulk is a high grade osteosarcoma. This research demonstrates the existence of CSCs also in human primary SCO and highlights the establishment of this particular stabilized cancer stem cell line. This will represent a first step into the study of the biology of these cells to discover new molecular targets molecules for new incisive therapeutic strategies against this highly aggressive OSA. PMID:27651797

  6. Establishment and Characterization of a Human Small Cell Osteosarcoma Cancer Stem Cell Line: A New Possible In Vitro Model for Discovering Small Cell Osteosarcoma Biology

    PubMed Central

    Zonefrati, Roberto; Mavilia, Carmelo; Franchi, Alessandro; Capanna, Rodolfo

    2016-01-01

    Osteosarcoma (OSA) is the most common primary malignant bone tumor, usually arising in the long bones of children and young adults. There are different subtypes of OSA, among which we find the conventional OS (also called medullary or central osteosarcoma) which has a high grade of malignancy and an incidence of 80%. There are different subtypes of high grade OS like chondroblastic, fibroblastic, osteoblastic, telangiectatic, and the small cell osteosarcoma (SCO). In this study, for the first time, we have isolated, established, and characterized a cell line of cancer stem cells (CSCs) from a human SCO. First of all, we have established a primary finite cell line of SCO, from which we have isolated the CSCs by the sphere formation assay. We have proved their in vitro mesenchymal and embryonic stem phenotype. Additionally, we have showed their neoplastic phenotype, since the original tumor bulk is a high grade osteosarcoma. This research demonstrates the existence of CSCs also in human primary SCO and highlights the establishment of this particular stabilized cancer stem cell line. This will represent a first step into the study of the biology of these cells to discover new molecular targets molecules for new incisive therapeutic strategies against this highly aggressive OSA. PMID:27651797

  7. 3D In Vitro Model of a Functional Epidermal Permeability Barrier from Human Embryonic Stem Cells and Induced Pluripotent Stem Cells

    PubMed Central

    Petrova, Anastasia; Celli, Anna; Jacquet, Laureen; Dafou, Dimitra; Crumrine, Debra; Hupe, Melanie; Arno, Matthew; Hobbs, Carl; Cvoro, Aleksandra; Karagiannis, Panagiotis; Devito, Liani; Sun, Richard; Adame, Lillian C.; Vaughan, Robert; McGrath, John A.; Mauro, Theodora M.; Ilic, Dusko

    2014-01-01

    Summary Cornification and epidermal barrier defects are associated with a number of clinically diverse skin disorders. However, a suitable in vitro model for studying normal barrier function and barrier defects is still lacking. Here, we demonstrate the generation of human epidermal equivalents (HEEs) from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). HEEs are structurally similar to native epidermis, with a functional permeability barrier. We exposed a pure population of hESC/iPSC-derived keratinocytes, whose transcriptome corresponds to the gene signature of normal primary human keratinocytes (NHKs), to a sequential high-to-low humidity environment in an air/liquid interface culture. The resulting HEEs had all of the cellular strata of the human epidermis, with skin barrier properties similar to those of normal skin. Such HEEs generated from disease-specific iPSCs will be an invaluable tool not only for dissecting molecular mechanisms that lead to epidermal barrier defects but also for drug development and screening. PMID:24936454

  8. A Novel Physiology-Based Mathematical Model to Estimate Red Blood Cell Lifespan in Different Human Age Groups.

    PubMed

    An, Guohua; Widness, John A; Mock, Donald M; Veng-Pedersen, Peter

    2016-09-01

    Direct measurement of red blood cell (RBC) survival in humans has improved from the original accurate but limited differential agglutination technique to the current reliable, safe, and accurate biotin method. Despite this, all of these methods are time consuming and require blood sampling over several months to determine the RBC lifespan. For situations in which RBC survival information must be obtained quickly, these methods are not suitable. With the exception of adults and infants, RBC survival has not been extensively investigated in other age groups. To address this need, we developed a novel, physiology-based mathematical model that quickly estimates RBC lifespan in healthy individuals at any age. The model is based on the assumption that the total number of RBC recirculations during the lifespan of each RBC (denoted by N max) is relatively constant for all age groups. The model was initially validated using the data from our prior infant and adult biotin-labeled red blood cell studies and then extended to the other age groups. The model generated the following estimated RBC lifespans in 2-year-old, 5-year-old, 8-year-old, and 10-year-old children: 62, 74, 82, and 86 days, respectively. We speculate that this model has useful clinical applications. For example, HbA1c testing is not reliable in identifying children with diabetes because HbA1c is directly affected by RBC lifespan. Because our model can estimate RBC lifespan in children at any age, corrections to HbA1c values based on the model-generated RBC lifespan could improve diabetes diagnosis as well as therapy in children.

  9. Lycopene attenuates Aβ1-42 secretion and its toxicity in human cell and Caenorhabditis elegans models of Alzheimer disease.

    PubMed

    Chen, Wei; Mao, Liuqun; Xing, Huanhuan; Xu, Lei; Fu, Xiang; Huang, Liyingzi; Huang, Dongling; Pu, Zhijun; Li, Qinghua

    2015-11-01

    Growing evidence suggests concentration of lycopene was reduced in plasma of patients with Alzheimer disease (AD). Lycopene, a member of the carotenoid family, has been identified as an antioxidant to attenuate oxidative damage and has neuroprotective role in several AD models. However, whether lycopene is involved in the pathogenesis of AD and molecular underpinnings are elusive. In this study, we found that lycopene can significantly delay paralysis in the Aβ1-42-transgenic Caenorhabditis elegans strain GMC101. Lycopene treatment reduced Aβ1-42 secretion in SH-SY5Y cells overexpressing the Swedish mutant form of human β-amyloid precursor protein (APPsw). Next, we found lycopene can down-regulate expression level of β-amyloid precursor protein(APP) in APPsw cells. Moreover, lycopene treatment can not change endogenous reactive oxygen species level and apoptosis in APPsw cells. However, lycopene treatment protected against H2O2-induced oxidative stress and copper-induced damage in APPsw cells. Collectively, our data support that elevated lycopene contributes to the lower pathogenesis of AD. Our findings suggest that increasing lycopene in neurons may be a novel approach to attenuate onset and development of AD.

  10. Human amniotic epithelial cells can differentiate into granulosa cells and restore folliculogenesis in a mouse model of chemotherapy-induced premature ovarian failure

    PubMed Central

    2013-01-01

    Introduction Ovarian dysfunction frequently occurs in female cancer patients after chemotherapy, but human amniotic epithelial cells (hAECs) that can differentiate into cell types that arise from all three germ layers may offer promise for restoration of such dysfunction. Previous studies confirmed that hAECs could differentiate into cells that express germ cell-specific markers, but at this time hAECs have not been shown to restore ovarian function. Methods To model premature ovarian failure, hAECs infected with lenti-virus carrying green fluorescent protein were injected into the tail vein of mice sterilized with cyclophosphamide and busulphan. hAECs migrated to the mouse ovaries and overall ovarian function was measured using immunohistochemical techniques. Results Seven days to two months after hAECs transplantation, ovarian cells were morphologically restored in sterilized mice. Hemotoxylin and eosin staining revealed that restored ovarian cells developed follicles at all stages. No follicles were observed in control mice at the same time period. Immunostaining with anti-human antigen antibodies and pre-transplantation labeling with green fluorescent protein (GFP) revealed that the grafted hAECs survived and migrated to mouse ovary, differentiating into granulosa cells. Furthermore, the ovarian function marker, anti-Müllerian hormone, was evident in treated mouse ovaries after hAEC transplantation. Conclusions Intravenously injected hAECs reached the ovaries of chemotherapy-treated mice and restored folliculogenesis, data which suggest promise for hAECs for promoting reproductive health and improving the quality of life for female cancer survivors. PMID:24406076

  11. Nucleolin antagonist triggers autophagic cell death in human glioblastoma primary cells and decreased in vivo tumor growth in orthotopic brain tumor model.

    PubMed

    Benedetti, Elisabetta; Antonosante, Andrea; d'Angelo, Michele; Cristiano, Loredana; Galzio, Renato; Destouches, Damien; Florio, Tiziana Marilena; Dhez, Anne Chloé; Astarita, Carlo; Cinque, Benedetta; Fidoamore, Alessia; Rosati, Floriana; Cifone, Maria Grazia; Ippoliti, Rodolfo; Giordano, Antonio; Courty, José; Cimini, Annamaria

    2015-12-01

    Nucleolin (NCL) is highly expressed in several types of cancer and represents an interesting therapeutic target. It is expressed at the plasma membrane of tumor cells, a property which is being used as a marker for several human cancer including glioblastoma. In this study we investigated targeting NCL as a new therapeutic strategy for the treatment of this pathology. To explore this possibility, we studied the effect of an antagonist of NCL, the multivalent pseudopeptide N6L using primary culture of human glioblastoma cells. In this system, N6L inhibits cell growth with different sensitivity depending to NCL localization. Cell cycle analysis indicated that N6L-induced growth reduction was due to a block of the G1/S transition with down-regulation of the expression of cyclin D1 and B2. By monitoring autophagy markers such as p62 and LC3II, we demonstrate that autophagy is enhanced after N6L treatment. In addition, N6L-treatment of mice bearing tumor decreased in vivo tumor growth in orthotopic brain tumor model and increase mice survival. The results obtained indicated an anti-proliferative and pro-autophagic effect of N6L and point towards its possible use as adjuvant agent to the standard therapeutic protocols presently utilized for glioblastoma.

  12. Multiple systemic transplantations of human amniotic mesenchymal stem cells exert therapeutic effects in an ALS mouse model.

    PubMed

    Sun, Haitao; Hou, Zongliu; Yang, Huaqiang; Meng, Mingyao; Li, Peng; Zou, Qingjian; Yang, Lujun; Chen, Yuxin; Chai, Huihui; Zhong, Huilin; Yang, Zara Zhuyun; Zhao, Jing; Lai, Liangxue; Jiang, Xiaodan; Xiao, Zhicheng

    2014-09-01

    Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive neurodegenerative disease involving degeneration of motor neurons in the central nervous system. Stem cell treatment is a potential therapy for this fatal disorder. The human amniotic membrane (HAM), an extremely rich and easily accessible tissue, has been proposed as an attractive material in cellular therapy and regenerative medicine because of its advantageous characteristics. In the present study, we evaluate the long-term effects of a cellular treatment by intravenous administration of human amniotic mesenchymal stem cells (hAMSCs) derived from HAM into a hSOD1(G93A) mouse model. The mice received systemic administration of hAMSCs or phosphate-buffered saline (PBS) at the onset, progression and symptomatic stages of the disease. hAMSCs were detected in the spinal cord at the final stage of the disease, in the form of isolates or clusters and were negative for β-tubulin III and GFAP. Compared with the treatment with PBS, multiple hAMSC transplantations significantly retarded disease progression, extended survival, improved motor function, prevented motor neuron loss and decreased neuroinflammation in mice. These findings demonstrate that hAMSC transplantation is a promising cellular treatment for ALS.

  13. An In Vitro Model of Latency and Reactivation of Varicella Zoster Virus in Human Stem Cell-Derived Neurons

    PubMed Central

    Markus, Amos; Lebenthal-Loinger, Ilana; Yang, In Hong; Kinchington, Paul R.; Goldstein, Ronald S.

    2015-01-01

    Varicella zoster virus (VZV) latency in sensory and autonomic neurons has remained enigmatic and difficult to study, and experimental reactivation has not yet been achieved. We have previously shown that human embryonic stem cell (hESC)-derived neurons are permissive to a productive and spreading VZV infection. We now demonstrate that hESC-derived neurons can also host a persistent non-productive infection lasting for weeks which can subsequently be reactivated by multiple experimental stimuli. Quiescent infections were established by exposing neurons to low titer cell-free VZV either by using acyclovir or by infection of axons in compartmented microfluidic chambers without acyclovir. VZV DNA and low levels of viral transcription were detectable by qPCR for up to seven weeks. Quiescently-infected human neuronal cultures were induced to undergo renewed viral gene and protein expression by growth factor removal or by inhibition of PI3-Kinase activity. Strikingly, incubation of cultures induced to reactivate at a lower temperature (34°C) resulted in enhanced VZV reactivation, resulting in spreading, productive infections. Comparison of VZV genome transcription in quiescently-infected to productively-infected neurons using RNASeq revealed preferential transcription from specific genome regions, especially the duplicated regions. These experiments establish a powerful new system for modeling the VZV latent state, and reveal a potential role for temperature in VZV reactivation and disease. PMID:26042814

  14. Divergent immunomodulating effects of probiotics on T cell responses to oral attenuated human rotavirus vaccine and virulent human rotavirus infection in a neonatal gnotobiotic piglet disease model.

    PubMed

    Chattha, Kuldeep S; Vlasova, Anastasia N; Kandasamy, Sukumar; Rajashekara, Gireesh; Saif, Linda J

    2013-09-01

    Rotaviruses (RVs) are a leading cause of childhood diarrhea. Current oral vaccines are not effective in impoverished countries where the vaccine is needed most. Therefore, alternative affordable strategies are urgently needed. Probiotics can alleviate diarrhea in children and enhance specific systemic and mucosal Ab responses, but the T cell responses are undefined. In this study, we elucidated the T cell and cytokine responses to attenuated human RV (AttHRV) and virulent human RV (HRV) in gnotobiotic pigs colonized with probiotics (Lactobacillus rhamnosus strain GG [LGG] and Bifidobacterium lactis Bb12 [Bb12]), mimicking gut commensals in breastfed infants. Neonatal gnotobiotic pigs are the only animal model susceptible to HRV diarrhea. Probiotic colonized and nonvaccinated (Probiotic) pigs had lower diarrhea and reduced virus shedding postchallenge compared with noncolonized and nonvaccinated pigs (Control). Higher protection in the Probiotic group coincided with higher ileal T regulatory cells (Tregs) before and after challenge, and higher serum TGF-β and lower serum and biliary proinflammatory cytokines postchallenge. Probiotic colonization in vaccinated pigs enhanced innate serum IFN-α, splenic and circulatory IFN-γ-producing T cells, and serum Th1 cytokines, but reduced serum Th2 cytokines compared with noncolonized vaccinated pigs (Vac). Thus, LGG+Bb12 induced systemic Th1 immunostimulatory effects on oral AttHRV vaccine that coincided with lower diarrhea severity and reduced virus shedding postchallenge in Vac+Pro compared with Vac pigs. Previously unreported intestinal CD8 Tregs were induced in vaccinated groups postchallenge. Thus, probiotics LGG+Bb12 exert divergent immunomodulating effects, with enhanced Th1 responses to oral AttHRV vaccine, whereas inducing Treg responses to virulent HRV. PMID:23918983

  15. Small airway epithelial cells exposure to printer-emitted engineered nanoparticles induces cellular effects on human microvascular endothelial cells in an alveolar-capillary co-culture model.

    PubMed

    Sisler, Jennifer D; Pirela, Sandra V; Friend, Sherri; Farcas, Mariana; Schwegler-Berry, Diane; Shvedova, Anna; Castranova, Vincent; Demokritou, Philip; Qian, Yong

    2015-01-01

    The printer is one of the most common office equipment. Recently, it was reported that toner formulations for printing equipment constitute nano-enabled products (NEPs) and contain engineered nanomaterials (ENMs) that become airborne during printing. To date, insufficient research has been performed to understand the potential toxicological properties of printer-emitted particles (PEPs) with several studies using bulk toner particles as test particles. These studies demonstrated the ability of toner particles to cause chronic inflammation and fibrosis in animal models. However, the toxicological implications of inhalation exposures to ENMs emitted from laser printing equipment remain largely unknown. The present study investigates the toxicological effects of PEPs using an in vitro alveolar-capillary co-culture model with Human Small Airway Epithelial Cells (SAEC) and Human Microvascular Endothelial Cells (HMVEC). Our data demonstrate that direct exposure of SAEC to low concentrations of PEPs (0.5 and 1.0 µg/mL) caused morphological changes of actin remodeling and gap formations within the endothelial monolayer. Furthermore, increased production of reactive oxygen species (ROS) and angiogenesis were observed in the HMVEC. Analysis of cytokine and chemokine levels demonstrates that interleukin (IL)-6 and MCP-1 may play a major role in the cellular communication observed between SAEC and HMVEC and the resultant responses in HMVEC. These data indicate that PEPs at low, non-cytotoxic exposure levels are bioactive and affect cellular responses in an alveolar-capillary co-culture model, which raises concerns for potential adverse health effects.

  16. Selection of a Relevant In Vitro Blood-Brain Barrier Model to Investigate Pro-Metastatic Features of Human Breast Cancer Cell Lines

    PubMed Central

    Drolez, Aurore; Vandenhaute, Elodie; Julien, Sylvain; Gosselet, Fabien; Burchell, Joy; Cecchelli, Roméo; Delannoy, Philippe; Dehouck, Marie-Pierre; Mysiorek, Caroline

    2016-01-01

    Around 7–17% of metastatic breast cancer patients will develop brain metastases, associated with a poor prognosis. To reach the brain parenchyma, cancer cells need to cross the highly restrictive endothelium of the Blood-Brain Barrier (BBB). As treatments for brain metastases are mostly inefficient, preventing cancer cells to reach the brain could provide a relevant and important strategy. For that purpose an in vitro approach is required to identify cellular and molecular interaction mechanisms between breast cancer cells and BBB endothelium, notably at the early steps of the interaction. However, while numerous studies are performed with in vitro models, the heterogeneity and the quality of BBB models used is a limitation to the extrapolation of the obtained results to in vivo context, showing that the choice of a model that fulfills the biological BBB characteristics is essential. Therefore, we compared pre-established and currently used in vitro models from different origins (bovine, mice, human) in order to define the most appropriate tool to study interactions between breast cancer cells and the BBB. On each model, the BBB properties and the adhesion capacities of breast cancer cell lines were evaluated. As endothelial cells represent the physical restriction site of the BBB, all the models consisted of endothelial cells from animal or human origins. Among these models, only the in vitro BBB model derived from human stem cells both displayed BBB properties and allowed measurement of meaningful different interaction capacities of the cancer cell lines. Importantly, the measured adhesion and transmigration were found to be in accordance with the cancer cell lines molecular subtypes. In addition, at a molecular level, the inhibition of ganglioside biosynthesis highlights the potential role of glycosylation in breast cancer cells adhesion capacities. PMID:26958843

  17. Selection of a Relevant In Vitro Blood-Brain Barrier Model to Investigate Pro-Metastatic Features of Human Breast Cancer Cell Lines.

    PubMed

    Drolez, Aurore; Vandenhaute, Elodie; Julien, Sylvain; Gosselet, Fabien; Burchell, Joy; Cecchelli, Roméo; Delannoy, Philippe; Dehouck, Marie-Pierre; Mysiorek, Caroline

    2016-01-01

    Around 7-17% of metastatic breast cancer patients will develop brain metastases, associated with a poor prognosis. To reach the brain parenchyma, cancer cells need to cross the highly restrictive endothelium of the Blood-Brain Barrier (BBB). As treatments for brain metastases are mostly inefficient, preventing cancer cells to reach the brain could provide a relevant and important strategy. For that purpose an in vitro approach is required to identify cellular and molecular interaction mechanisms between breast cancer cells and BBB endothelium, notably at the early steps of the interaction. However, while numerous studies are performed with in vitro models, the heterogeneity and the quality of BBB models used is a limitation to the extrapolation of the obtained results to in vivo context, showing that the choice of a model that fulfills the biological BBB characteristics is essential. Therefore, we compared pre-established and currently used in vitro models from different origins (bovine, mice, human) in order to define the most appropriate tool to study interactions between breast cancer cells and the BBB. On each model, the BBB properties and the adhesion capacities of breast cancer cell lines were evaluated. As endothelial cells represent the physical restriction site of the BBB, all the models consisted of endothelial cells from animal or human origins. Among these models, only the in vitro BBB model derived from human stem cells both displayed BBB properties and allowed measurement of meaningful different interaction capacities of the cancer cell lines. Importantly, the measured adhesion and transmigration were found to be in accordance with the cancer cell lines molecular subtypes. In addition, at a molecular level, the inhibition of ganglioside biosynthesis highlights the potential role of glycosylation in breast cancer cells adhesion capacities.

  18. Generation of improved humanized mouse models for human infectious diseases

    PubMed Central

    Brehm, Michael A.; Wiles, Michael V.; Greiner, Dale L.; Shultz, Leonard D.

    2014-01-01

    The study of human-specific infectious agents has been hindered by the lack of optimal small animal models. More recently development of novel strains of immunodeficient mice has begun to provide the opportunity to utilize small animal models for the study of many human-specific infectious agents. The introduction of a targeted mutation in the IL2 receptor common gamma chain gene (IL2rgnull) in mice already deficient in T and B cells led to a breakthrough in the ability to engraft hematopoietic stem cells, as well as functional human lymphoid cells and tissues, effectively creating human immune systems in immunodeficient mice. These humanized mice are becoming increasingly important as pre-clinical models for the study of human immunodeficiency virus-1 (HIV-1) and other human-specific infectious agents. However, there remain a number of opportunities to further improve humanized mouse models for the study of human-specific infectious agents. This is being done by the implementation of innovative technologies, which collectively will accelerate the development of new models of genetically modified mice, including; i) modifications of the host to reduce innate immunity, which impedes human cell engraftment; ii) genetic modification to provide human-specific growth factors and cytokines required for optimal human cell growth and function; iii) and new cell and tissue engraftment protocols. The development of “next generation” humanized mouse models continues to provide exciting opportunities for the establishment of robust small animal models to study the pathogenesis of human-specific infectious agents, as well as for testing the efficacy of therapeutic agents and experimental vaccines. PMID:24607601

  19. Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell- and patient-derived tumor organoids.

    PubMed

    Huang, Ling; Holtzinger, Audrey; Jagan, Ishaan; BeGora, Michael; Lohse, Ines; Ngai, Nicholas; Nostro, Cristina; Wang, Rennian; Muthuswamy, Lakshmi B; Crawford, Howard C; Arrowsmith, Cheryl; Kalloger, Steve E; Renouf, Daniel J; Connor, Ashton A; Cleary, Sean; Schaeffer, David F; Roehrl, Michael; Tsao, Ming-Sound; Gallinger, Steven; Keller, Gordon; Muthuswamy, Senthil K

    2015-11-01

    There are few in vitro models of exocrine pancreas development and primary human pancreatic adenocarcinoma (PDAC). We establish three-dimensional culture conditions to induce the differentiation of human pluripotent stem cells into exocrine progenitor organoids that form ductal and acinar structures in culture and in vivo. Expression of mutant KRAS or TP53 in progenitor organoids induces mutation-specific phenotypes in culture and in vivo. Expression of TP53(R175H) induces cytosolic SOX9 localization. In patient tumors bearing TP53 mutations, SOX9 was cytoplasmic and associated with mortality. We also define culture conditions for clonal generation of tumor organoids from freshly resected PDAC. Tumor organoids maintain the differentiation status, histoarchitecture and phenotypic heterogeneity of the primary tumor and retain patient-specific physiological changes, including hypoxia, oxygen consumption, epigenetic marks and differences in sensitivity to inhibition of the histone methyltransferase EZH2. Thus, pancreatic progenitor organoids and tumor organoids can be used to model PDAC and for drug screening to identify precision therapy strategies.

  20. Ductal pancreatic cancer modeling and drug screening using human pluripotent stem cell and patient-derived tumor organoids

    PubMed Central

    Huang, Ling; Holtzinger, Audrey; Jagan, Ishaan; BeGora, Michael; Lohse, Ines; Ngai, Nicholas; Nostro, Cristina; Wang, Rennian; Muthuswamy, Lakshmi B.; Crawford, Howard C.; Arrowsmith, Cheryl; Kalloger, Steve E.; Renouf, Daniel J.; Connor, Ashton A; Cleary, Sean; Schaeffer, David F.; Roehrl, Michael; Tsao, Ming-Sound; Gallinger, Steven; Keller, Gordon; Muthuswamy, Senthil K.

    2016-01-01

    There are few in vitro models of exocrine pancreas development and primary human pancreatic adenocarcinoma (PDAC). We establish three-dimensional culture conditions to induce the differentiation of human pluripotent stem cells (PSCs) into exocrine progenitor organoids that form ductal and acinar structures in culture and in vivo. Expression of mutant KRAS or TP53 in progenitor organoids induces mutation-specific phenotypes in culture and in vivo. Expression of TP53R175H induced cytosolic SOX9 localization. In patient tumors bearing TP53 mutations, SOX9 was cytoplasmic and associated with mortality. Culture conditions are also defined for clonal generation of tumor organoids from freshly resected PDAC. Tumor organoids maintain the differentiation status, histoarchitecture, phenotypic heterogeneity of the primary tumor, and retain patient-specific physiologic changes including hypoxia, oxygen consumption, epigenetic marks, and differential sensitivity to EZH2 inhibition. Thus, pancreatic progenitor organoids and tumor organoids can be used to model PDAC and for drug screening to identify precision therapy strategies. PMID:26501191

  1. Urine-sample-derived human induced pluripotent stem cells as a model to study PCSK9-mediated autosomal dominant hypercholesterolemia.

    PubMed

    Si-Tayeb, Karim; Idriss, Salam; Champon, Benoite; Caillaud, Amandine; Pichelin, Matthieu; Arnaud, Lucie; Lemarchand, Patricia; Le May, Cédric; Zibara, Kazem; Cariou, Bertrand

    2016-01-01

    Proprotein convertase subtilisin kexin type 9 (PCSK9) is a critical modulator of cholesterol homeostasis. Whereas PCSK9 gain-of-function (GOF) mutations are associated with autosomal dominant hypercholesterolemia (ADH) and premature atherosclerosis, PCSK9 loss-of-function (LOF) mutations have a cardio-protective effect and in some cases can lead to familial hypobetalipoproteinemia (FHBL). However, limitations of the currently available cellular models preclude deciphering the consequences of PCSK9 mutation further. We aimed to validate urine-sample-derived human induced pluripotent stem cells (UhiPSCs) as an appropriate tool to model PCSK9-mediated ADH and FHBL. To achieve our goal, urine-sample-derived somatic cells were reprogrammed into hiPSCs by using episomal vectors. UhiPSC were efficiently differentiated into hepatocyte-like cells (HLCs). Compared to control cells, cells originally derived from an individual with ADH (HLC-S127R) secreted less PCSK9 in the media (-38.5%; P=0.038) and had a 71% decrease (P<0.001) of low-density lipoprotein (LDL) uptake, whereas cells originally derived from an individual with FHBL (HLC-R104C/V114A) displayed a strong decrease in PCSK9 secretion (-89.7%; P<0.001) and had a 106% increase (P=0.0104) of LDL uptake. Pravastatin treatment significantly enhanced LDL receptor (LDLR) and PCSK9 mRNA gene expression, as well as PCSK9 secretion and LDL uptake in both control and S127R HLCs. Pravastatin treatment of multiple clones led to an average increase of LDL uptake of 2.19 ± 0.77-fold in HLC-S127R compared to 1.38 ± 0.49 fold in control HLCs (P<0.01), in line with the good response to statin treatment of individuals carrying the S127R mutation (mean LDL cholesterol reduction=60.4%, n=5). In conclusion, urine samples provide an attractive and convenient source of somatic cells for reprogramming and hepatocyte differentiation, but also a powerful tool to further decipher PCSK9 mutations and function.

  2. Urine-sample-derived human induced pluripotent stem cells as a model to study PCSK9-mediated autosomal dominant hypercholesterolemia

    PubMed Central

    Si-Tayeb, Karim; Idriss, Salam; Champon, Benoite; Caillaud, Amandine; Pichelin, Matthieu; Arnaud, Lucie; Lemarchand, Patricia; Le May, Cédric; Zibara, Kazem; Cariou, Bertrand

    2016-01-01

    ABSTRACT Proprotein convertase subtilisin kexin type 9 (PCSK9) is a critical modulator of cholesterol homeostasis. Whereas PCSK9 gain-of-function (GOF) mutations are associated with autosomal dominant hypercholesterolemia (ADH) and premature atherosclerosis, PCSK9 loss-of-function (LOF) mutations have a cardio-protective effect and in some cases can lead to familial hypobetalipoproteinemia (FHBL). However, limitations of the currently available cellular models preclude deciphering the consequences of PCSK9 mutation further. We aimed to validate urine-sample-derived human induced pluripotent stem cells (UhiPSCs) as an appropriate tool to model PCSK9-mediated ADH and FHBL. To achieve our goal, urine-sample-derived somatic cells were reprogrammed into hiPSCs by using episomal vectors. UhiPSC were efficiently differentiated into hepatocyte-like cells (HLCs). Compared to control cells, cells originally derived from an individual with ADH (HLC-S127R) secreted less PCSK9 in the media (−38.5%; P=0.038) and had a 71% decrease (P<0.001) of low-density lipoprotein (LDL) uptake, whereas cells originally derived from an individual with FHBL (HLC-R104C/V114A) displayed a strong decrease in PCSK9 secretion (−89.7%; P<0.001) and had a 106% increase (P=0.0104) of LDL uptake. Pravastatin treatment significantly enhanced LDL receptor (LDLR) and PCSK9 mRNA gene expression, as well as PCSK9 secretion and LDL uptake in both control and S127R HLCs. Pravastatin treatment of multiple clones led to an average increase of LDL uptake of 2.19±0.77-fold in HLC-S127R compared to 1.38±0.49 fold in control HLCs (P<0.01), in line with the good response to statin treatment of individuals carrying the S127R mutation (mean LDL cholesterol reduction=60.4%, n=5). In conclusion, urine samples provide an attractive and convenient source of somatic cells for reprogramming and hepatocyte differentiation, but also a powerful tool to further decipher PCSK9 mutations and function. PMID:26586530

  3. Mechanisms of pathogenesis induced by bovine leukemia virus as a model for human T-cell leukemia virus

    PubMed Central

    Aida, Yoko; Murakami, Hironobu; Takahashi, Masahiko; Takeshima, Shin-Nosuke

    2013-01-01

    Bovine leukemia virus (BLV) and human T-cell leukemia virus type 1 (HTLV-1) make up a unique retrovirus family. Both viruses induce chronic lymphoproliferative diseases with BLV affecting the B-cell lineage and HTLV-1 affecting the T-cell lineage. The pathologies of BLV- and HTLV-induced infections are notably similar, with an absence of chronic viraemia and a long latency period. These viruses encode at least two regulatory proteins, namely, Tax and Rex, in the pX region located between the env gene and the 3′ long terminal repeat. The Tax protein is a key contributor to the oncogenic potential of the virus, and is also the key protein involved in viral replication. However, BLV infection is not sufficient for leukemogenesis, and additional events such as gene mutations must take place. In this review, we first summarize the similarities between the two viruses in terms of genomic organization, virology, and pathology. We then describe the current knowledge of the BLV model, which may also be relevant for the understanding of leukemogenesis caused by HTLV-1. In addition, we address our improved understanding of Tax functions through the newly identified BLV Tax mutants, which have a substitution between amino acids 240 and 265. PMID:24265629

  4. [Regulatory B cells in human autoimmune diseases].

    PubMed

    Miyagaki, Tomomitsu

    2015-01-01

    B cells have been generally considered to be positive regulators of immune responses because of their ability to produce antigen-specific antibodies and to activate T cells through antigen presentation. Impairment of B cell development and function may cause autoimmune diseases. Recently, specific B cell subsets that can negatively regulate immune responses have been described in mouse models of a wide variety of autoimmune diseases. The concept of those B cells, termed regulatory B cells, is now recognized as important in the murine immune system. Among several regulatory B cell subsets, IL-10-producing regulatory B cells are the most widely investigated. On the basis of discoveries from studies of such mice, human regulatory B cells that produce IL-10 in most cases are becoming an active area of research. There have been emerging data suggesting the importance of human regulatory B cells in various diseases. Revealing the immune regulation mechanisms of human regulatory B cells in human autoimmune diseases could lead to the development of novel B cell targeted therapies. This review highlights the current knowledge on regulatory B cells, mainly IL-10-producing regulatory B cells, in clinical research using human samples. PMID:26725860

  5. A new in vitro model of Entamoeba histolytica adhesion, using the human colon carcinoma cell line Caco-2: scanning electron microscopic study.

    PubMed Central

    Rigothier, M C; Coconnier, M H; Servin, A L; Gayral, P

    1991-01-01

    The human colon carcinoma cell line Caco-2, which is widely used to study the adhesion and cytotoxicity of enterobacteria, was used to investigate the adhesion of the trophozoites of Entamoeba histolytica. We observed a high percentage of adhesion of amoebae to Caco-2 cells. Scanning electron microscopy showed that amoebial membrane structures were involved in adhesion and the cytolytic action. These differentiated cells should prove to be a useful model system for investigation of the pathogenic action of amoebae. Images PMID:1937772

  6. Atrial-like cardiomyocytes from human pluripotent stem cells are a robust preclinical model for assessing atrial-selective pharmacology

    PubMed Central

    Devalla, Harsha D; Schwach, Verena; Ford, John W; Milnes, James T; El-Haou, Said; Jackson, Claire; Gkatzis, Konstantinos; Elliott, David A; Chuva de Sousa Lopes, Susana M; Mummery, Christine L; Verkerk, Arie O; Passier, Robert

    2015-01-01

    Drugs targeting atrial-specific ion channels, Kv1.5 or Kir3.1/3.4, are being developed as new therapeutic strategies for atrial fibrillation. However, current preclinical studies carried out in non-cardiac cell lines or animal models may not accurately represent the physiology of a human cardiomyocyte (CM). In the current study, we tested whether human embryonic stem cell (hESC)-derived atrial CMs could predict atrial selectivity of pharmacological compounds. By modulating retinoic acid signaling during hESC differentiation, we generated atrial-like (hESC-atrial) and ventricular-like (hESC-ventricular) CMs. We found the expression of atrial-specific ion channel genes, KCNA5 (encoding Kv1.5) and KCNJ3 (encoding Kir 3.1), in hESC-atrial CMs and further demonstrated that these ion channel genes are regulated by COUP-TF transcription factors. Moreover, in response to multiple ion channel blocker, vernakalant, and Kv1.5 blocker, XEN-D0101, hESC-atrial but not hESC-ventricular CMs showed action potential (AP) prolongation due to a reduction in early repolarization. In hESC-atrial CMs, XEN-R0703, a novel Kir3.1/3.4 blocker restored the AP shortening caused by CCh. Neither CCh nor XEN-R0703 had an effect on hESC-ventricular CMs. In summary, we demonstrate that hESC-atrial CMs are a robust model for pre-clinical testing to assess atrial selectivity of novel antiarrhythmic drugs. PMID:25700171

  7. Cell proliferation in cancer prevention; effects of preventive agents on estrogen-related endometrial carcinogenesis model and on an in vitro model in human colorectal cells.

    PubMed

    Mori, H; Niwa, K; Zheng, Q; Yamada, Y; Sakata, K; Yoshimi, N

    2001-09-01

    Proto-oncogenes such as c-fos, c-jun and c-myc are known to relate to cell proliferation and differentiation. Some oriental herbal medicines like Glycyrrhizae radix or Juzen-taiho-to were found to suppress estradiol-17 beta (E2)-induced expression of c-fos/jun in uterine corpus and inhibited N-methyl-N-nitrosourea and E2-induced endometrial carcinogenesis in mice. It is suggested that the effects of such oriental drugs are exerted probably through suppression of estrogen-induced c-fos/jun expression and they are promising preventing agents for endometrial cancers. In the combined in vitro assay for cell proliferation (MTS assay) and apoptosis (DNA fragmentation) in human colorectal cancer cells (Colo 320), a number of naturally occurring chemopreventive agents such as curcumin, quercetin, auraptene, 1'-acetoxychavicol acetate (ACA) and indole-3-carbinol were shown to generate apoptosis as well as to inhibit cell proliferation. The results suggest a mode of action of these chemopreventive agents and also imply that such in vitro short term assay is useful for detection of new agents for cancer prevention.

  8. Applications of induced pluripotent stem cells in the modeling of human inflammatory bowel diseases.

    PubMed

    Liu, Jingquan; Shi, Bin; Shi, Kai; Zhang, Hongze

    2015-01-01

    Inflammatory bowel diseases (IBDs) are chronic and involve the gastrointestinal tract; the two primary IBDs are ulcerative colitis and Crohn's disease. Existing treatments for IBD include control of active inflammation and regulation of immune disorders, and commonly used drugs include salicylates, corticosteroids, and immunosuppressants. At the same time, an in-depth study of IBD pathogenesis promoted the acceptance of bioimmunotherapy by increasing numbers of people. However, long-term use of these drugs can cause adverse reactions that are difficult for patients to overcome, with limited efficacy for critically ill patients. Recent studies have found that stem cell transplantation is a new and effective therapy and IBD treatment, particularly for refractory cases. Stem cells, especially induced pluripotent stem cells (iPSCs), can differentiate into functional intestinal epithelia and their use avoids ethical issues arising from embryonic stem cells, providing a new kind of seed cell for alternative treatments for IBD. This paper reviews iPSCs as a potential new treatment for IBDs in order to provide an experimental and clinical reference.

  9. Trophoblast lineage cells derived from human induced pluripotent stem cells

    SciTech Connect

    Chen, Ying; Wang, Kai; Chandramouli, Gadisetti V.R.; Knott, Jason G.; Leach, Richard

    2013-07-12

    Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.

  10. Silencing of APE1 enhances sensitivity of human hepatocellular carcinoma cells to radiotherapy in vitro and in a xenograft model.

    PubMed

    Cun, Yanping; Dai, Nan; Xiong, Chengjie; Li, Mengxia; Sui, Jiangdong; Qian, Chengyuan; Li, Zheng; Wang, Dong

    2013-01-01

    Resistance to radiotherapy is a key limitation for the treatment of human hepatocellular carcinoma (HCC). To overcome this problem, we investigated the correlation between radioresistance and the human apurinic/apyrimidinic endonuclease (APE1), a bifunctional protein, which plays an important role in DNA repair and redox regulation activity of transcription factors. In the present study, we examined the radiosensitivity profiles of three human HCC cell lines, HepG2, Hep3B, and MHCC97L, using the adenoviral vector Ad5/F35-mediated APE1 siRNA (Ad5/F35-siAPE1). The p53 mutant cell lines MHCC97L showed radioresistance, compared with HepG2 and Hep3B cells. APE1 was strongly expressed in MHCC97L cells and was induced by irradiation in a dose-dependent manner, and Ad5/F35-siAPE1 effectively inhibited irradiation-induced APE1 and p53 expression. Moreover, silencing of APE1 significantly potentiated the growth inhibition and apoptosis induction by irradiation in all tested human HCC cell lines. In addition, Ad5/F35-siAPE1 significantly enhanced inhibition of tumor growth and potentiated cell apoptosis by irradiation both in HepG2 and MHCC97L xenografts. In conclusion, down regulation of APE1 could enhance sensitivity of human HCC cells to radiotherapy in vitro and in vivo.

  11. Human Biomarker Discovery and Predictive Models for Disease Progression for Idiopathic Pneumonia Syndrome Following Allogeneic Stem Cell Transplantation*

    PubMed Central

    Schlatzer, Daniela M.; Dazard, Jean-Eudes; Ewing, Rob M.; Ilchenko, Serguei; Tomcheko, Sara E.; Eid, Saada; Ho, Vincent; Yanik, Greg; Chance, Mark R.; Cooke, Kenneth R.

    2012-01-01

    Allogeneic hematopoietic stem cell transplantation (SCT) is the only curative therapy for many malignant and nonmalignant conditions. Idiopathic pneumonia syndrome (IPS) is a frequently fatal complication that limits successful outcomes. Preclinical models suggest that IPS represents an immune mediated attack on the lung involving elements of both the adaptive and the innate immune system. However, the etiology of IPS in humans is less well understood. To explore the disease pathway and uncover potential biomarkers of disease, we performed two separate label-free, proteomics experiments defining the plasma protein profiles of allogeneic SCT patients with IPS. Samples obtained from SCT recipients without complications served as controls. The initial discovery study, intended to explore the disease pathway in humans, identified a set of 81 IPS-associated proteins. These data revealed similarities between the known IPS pathways in mice and the condition in humans, in particular in the acute phase response. In addition, pattern recognition pathways were judged to be significant as a function of development of IPS, and from this pathway we chose the lipopolysaccaharide-binding protein (LBP) protein as a candidate molecular diagnostic for IPS, and verified its increase as a function of disease using an ELISA assay. In a separately designed study, we identified protein-based classifiers that could predict, at day 0 of SCT, patients who: 1) progress to IPS and 2) respond to cytokine neutralization therapy. Using cross-validation strategies, we built highly predictive classifier models of both disease progression and therapeutic response. In sum, data generated in this report confirm previous clinical and experimental findings, provide new insights into the pathophysiology of IPS, identify potential molecular classifiers of the condition, and uncover a set of markers potentially of interest for patient stratification as a basis for individualized therapy. PMID:22337588

  12. Glutathione metabolism in the HaCaT cell line as a model for the detoxification of the model sensitisers 2,4-dinitrohalobenzenes in human skin.

    PubMed

    Jacquoilleot, Sandrine; Sheffield, David; Olayanju, Adedamola; Sison-Young, Rowena; Kitteringham, Neil R; Naisbitt, Dean J; Aleksic, Maja

    2015-08-19

    Glutathione (GSH) is the most prominent antioxidant in cells and the co-factor of an important set of enzymes involved in the skin metabolic clearance system, glutathione S-transferases (GST). Here, we describe an LC-MS (liquid chromatography-mass spectroscopy) method to measure GSH and its disulfide form (GSSG) in HaCaT cells and a 3D Reconstructed Human Epidermis (RHE) model. In our assay, the basal level of GSH in both systems was in the low nmol/mg soluble protein range, while the level of GSSG was systematically below our limit of quantification (0.1 μM). We found that 2,4-dinitrohalobenzenes deplete the GSH present in HaCaT cells within the first hour of exposure, in a dose dependent manner. The level of GSH in HaCaT cells treated with a single non-toxic dose of 10 μM of dinitrohalobenzene was also shown to increase after two hours. While cells treated with 1-chloro-2,4-dinitrobenzene (DNCB) and 1-fluoro-2,4-dinitrobenzene (DNFB) repleted GSH to levels similar to untreated control cells within 24h, 1-bromo-2,4-dinitrobenzene (DNBB) seemed to prevent such a repletion and appeared to be the most toxic compound in all assays. A mathematical modelling of experimental results was performed to further rationalise the differences observed between test chemicals. For this purpose the biological phenomena observed were simplified into two sequential events: the initial depletion of the GSH stock after chemical treatment followed by the repletion of the GSH once the chemical was cleared. Activation of the nuclear factor E2-related factor 2 (Nrf2) pathway was observed with all compounds within two hours, and at concentrations less than 10 μM. These data show that GSH depletion and repletion occur rapidly in skin cells and emphasize the importance of conducting kinetic studies when performing in vitro experiments exploring skin sensitization.

  13. Combinatorial DNA Damage Pairing Model Based on X-Ray-Induced Foci Predicts the Dose and LET Dependence of Cell Death in Human Breast Cells

    SciTech Connect

    Vadhavkar, Nikhil; Pham, Christopher; Georgescu, Walter; Deschamps, Thomas; Heuskin, Anne-Catherine; Tang, Jonathan; Costes, Sylvain V.

    2014-09-01

    In contrast to the classic view of static DNA double-strand breaks (DSBs) being repaired at the site of damage, we hypothesize that DSBs move and merge with each other over large distances (m). As X-ray dose increases, the probability of having DSB clusters increases as does the probability of misrepair and cell death. Experimental work characterizing the X-ray dose dependence of radiation-induced foci (RIF) in nonmalignant human mammary epithelial cells (MCF10A) is used here to validate a DSB clustering model. We then use the principles of the local effect model (LEM) to predict the yield of DSBs at the submicron level. Two mechanisms for DSB clustering, namely random coalescence of DSBs versus active movement of DSBs into repair domains are compared and tested. Simulations that best predicted both RIF dose dependence and cell survival after X-ray irradiation favored the repair domain hypothesis, suggesting the nucleus is divided into an array of regularly spaced repair domains of ~;;1.55 m sides. Applying the same approach to high-linear energy transfer (LET) ion tracks, we are able to predict experimental RIF/m along tracks with an overall relative error of 12percent, for LET ranging between 30 350 keV/m and for three different ions. Finally, cell death was predicted by assuming an exponential dependence on the total number of DSBs and of all possible combinations of paired DSBs within each simulated RIF. Relative biological effectiveness (RBE) predictions for cell survival of MCF10A exposed to high-LET showed an LET dependence that matches previous experimental results for similar cell types. Overall, this work suggests that microdosimetric properties of ion tracks at the submicron level are sufficient to explain both RIF data and survival curves for any LET, similarly to the LEM assumption. Conversely, high-LET death mechanism does not have to infer linear-quadratic dose formalism as done in the LEM. In addition, the size of repair domains derived in our model

  14. Cartilage repair by human umbilical cord blood-derived mesenchymal stem cells with different hydrogels in a rat model.

    PubMed

    Park, Yong-Beom; Song, Minjung; Lee, Choong-Hee; Kim, Jin-A; Ha, Chul-Won

    2015-11-01

    This study was carried out to assess the feasibility of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in articular cartilage repair and to further determine a suitable delivering hydrogel in a rat model. Critical sized full thickness cartilage defects were created. The hUCB-MSCs and three different hydrogel composites (hydrogel A; 4% hyaluronic acid/30% pluronic (1:1, v/v), hydrogel B; 4% hyaluronic acid, and hydrogel C; 4% hyaluronic acid/30% pluronic/chitosan (1:1:2, v/v)) were implanted into the experimental knee (right knee) and hydrogels without hUCB-MSCs were implanted into the control knee (left knee). Defects were evaluated after 8 weeks. The hUCB-MSCs with hydrogels composites resulted in a better repair as seen by gross and histological evaluation compared with hydrogels without hUCB-MSCs. Among the three different hydrogels, the 4% hyaluronic acid hydrogel composite (hydrogel B) showed the best result in cartilage repair as seen by the histological evaluation compared with the other hydrogel composites (hydrogel A and C). The results of this study suggest that hUCB-MSCs may be a promising cell source in combination with 4% hyaluronic acid hydrogels in the in vivo repair of cartilage defects.

  15. Human mesenchymal stem cells resolve airway inflammation, hyperreactivity, and histopathology in a mouse model of occupational asthma.

    PubMed

    Martínez-González, Itziar; Cruz, Maria-Jesús; Moreno, Rafael; Morell, Ferran; Muñoz, Xavier; Aran, Josep M

    2014-10-01

    Occupational asthma (OA) is characterized by allergic airway inflammation and hyperresponsiveness, leading to progressive airway remodeling and a concomitant decline in lung function. The management of OA remains suboptimal in clinical practice. Thus, establishing effective therapies might overcome the natural history of the disease. We evaluated the ability of human adipose-tissue-derived mesenchymal stem cells (hASCs), either unmodified or engineered to secrete the IL-33 decoy receptor sST2, to attenuate the inflammatory and respiratory symptoms in a previously validated mouse model of OA to ammonium persulfate (AP). Twenty-four hours after a dermal AP sensitization and intranasal challenge regimen, the animals received intravenously 1 × 10(6) cells (either hASCs or hASCs overexpressing sST2) or saline and were analyzed at 1, 3, and 6 days after treatment. The infused hASCs induced an anti-inflammatory and restorative program upon reaching the AP-injured, asthmatic lungs, leading to early reduction of neutrophilic inflammation and total IgE production, preserved alveolar architecture with nearly absent lymphoplasmacytic infiltrates, negligible smooth muscle hyperplasia/hypertrophy in the peribronchiolar areas, and baseline airway hyperreactivity (AHR) to methacholine. Local sST2 overexpression barely increased the substantial efficacy displayed by unmodified hASCs. Thus, hASCs may represent a viable multiaction therapeutic capable to adequately respond to the AP-injured lung environment by resolving inflammation, tissue remodeling, and bronchial hyperresponsiveness typical of OA. PMID:24798370

  16. Human Mesenchymal Stem Cells Resolve Airway Inflammation, Hyperreactivity, and Histopathology in a Mouse Model of Occupational Asthma

    PubMed Central

    Martínez-González, Itziar; Moreno, Rafael; Morell, Ferran; Muñoz, Xavier

    2014-01-01

    Occupational asthma (OA) is characterized by allergic airway inflammation and hyperresponsiveness, leading to progressive airway remodeling and a concomitant decline in lung function. The management of OA remains suboptimal in clinical practice. Thus, establishing effective therapies might overcome the natural history of the disease. We evaluated the ability of human adipose-tissue-derived mesenchymal stem cells (hASCs), either unmodified or engineered to secrete the IL-33 decoy receptor sST2, to attenuate the inflammatory and respiratory symptoms in a previously validated mouse model of OA to ammonium persulfate (AP). Twenty-four hours after a dermal AP sensitization and intranasal challenge regimen, the animals received intravenously 1×106 cells (either hASCs or hASCs overexpressing sST2) or saline and were analyzed at 1, 3, and 6 days after treatment. The infused hASCs induced an anti-inflammatory and restorative program upon reaching the AP-injured, asthmatic lungs, leading to early reduction of neutrophilic inflammation and total IgE production, preserved alveolar architecture with nearly absent lymphoplasmacytic infiltrates, negligible smooth muscle hyperplasia/hypertrophy in the peribronchiolar areas, and baseline airway hyperreactivity (AHR) to methacholine. Local sST2 overexpression barely increased the substantial efficacy displayed by unmodified hASCs. Thus, hASCs may represent a viable multiaction therapeutic capable to adequately respond to the AP-injured lung environment by resolving inflammation, tissue remodeling, and bronchial hyperresponsiveness typical of OA. PMID:24798370

  17. Biodistribution and in vivo efficacy of genetically modified human mesenchymal stem cells systemically transplanted into a mouse bone fracture model.

    PubMed

    Kang, Jin Wook; Park, Ki Dae; Choi, Youngju; Baek, Dae Hyun; Cho, Wan-Seob; Choi, Mina; Park, Jae Hyun; Choi, Kyoung Suk; Kim, Hyung Soo; Yoo, Tae Moo

    2013-08-01

    Human mesenchymal stem cells (hMSCs) have generated a great deal of interest in clinical application due to their ability to undergo multi-lineage differentiation. Recently, ex vivo genetic modification of hMSCs was attempted to increase their differentiation potential. The present study was conducted to evaluate the biodistribution and in vivo efficacy of genetically modified hMSCs. To accomplish this, Runx2, which is a key transcription factor associated with osteoblast differentiation, was transduced into hMSCs using lentiviral vectors expressing green fluorescent protein (GFP) or luciferase. Here, we developed an experimental fracture in mice femur to investigate the effects of Runx2-transduced hMSCs on bone healing and migration into injury site. We conducted bio-luminescence imaging (BLI) using luciferase-tagged vector and quantitative real-time PCR using GFP probe to investigate the biodistribution of Runx2-transduced hMSCs in the fracture model. The biodistribution of hMSC cells in the fractured femur was observed at 14 days post-transplantation upon both BLI imaging and real-time PCR. Moreover, the fractured mice transplanted with Runx2-transduced hMSCs showed superior bone healing when compared to mock-transduced hMSC and MRC5 fibroblasts which were used as control. These data suggested that transplanted genetically modified hMSCs systemically migrate to the fractured femur, where they contribute to bone formation in vivo.

  18. KR-31543 reduces the production of proinflammatory molecules in human endothelial cells and monocytes and attenuates atherosclerosis in mouse model.

    PubMed

    Choi, Jae-Hoon; Yoo, Ji-Young; Kim, Sun-Ok; Yoo, Sung-Eun; Oh, Goo Taeg

    2012-12-31

    KR-31543, (2S, 3R, 4S)-6-amino-4-[N-(4-chlorophenyl)- N-(2-methyl-2H-tetrazol-5-ylmethyl) amino]-3,4-dihydro- 2-dimethyoxymethyl-3-hydroxy-2-methyl-2H-1-benz opyran is a new neuroprotective agent for ischemiareperfusion damage. It has also been reported that KR-31543 has protective effects on lipid peroxidation and H₂O₂-induced reactive oxygen species production. In this study, we investigated the anti-inflammatory and anti-atherogenic properties of KR-31543. We observed that KR-31543 treatment reduced the production of MCP-1, IL-8, and VCAM-1 in HUVECs, and of MCP-1 and IL-6 in THP-1 human monocytes. We also examined the effect of KR-31543 on monocytes migration in vitro. KR-31543 treatment effectively reduced the migration of THP-1 human monocytes to the HUVEC monolayer in a dose-dependent manner. We next examined the effects of this compound on atherogenesis in LDL receptor deficient (Ldlr ⁻/⁻) mice. After 10 weeks of western diet, the formation of atherosclerotic lesion in aorta was reduced in the KR-31543-treated group compared to the control group. The accumulation of macrophages in lesion was also reduced in KR-31543 treated group. However, the plasma levels of total cholesterol, HDL, LDL, and triglyceride were not affected by KR-31543 treatment. Taken together, these results show that KR-31543 has anti-inflammatory properties on human monocytes and endothelial cells, and inhibits fatty streak lesion formation in mouse model of atherosclerosis, suggesting the potential of KR-31543 for the treatment for atherosclerosis.

  19. Novel scalable 3D cell based model for in vitro neurotoxicity testing: Combining human differentiated neurospheres with gene expression and functional endpoints.

    PubMed

    Terrasso, Ana Paula; Pinto, Catarina; Serra, Margarida; Filipe, Augusto; Almeida, Susana; Ferreira, Ana Lúcia; Pedroso, Pedro; Brito, Catarina; Alves, Paula Marques

    2015-07-10

    There is an urgent need for new in vitro strategies to identify neurotoxic agents with speed, reliability and respect for animal welfare. Cell models should include distinct brain cell types and represent brain microenvironment to attain higher relevance. The main goal of this study was to develop and validate a human 3D neural model containing both neurons and glial cells, applicable for toxicity testing in high-throughput platforms. To achieve this, a scalable bioprocess for neural differentiation of human NTera2/cl.D1 cells in stirred culture systems was developed. Endpoints based on neuronal- and astrocytic-specific gene expression and functionality in 3D were implemented in multi-well format and used for toxicity assessment. The prototypical neurotoxicant acrylamide affected primarily neurons, impairing synaptic function; our results suggest that gene expression of the presynaptic marker synaptophysin can be used as sensitive endpoint. Chloramphenicol, described as neurotoxicant affected both cell types, with cytoskeleton markers' expression significantly reduced, particularly in astrocytes. In conclusion, a scalable and reproducible process for production of differentiated neurospheres enriched in mature neurons and functional astrocytes was obtained. This 3D approach allowed efficient production of large numbers of human differentiated neurospheres, which in combination with gene expression and functional endpoints are a powerful cell model to evaluate human neuronal and astrocytic toxicity.

  20. Model experiments for immunomagnetic elimination of leukemic cells from human bone marrow. Presentation of a novel magnetic separation system.

    PubMed

    Gruhn, B; Häfer, R; Müller, A; Andrä, W; Danan, H; Zintl, F

    1991-11-01

    Optimal conditions for removing leukemic cells from human bone marrow with monoclonal antibodies (mAb) and magnetic immunobeads were investigated. Monodisperse 3 microns polystyrene microspheres containing magnetite were coated with affinity-purified rabbit antimouse IgG at 4 degrees C, pH 9.6 for 18 h. SKW-3 cells (T-CLL cell line) were marked with the supravital DNA stain Hoechst 33342, seeded into normal human bone marrow, and then incubated with the mAb CD1, CD6, and CD8 at 4 degrees C for 30 min. In preliminary experiments REH cells (cALL cells) and mouse anti-REH cell antibodies were used to find the most favorable conditions for the binding of magnetic beads to tumor cells. Optimal formation of cell-bead rosettes was achieved by rotating beads and tumor cells together at room temperature at a concentration of 1 x 10(7) cells/ml, a bead: tumor cell ratio of 100:1 and an incubation time of one hour. The novel magnetic separation apparatus consists of three polystyrene chambers connected by silicone rubber tubing. The chambers contain four steel inserts each equipped with 32 nickel wires, which are magnetized by permanent magnets in such a way that the inhomogeneous high gradient magnetic field could be established within the cell suspension containing the cells to be depleted. The fluid flow was established by a peristaltic pump. At a flow rate of 1.5 ml/min and a field strength of 160 kA/m, no beads could be detected in the purged marrow. A cocktail of the three mAb was more effective than any single antibody in forming bead-cell rosettes. Two sequential purging cycles were superior to one. The marrow recovered was highly viable as assessed by trypan blue dye exclusion and by growth of CFU-GM. PMID:1786986

  1. Model experiments for immunomagnetic elimination of leukemic cells from human bone marrow. Presentation of a novel magnetic separation system.

    PubMed

    Gruhn, B; Häfer, R; Müller, A; Andrä, W; Danan, H; Zintl, F

    1991-11-01

    Optimal conditions for removing leukemic cells from human bone marrow with monoclonal antibodies (mAb) and magnetic immunobeads were investigated. Monodisperse 3 microns polystyrene microspheres containing magnetite were coated with affinity-purified rabbit antimouse IgG at 4 degrees C, pH 9.6 for 18 h. SKW-3 cells (T-CLL cell line) were marked with the supravital DNA stain Hoechst 33342, seeded into normal human bone marrow, and then incubated with the mAb CD1, CD6, and CD8 at 4 degrees C for 30 min. In preliminary experiments REH cells (cALL cells) and mouse anti-REH cell antibodies were used to find the most favorable conditions for the binding of magnetic beads to tumor cells. Optimal formation of cell-bead rosettes was achieved by rotating beads and tumor cells together at room temperature at a concentration of 1 x 10(7) cells/ml, a bead: tumor cell ratio of 100:1 and an incubation time of one hour. The novel magnetic separation apparatus consists of three polystyrene chambers connected by silicone rubber tubing. The chambers contain four steel inserts each equipped with 32 nickel wires, which are magnetized by permanent magnets in such a way that the inhomogeneous high gradient magnetic field could be established within the cell suspension containing the cells to be depleted. The fluid flow was established by a peristaltic pump. At a flow rate of 1.5 ml/min and a field strength of 160 kA/m, no beads could be detected in the purged marrow. A cocktail of the three mAb was more effective than any single antibody in forming bead-cell rosettes. Two sequential purging cycles were superior to one. The marrow recovered was highly viable as assessed by trypan blue dye exclusion and by growth of CFU-GM.

  2. Human Lung Cancer Cells Grown in an Ex Vivo 3D Lung Model Produce Matrix Metalloproteinases Not Produced in 2D Culture

    PubMed Central

    Mishra, Dhruva K.; Sakamoto, Jason H.; Thrall, Michael J.; Baird, Brandi N.; Blackmon, Shanda H.; Ferrari, Mauro; Kurie, Jonathan M.; Kim, Min P.

    2012-01-01

    We compared the growth of human lung cancer cells in an ex vivo three-dimensional (3D) lung model and 2D culture to determine which better mimics lung cancer growth in patients. A549 cells were grown in an ex vivo 3D lung model and in 2D culture for 15 days. We measured the size and formation of tumor nodules and counted the cells after 15 days. We also stained the tissue/cells for Ki-67, and Caspase-3. We measured matrix metalloproteinase (MMP) levels in the conditioned media and in blood plasma from patients with adenocarcinoma of the lung. Organized tumor nodules with intact vascular space formed in the ex vivo 3D lung model but not in 2D culture. Proliferation and apoptosis were greater in the ex vivo 3D lung model compared to the 2D culture. After 15 days, there were significantly more cells in the 2D culture than the 3D model. MMP-1, MMP-9, and MMP-10 production were significantly greater in the ex vivo 3D lung model. There was no production of MMP-9 in the 2D culture. The patient samples contained MMP-1, MMP-2, MMP-9, and MMP-10. The human lung cancer cells grown on ex vivo 3D model form perfusable nodules that grow over time. It also produced MMPs that were not produced in 2D culture but seen in human lung cancer patients. The ex vivo 3D lung model may more closely mimic the biology of human lung cancer development than the 2D culture. PMID:23028922

  3. Deregulation of energy metabolism promotes antifibrotic effects in human hepatic stellate cells and prevents liver fibrosis in a mouse model.

    PubMed

    Karthikeyan, Swathi; Potter, James J; Geschwind, Jean-Francois; Sur, Surojit; Hamilton, James P; Vogelstein, Bert; Kinzler, Kenneth W; Mezey, Esteban; Ganapathy-Kanniappan, Shanmugasundaram

    2016-01-15

    Liver fibrosis and cirrhosis result from uncontrolled secretion and accumulation of extracellular matrix (ECM) proteins by hepatic stellate cells (HSCs) that are activated by liver injury and inflammation. Despite the progress in understanding the biology liver fibrogenesis and the identification of potential targets for treating fibrosis, development of an effective therapy remains elusive. Since an uninterrupted supply of intracellular energy is critical for the activated-HSCs to maintain constant synthesis and secretion of ECM, we hypothesized that interfering with energy metabolism could affect ECM secretion. Here we report that a sublethal dose of the energy blocker, 3-bromopyruvate (3-BrPA) facilitates phenotypic alteration of activated LX-2 (a human hepatic stellate cell line), into a less-active form. This treatment-dependent reversal of activated-LX2 cells was evidenced by a reduction in α-smooth muscle actin (α-SMA) and collagen secretion, and an increase in activity of matrix metalloproteases. Mechanistically, 3-BrPA-dependent antifibrotic effects involved down-regulation of the mitochondrial metabolic enzyme, ATP5E, and up-regulation of glycolysis, as evident by elevated levels of lactate dehydrogenase, lactate production and its transporter, MCT4. Finally, the antifibrotic effects of 3-BrPA were validated in vivo in a mouse model of carbon tetrachloride-induced liver fibrosis. Results from histopathology & histochemical staining for collagen and α-SMA substantiated that 3-BrPA promotes antifibrotic effects in vivo. Taken together, our data indicate that sublethal, metronomic treatment with 3-BrPA blocks the progression of liver fibrosis suggesting its potential as a novel therapeutic for treating liver fibrosis.

  4. A biomathematical model of time-delayed feedback in the human male hypothalamic-pituitary-Leydig cell axis.

    PubMed

    Keenan, D M; Veldhuis, J D

    1998-07-01

    We develop, implement, and test a feedback and feedforward biomathematical construct of the male hypothalamic [gonadotropin-releasing hormone (GnRH)]-pituitary [luteinizing hormone (LH)]-gonadal [testosterone (Te)] axis. This stochastic differential equation formulation consists of a nonstationary stochastic point process responsible for generating episodic release of GnRH, which is modulated negatively by short-loop (GnRH) and long-loop (Te) feedback. Pulsatile GnRH release in turn drives bursts of LH secretion via an agonistic dose-response curve that is partially damped by Te negative feedback. Circulating LH stimulates (feedforward) Te synthesis and release by a second dose response. Te acts via negative dose-responsive feedback on GnRH and LH output, thus fulfilling conditions of a closed-loop control system. Four computer simulations document expected feedback performance, as published earlier for the human male GnRH-LH-Te axis. Six other simulations test distinct within-model coupling mechanisms to link a circadian modulatory input to a pulsatile control node so as to explicate the known 24-h variations in Te and, to a lesser extent, LH. We conclude that relevant dynamic function, internodal dose-dependent regulatory connections, and within-system time-delayed coupling together provide a biomathematical basis for a nonlinear feedback-feedforward control model with combined pulsatile and circadian features that closely emulate the measurable output activities of the male hypothalamic-pituitary-Leydig cell axis.

  5. Modeling cell response to low doses of photon irradiation: Part 2--application to radiation-induced chromosomal aberrations in human carcinoma cells.

    PubMed

    Cunha, Micaela; Testa, Etienne; Komova, Olga V; Nasonova, Elena A; Mel'nikova, Larisa A; Shmakova, Nina L; Beuve, Michaël

    2016-03-01

    The biological phenomena observed at low doses of ionizing radiation (adaptive response, bystander effects, genomic instability, etc.) are still not well understood. While at high irradiation doses, cellular death may be directly linked to DNA damage, at low doses, other cellular structures may be involved in what are known as non-(DNA)-targeted effects. Mitochondria, in particular, may play a crucial role through their participation in a signaling network involving oxygen/nitrogen radical species. According to the size of the implicated organelles, the fluctuations in the energy deposited into these target structures may impact considerably the response of cells to low doses of ionizing irradiation. Based on a recent simulation of these fluctuations, a theoretical framework was established to have further insight into cell responses to low doses of photon irradiation, namely the triggering of radioresistance mechanisms by energy deposition into specific targets. Three versions of a model are considered depending on the target size and on the number of targets that need to be activated by energy deposition to trigger radioresistance mechanisms. These model versions are applied to the fraction of radiation-induced chromosomal aberrations measured at low doses in human carcinoma cells (CAL51). For this cell line, it was found in the present study that the mechanisms of radioresistance could not be triggered by the activation of a single small target (nanometric size, 100 nm), but could instead be triggered by the activation of a large target (micrometric, 10 μm) or by the activation of a great number of small targets. The mitochondria network, viewed either as a large target or as a set of small units, might be concerned by these low-dose effects. PMID:26708100

  6. Activation of Human T Cells in Hypertension: Studies of Humanized Mice and Hypertensive Humans.

    PubMed

    Itani, Hana A; McMaster, William G; Saleh, Mohamed A; Nazarewicz, Rafal R; Mikolajczyk, Tomasz P; Kaszuba, Anna M; Konior, Anna; Prejbisz, Aleksander; Januszewicz, Andrzej; Norlander, Allison E; Chen, Wei; Bonami, Rachel H; Marshall, Andrew F; Poffenberger, Greg; Weyand, Cornelia M; Madhur, Meena S; Moore, Daniel J; Harrison, David G; Guzik, Tomasz J

    2016-07-01

    Emerging evidence supports an important role for T cells in the genesis of hypertension. Because this work has predominantly been performed in experimental animals, we sought to determine whether human T cells are activated in hypertension. We used a humanized mouse model in which the murine immune system is replaced by the human immune system. Angiotensin II increased systolic pressure to 162 versus 116 mm Hg for sham-treated animals. Flow cytometry of thoracic lymph nodes, thoracic aorta, and kidney revealed increased infiltration of human leukocytes (CD45(+)) and T lymphocytes (CD3(+) and CD4(+)) in response to angiotensin II infusion. Interestingly, there was also an increase in the memory T cells (CD3(+)/CD45RO(+)) in the aortas and lymph nodes. Prevention of hypertension using hydralazine and hydrochlorothiazide prevented the accumulation of T cells in these tissues. Studies of isolated human T cells and monocytes indicated that angiotensin II had no direct effect on cytokine production by T cells or the ability of dendritic cells to drive T-cell proliferation. We also observed an increase in circulating interleukin-17A producing CD4(+) T cells and both CD4(+) and CD8(+) T cells that produce interferon-γ in hypertensive compared with normotensive humans. Thus, human T cells become activated and invade critical end-organ tissues in response to hypertension in a humanized mouse model. This response likely reflects the hypertensive milieu encountered in vivo and is not a direct effect of the hormone angiotensin II.

  7. Activation of Human T Cells in Hypertension: Studies of Humanized Mice and Hypertensive Humans.

    PubMed

    Itani, Hana A; McMaster, William G; Saleh, Mohamed A; Nazarewicz, Rafal R; Mikolajczyk, Tomasz P; Kaszuba, Anna M; Konior, Anna; Prejbisz, Aleksander; Januszewicz, Andrzej; Norlander, Allison E; Chen, Wei; Bonami, Rachel H; Marshall, Andrew F; Poffenberger, Greg; Weyand, Cornelia M; Madhur, Meena S; Moore, Daniel J; Harrison, David G; Guzik, Tomasz J

    2016-07-01

    Emerging evidence supports an important role for T cells in the genesis of hypertension. Because this work has predominantly been performed in experimental animals, we sought to determine whether human T cells are activated in hypertension. We used a humanized mouse model in which the murine immune system is replaced by the human immune system. Angiotensin II increased systolic pressure to 162 versus 116 mm Hg for sham-treated animals. Flow cytometry of thoracic lymph nodes, thoracic aorta, and kidney revealed increased infiltration of human leukocytes (CD45(+)) and T lymphocytes (CD3(+) and CD4(+)) in response to angiotensin II infusion. Interestingly, there was also an increase in the memory T cells (CD3(+)/CD45RO(+)) in the aortas and lymph nodes. Prevention of hypertension using hydralazine and hydrochlorothiazide prevented the accumulation of T cells in these tissues. Studies of isolated human T cells and monocytes indicated that angiotensin II had no direct effect on cytokine production by T cells or the ability of dendritic cells to drive T-cell proliferation. We also observed an increase in circulating interleukin-17A producing CD4(+) T cells and both CD4(+) and CD8(+) T cells that produce interferon-γ in hypertensive compared with normotensive humans. Thus, human T cells become activated and invade critical end-organ tissues in response to hypertension in a humanized mouse model. This response likely reflects the hypertensive milieu encountered in vivo and is not a direct effect of the hormone angiotensin II. PMID:27217403

  8. A calibrated agent-based computer model of stochastic cell dynamics in normal human colon crypts useful for in silico experiments

    PubMed Central

    2013-01-01

    Background Normal colon crypts consist of stem cells, proliferating cells, and differentiated cells. Abnormal rates of proliferation and differentiation can initiate colon cancer. We have measured the variation in the number of each of these cell types in multiple crypts in normal human biopsy specimens. This has provided the opportunity to produce a calibrated computational model that simulates cell dynamics in normal human crypts, and by changing model parameter values, to simulate the initiation and treatment of colon cancer. Results An agent-based model of stochastic cell dynamics in human colon crypts was developed in the multi-platform open-source application NetLogo. It was assumed that each cell’s probability of proliferation and probability of death is determined by its position in two gradients along the crypt axis, a divide gradient and in a die gradient. A cell’s type is not intrinsic, but rather is determined by its position in the divide gradient. Cell types are dynamic, plastic, and inter-convertible. Parameter values were determined for the shape of each of the gradients, and for a cell’s response to the gradients. This was done by parameter sweeps that indicated the values that reproduced the measured number and variation of each cell type, and produced quasi-stationary stochastic dynamics. The behavior of the model was verified by its ability to reproduce the experimentally observed monocolonal conversion by neutral drift, the formation of adenomas resulting from mutations either at the top or bottom of the crypt, and by the robust ability of crypts to recover from perturbation by cytotoxic agents. One use of the virtual crypt model was demonstrated by evaluating different cancer chemotherapy and radiation scheduling protocols. Conclusions A virtual crypt has been developed that simulates the quasi-stationary stochastic cell dynamics of normal human colon crypts. It is unique in that it has been calibrated with measurements of human biopsy

  9. Carbon Nanotubes and Human Cells?

    ERIC Educational Resources Information Center

    King, G. Angela

    2005-01-01

    Single-walled carbon nanotubes that were chemically altered to be water soluble are shown to enter fibroblasts, T cells, and HL60 cells. Nanoparticles adversely affect immortalized HaCaT human keratinocyte cultures, indicating that they may enter cells.

  10. Cell motility in models of wounded human skin is improved by Gap27 despite raised glucose, insulin and IGFBP-5

    SciTech Connect

    Wright, Catherine S.; Berends, Rebecca F.; Flint, David J.; Martin, Patricia E.M.

    2013-02-15

    Reducing Cx43 expression stimulates skin wound healing. This is mimicked in models when Cx43 function is blocked by the connexin mimetic peptide Gap27. IGF-I also stimulates wound healing with IGFBP-5 attenuating its actions. Further, the IGF-I to IGFBP-5 ratio is altered in diabetic skin, where wound closure is impaired. We investigated whether Gap27 remains effective in augmenting scrape-wound closure in human skin wound models simulating diabetes-induced changes, using culture conditions with raised glucose, insulin and IGFBP-5. Gap27 increased scrape-wound closure in normal glucose and insulin (NGI) and to a lesser extent in high glucose and insulin (HGI). IGF-I enhanced scrape-wound closure in keratinocytes whereas IGFBP-5 inhibited this response. Gap27 overcame the inhibitory effects of IGFBP-5 on IGF-I activity. Connexin-mediated communication (CMC) was reduced in HGI, despite raised Cx43, and Gap27 significantly decreased CMC in NGI and HGI. IGF-I and IGFBP-5 did not affect CMC. IGF-I increased keratinocyte proliferation in NGI, and Gap27 increased proliferation in NGI to a greater extent than in HGI. We conclude that IGF-I and Gap27 stimulate scrape-wound closure by independent mechanisms with Gap27 inhibiting Cx43 function. Gap27 can enhance wound closure in diabetic conditions, irrespective of the IGF-I:IGFBP-5 balance. - Highlights: ► Human organotypic and keratinocyte ‘diabetic’ skin models were used to demonstrate the ability of Gap27 to improve scrape-wound closure. ► Gap27 enhanced scrape-wound closure by reducing Cx43-mediated communication, whereas IGFBP-5 retarded cell migration. ► IGF-I and IGFBP-5 did not affect connexin-mediated pathways. ► Gap27 can override altered glucose, insulin, IGF-I, and IGFBP-5 in ‘diabetic’ skin models and thus has therapeutic potential.

  11. Effect of human milk on blood and bone marrow cells in a malnourished mice model; comparative study with cow milk.

    PubMed

    García, Isabel; Salva, Susana; Zelaya, Hortensia; Villena, Julio; Agüero, Graciela

    2013-11-01

    We studied the impact of human (HM) and cow (CM) milk on the recovery of blood and bone marrow cells in malnourished mice. Results: both milks normalized serum albumin levels and improved thymus weight. HM was less effective than CM to increase body weight and serum transferrin levels. In contrast, HM was more effective than CM to increase the number of leukocytes and lymphocytes in peripheral blood. Both milks induced an increment in mitotic pool cells in bone marrow and α-naphthyl butyrate esterase positive cells in peripheral blood. They also normalized phagocytic function in blood neutrophils and oxidative burst in peritoneal cells. Conclusion: both milks were equally effective to exert favorable effects on the number of the bone marrow cells and the functions of the blood and peritoneal cells involved in immune response. However, only HM normalized the number of leukocytes and increased the number of neutrophils in peripheral blood.

  12. Vascular Potential of Human Pluripotent Stem Cells

    PubMed Central

    Iacobas, Ionela; Vats, Archana; Hirschi, Karen K.

    2010-01-01

    Cardiovascular disease is the number one cause of death and disability in the US. Understanding the biological activity of stem and progenitor cells, and their ability to contribute to the repair, regeneration and remodeling of the heart and blood vessels affected by pathologic processes is an essential part of the paradigm in enabling us to achieve a reduction in related deaths. Both human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells are promising sources of cells for clinical cardiovascular therapies. Additional in vitro studies are needed, however, to understand their relative phenotypes and molecular regulation toward cardiovascular cell fates. Further studies in translational animal models are also needed to gain insights into the potential and function of both human ES- and iPS-derived cardiovascular cells, and enable translation from experimental and pre-clinical studies to human trials. PMID:20453170

  13. Comparative Proteomic Characterization of 4 Human Liver-Derived Single Cell Culture Models Reveals Significant Variation in the Capacity for Drug Disposition, Bioactivation, and Detoxication

    PubMed Central

    Sison-Young, Rowena L. C.; Mitsa, Dimitra; Jenkins, Rosalind E.; Mottram, David; Alexandre, Eliane; Richert, Lysiane; Aerts, Hélène; Weaver, Richard J.; Jones, Robert P.; Johann, Esther; Hewitt, Philip G.; Ingelman-Sundberg, Magnus; Goldring, Christopher E. P.; Kitteringham, Neil R.; Park, B. Kevin

    2015-01-01

    In vitro preclinical models for the assessment of drug-induced liver injury (DILI) are usually based on cryopreserved primary human hepatocytes (cPHH) or human hepatic tumor-derived cell lines; however, it is unclear how well such cell models reflect the normal function of liver cells. The physiological, pharmacological, and toxicological phenotyping of available cell-based systems is necessary in order to decide the testing purpose for which they are fit. We have therefore undertaken a global proteomic analysis of 3 human-derived hepatic cell lines (HepG2, Upcyte, and HepaRG) in comparison with cPHH with a focus on drug metabolizing enzymes and transport proteins (DMETs), as well as Nrf2-regulated proteins. In total, 4946 proteins were identified, of which 2722 proteins were common across all cell models, including 128 DMETs. Approximately 90% reduction in expression of cytochromes P450 was observed in HepG2 and Upcyte cells, and approximately 60% in HepaRG cells relative to cPHH. Drug transporter expression was also lower compared with cPHH with the exception of MRP3 and P-gp (MDR1) which appeared to be significantly expressed in HepaRG cells. In contrast, a high proportion of Nrf2-regulated proteins were more highly expressed in the cell lines compared with cPHH. The proteomic database derived here will provide a rational basis for the context-specific selection of the most appropriate ‘hepatocyte-like’ cell for the evaluation of particular cellular functions associated with DILI and, at the same time, assist in the construction of a testing paradigm which takes into account the in vivo disposition of a new drug. PMID:26160117

  14. Comparative Proteomic Characterization of 4 Human Liver-Derived Single Cell Culture Models Reveals Significant Variation in the Capacity for Drug Disposition, Bioactivation, and Detoxication.

    PubMed

    Sison-Young, Rowena L C; Mitsa, Dimitra; Jenkins, Rosalind E; Mottram, David; Alexandre, Eliane; Richert, Lysiane; Aerts, Hélène; Weaver, Richard J; Jones, Robert P; Johann, Esther; Hewitt, Philip G; Ingelman-Sundberg, Magnus; Goldring, Christopher E P; Kitteringham, Neil R; Park, B Kevin

    2015-10-01

    In vitro preclinical models for the assessment of drug-induced liver injury (DILI) are usually based on cryopreserved primary human hepatocytes (cPHH) or human hepatic tumor-derived cell lines; however, it is unclear how well such cell models reflect the normal function of liver cells. The physiological, pharmacological, and toxicological phenotyping of available cell-based systems is necessary in order to decide the testing purpose for which they are fit. We have therefore undertaken a global proteomic analysis of 3 human-derived hepatic cell lines (HepG2, Upcyte, and HepaRG) in comparison with cPHH with a focus on drug metabolizing enzymes and transport proteins (DMETs), as well as Nrf2-regulated proteins. In total, 4946 proteins were identified, of which 2722 proteins were common across all cell models, including 128 DMETs. Approximately 90% reduction in expression of cytochromes P450 was observed in HepG2 and Upcyte cells, and approximately 60% in HepaRG cells relative to cPHH. Drug transporter expression was also lower compared with cPHH with the exception of MRP3 and P-gp (MDR1) which appeared to be significantly expressed in HepaRG cells. In contrast, a high proportion of Nrf2-regulated proteins were more highly expressed in the cell lines compared with cPHH. The proteomic database derived here will provide a rational basis for the context-specific selection of the most appropriate 'hepatocyte-like' cell for the evaluation of particular cellular functions associated with DILI and, at the same time, assist in the construction of a testing paradigm which takes into account the in vivo disposition of a new drug.

  15. Ex Vivo Gene Therapy Using Human Mesenchymal Stem Cells to Deliver Growth Factors in the Skeletal Muscle of a Familial ALS Rat Model.

    PubMed

    Suzuki, Masatoshi; Svendsen, Clive N

    2016-01-01

    Therapeutic protein and molecule delivery to target sites by transplanted human stem cells holds great promise for ex vivo gene therapy. Our group has demonstrated the therapeutic benefits of ex vivo gene therapy targeting the skeletal muscles in a transgenic rat model of familial amyotrophic lateral sclerosis (ALS). We used human mesenchymal stem cells (hMSCs) and genetically modified them to release neuroprotective growth factors such as glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF). Intramuscular growth factor delivery via hMSCs can enhance neuromuscular innervation and motor neuron survival in a rat model of ALS (SOD1(G93A) transgenic rats). Here, we describe the protocol of ex vivo delivery of growth factors via lentiviral vector-mediated genetic modification of hMSCs and hMSC transplantation into the skeletal muscle of a familial ALS rat model.

  16. Single-agent cytarabine is insufficient for the treatment of human mantle cell lymphoma in mouse xenograft model.

    PubMed

    Klanova, M; Soukup, T; Molinsky, J; Lateckova, L; Vockova, P; Alam, M; Zivny, J; Trneny, M; Klener, P

    2016-01-01

    Mantle cell lymphoma is an aggressive type of B-cell non-Hodgkin lymphoma with adverse prognosis. It was demonstrated that alternation of CHOP and DHAP chemotherapy improved outcome of mantle cell lymphoma patients. However, which components of DHAP, cisplatin, cytarabine, or both, were responsible for the improved outcome remained unclear. To answer this question, antitumor efficacies of equally toxic doses of cytarabine, cisplatin, and three different combinations were compared in vivo using mouse xenograft models of mantle cell lymphoma. We demonstrated that cisplatin, alone or with cytarabine, is significantly superior to single-agent cytarabine in both eliminating lymphoma cells and suppressing their proliferation rate. PMID:27468882

  17. Cell-specific biotransformation of benzophenone-2 and bisphenol-s in zebrafish and human in vitro models used for toxicity and estrogenicity screening.

    PubMed

    Le Fol, Vincent; Aït-Aïssa, Selim; Cabaton, Nicolas; Dolo, Laurence; Grimaldi, Marina; Balaguer, Patrick; Perdu, Elisabeth; Debrauwer, Laurent; Brion, François; Zalko, Daniel

    2015-03-17

    Several human and fish bioassays have been designed to characterize the toxicity and the estrogenic activity of chemicals. However, their biotransformation capability (bioactivation/detoxification processes) is rarely reported, although this can influence the estrogenic potency of test compounds. The fate of two estrogenic chemicals, the UV filter benzophenone-2 (BP2) and the bisphenol A substitute bisphenol S (BPS) was deciphered in eight human and zebrafish in vitro cell models, encompassing hepatic and mammary cellular contexts. BP2 and BPS were metabolized into a variety of gluco- and sulfo-conjugated metabolites. Similar patterns of BP2 and BPS biotransformation were observed among zebrafish models (primary hepatocytes, ZFL and ZELH-zfER cell lines). Interestingly, metabolic patterns in zebrafish models and in the human hepatic cell line HepaRG shared many similarities, while biotransformation rates in cell lines widely used for estrogenicity testing (MELN and T47D-KBLuc) were quantitatively low and qualitatively different. This study provides new data on the comparative metabolism of BP2 and BPS in human and fish cellular models that will help characterize their metabolic capabilities, and underlines the relevance of using in vitro zebrafish-based bioassays when screening for endocrine disrupting chemicals.

  18. High prevalence of side population in human cancer cell lines

    PubMed Central

    Boesch, Maximilian; Zeimet, Alain G.; Fiegl, Heidi; Wolf, Barbara; Huber, Julia; Klocker, Helmut; Gastl, Guenther

    2016-01-01

    Cancer cell lines are essential platforms for performing cancer research on human cells. We here demonstrate that, across tumor entities, human cancer cell lines harbor minority populations of putative stem-like cells, molecularly defined by dye extrusion resulting in the side population phenotype. These findings establish a heterogeneous nature of human cancer cell lines and argue for their stem cell origin. This should be considered when interpreting research involving these model systems. PMID:27226981

  19. Toward preclinical predictive drug testing for metabolism and hepatotoxicity by using in vitro models derived from human embryonic stem cells and human cell lines - a report on the Vitrocellomics EU-project.

    PubMed

    Mandenius, Carl-Fredrik; Andersson, Tommy B; Alves, Paula M; Batzl-Hartmann, Christine; Björquist, Petter; Carrondo, Manuel J T; Chesne, Christophe; Coecke, Sandra; Edsbagge, Josefina; Fredriksson, J Magnus; Gerlach, Jörg C; Heinzle, Elmar; Ingelman-Sundberg, Magnus; Johansson, Inger; Küppers-Munther, Barbara; Müller-Vieira, Ursula; Noor, Fozia; Zeilinger, Katrin

    2011-05-01

    Drug-induced liver injury is a common reason for drug attrition in late clinical phases, and even for post-launch withdrawals. As a consequence, there is a broad consensus in the pharmaceutical industry, and within regulatory authorities, that a significant improvement of the current in vitro test methodologies for accurate assessment and prediction of such adverse effects is needed. For this purpose, appropriate in vivo-like hepatic in vitro models are necessary, in addition to novel sources of human hepatocytes. In this report, we describe recent and ongoing research toward the use of human embryonic stem cell (hESC)-derived hepatic cells, in conjunction with new and improved test methods, for evaluating drug metabolism and hepatotoxicity. Recent progress on the directed differentiation of human embryonic stem cells to the functional hepatic phenotype is reported, as well as the development and adaptation of bioreactors and toxicity assay technologies for the testing of hepatic cells. The aim of achieving a testing platform for metabolism and hepatotoxicity assessment, based on hESC-derived hepatic cells, has advanced markedly in the last 2-3 years. However, great challenges still remain, before such new test systems could be routinely used by the industry. In particular, we give an overview of results from the Vitrocellomics project (EU Framework 6) and discuss these in relation to the current state-of-the-art and the remaining difficulties, with suggestions on how to proceed before such in vitro systems can be implemented in industrial discovery and development settings and in regulatory acceptance.

  20. Human umbilical cord blood mononuclear cells and chorionic plate-derived mesenchymal stem cells promote axon survival in a rat model of optic nerve crush injury

    PubMed Central

    CHUNG, SOKJOONG; RHO, SEUNGSOO; KIM, GIJIN; KIM, SO-RA; BAEK, KWANG-HYUN; KANG, MYUNGSEO; LEW, HELEN

    2016-01-01

    The use of mesenchymal stem cells (MSCs) in cell therapy in regenerative medicine has great potential, particularly in the treatment of nerve injury. Umbilical cord blood (UCB) reportedly contains stem cells, which have been widely used as a hematopoietic source and may have therapeutic potential for neurological impairment. Although ongoing research is dedicated to the management of traumatic optic nerve injury using various measures, novel therapeutic strategies based on the complex underlying mechanisms responsible for optic nerve injury, such as inflammation and/or ischemia, are required. In the present study, a rat model of optic nerve crush (ONC) injury was established in order to examine the effects of transplanting human chorionic plate-derived MSCs (CP-MSCs) isolated from the placenta, as well as human UCB mononuclear cells (CB-MNCs) on compressed rat optic nerves. Expression markers for inflammation, apoptosis, and optic nerve regeneration were analyzed, as well as the axon survival rate by direct counting. Increased axon survival rates were observed following the injection of CB-MNCs at at 1 week post-transplantation compared with the controls. The levels of growth-associated protein-43 (GAP-43) were increased after the injection of CB-MNCs or CP-MSCs compared with the controls, and the expression levels of hypoxia-inducible factor-1α (HIF-1α) were also significantly increased following the injection of CB-MNCs or CP-MSCs. ERM-like protein (ERMN) and SLIT-ROBO Rho GTPase activating protein 2 (SRGAP2) were found to be expressed in the optic nerves of the CP-MSC-injected rats with ONC injury. The findings of our study suggest that the administration of CB-MNCs or CP-MSCs may promote axon survival through systemic concomitant mechanisms involving GAP-43 and HIF-1α. Taken together, these findings provide further understanding of the mechanisms repsonsible for optic nerve injury and may aid in the development of novel cell-based therapeutic strategies with

  1. Satellite cells in human skeletal muscle plasticity

    PubMed Central

    Snijders, Tim; Nederveen, Joshua P.; McKay, Bryon R.; Joanisse, Sophie; Verdijk, Lex B.; van Loon, Luc J. C.; Parise, Gianni

    2015-01-01

    Skeletal muscle satellite cells are considered to play a crucial role in muscle fiber maintenance, repair and remodeling. Our knowledge of the role of satellite cells in muscle fiber adaptation has traditionally relied on in vitro cell and in vivo animal models. Over the past decade, a genuine effort has been made to translate these results to humans under physiological conditions. Findings from in vivo human studies suggest that satellite cells play a key role in skeletal muscle fiber repair/remodeling in response to exercise. Mounting evidence indicates that aging has a profound impact on the regulation of satellite cells in human skeletal muscle. Yet, the precise role of satellite cells in the development of muscle fiber atrophy with age remains unresolved. This review seeks to integrate recent results from in vivo human studies on satellite cell function in muscle fiber repair/remodeling in the wider context of satellite cell biology whose literature is largely based on animal and cell models. PMID:26557092

  2. Human Factors Model

    NASA Technical Reports Server (NTRS)

    1993-01-01

    Jack is an advanced human factors software package that provides a three dimensional model for predicting how a human will interact with a given system or environment. It can be used for a broad range of computer-aided design applications. Jack was developed by the computer Graphics Research Laboratory of the University of Pennsylvania with assistance from NASA's Johnson Space Center, Ames Research Center and the Army. It is the University's first commercial product. Jack is still used for academic purposes at the University of Pennsylvania. Commercial rights were given to Transom Technologies, Inc.

  3. Early Developmental Perturbations in a Human Stem Cell Model of MODY5/HNF1B Pancreatic Hypoplasia

    PubMed Central

    Teo, Adrian Kee Keong; Lau, Hwee Hui; Valdez, Ivan Achel; Dirice, Ercument; Tjora, Erling; Raeder, Helge; Kulkarni, Rohit N.

    2016-01-01

    Summary Patients with an HNF1BS148L/+ mutation (MODY5) typically exhibit pancreatic hypoplasia. However, the molecular mechanisms are unknown due to inaccessibility of patient material and because mouse models do not fully recapitulate MODY5. Here, we differentiated MODY5 human-induced pluripotent stem cells (hiPSCs) into pancreatic progenitors, and show that the HNF1BS148L/+ mutation causes a compensatory increase in several pancreatic transcription factors, and surprisingly, a decrease in PAX6 pancreatic gene expression. The lack of suppression of PDX1, PTF1A, GATA4, and GATA6 indicates that MODY5-mediated pancreatic hypoplasia is mechanistically independent. Overexpression studies demonstrate that a compensatory increase in PDX1 gene expression is due to mutant HNF1BS148L/+ but not wild-type HNF1B or HNF1A. Furthermore, HNF1B does not appear to directly regulate PAX6 gene expression necessary for glucose tolerance. Our results demonstrate compensatory mechanisms in the pancreatic transcription factor network due to mutant HNF1BS148L/+ protein. Thus, patients typically develop MODY5 but not neonatal diabetes despite exhibiting pancreatic hypoplasia. PMID:26876668

  4. Nutraceutical Improvement Increases the Protective Activity of Broccoli Sprout Juice in a Human Intestinal Cell Model of Gut Inflammation

    PubMed Central

    Ferruzza, Simonetta; Natella, Fausta; Ranaldi, Giulia; Murgia, Chiara; Rossi, Carlotta; Trošt, Kajetan; Mattivi, Fulvio; Nardini, Mirella; Maldini, Mariateresa; Giusti, Anna Maria; Moneta, Elisabetta; Scaccini, Cristina; Sambuy, Yula; Morelli, Giorgio; Baima, Simona

    2016-01-01

    Benefits to health from a high consumption of fruits and vegetables are well established and have been attributed to bioactive secondary metabolites present in edible plants. However, the effects of specific health-related phytochemicals within a complex food matrix are difficult to assess. In an attempt to address this problem, we have used elicitation to improve the nutraceutical content of seedlings of Brassica oleracea grown under controlled conditions. Analysis, by LC-MS, of the glucosinolate, isothiocyanate and phenolic compound content of juices obtained from sprouts indicated that elicitation induces an enrichment of several phenolics, particularly of the anthocyanin fraction. To test the biological activity of basal and enriched juices we took advantage of a recently developed in vitro model of inflamed human intestinal epithelium. Both sprouts’ juices protected intestinal barrier integrity in Caco-2 cells exposed to tumor necrosis factor α under marginal zinc deprivation, with the enriched juice showing higher protection. Multivariate regression analysis indicated that the extent of rescue from stress-induced epithelial dysfunction correlated with the composition in bioactive molecules of the juices and, in particular, with a group of phenolic compounds, including several anthocyanins, quercetin-3-Glc, cryptochlorogenic, neochlorogenic and cinnamic acids. PMID:27529258

  5. Nutraceutical Improvement Increases the Protective Activity of Broccoli Sprout Juice in a Human Intestinal Cell Model of Gut Inflammation.

    PubMed

    Ferruzza, Simonetta; Natella, Fausta; Ranaldi, Giulia; Murgia, Chiara; Rossi, Carlotta; Trošt, Kajetan; Mattivi, Fulvio; Nardini, Mirella; Maldini, Mariateresa; Giusti, Anna Maria; Moneta, Elisabetta; Scaccini, Cristina; Sambuy, Yula; Morelli, Giorgio; Baima, Simona

    2016-01-01

    Benefits to health from a high consumption of fruits and vegetables are well established and have been attributed to bioactive secondary metabolites present in edible plants. However, the effects of specific health-related phytochemicals within a complex food matrix are difficult to assess. In an attempt to address this problem, we have used elicitation to improve the nutraceutical content of seedlings of Brassica oleracea grown under controlled conditions. Analysis, by LC-MS, of the glucosinolate, isothiocyanate and phenolic compound content of juices obtained from sprouts indicated that elicitation induces an enrichment of several phenolics, particularly of the anthocyanin fraction. To test the biological activity of basal and enriched juices we took advantage of a recently developed in vitro model of inflamed human intestinal epithelium. Both sprouts' juices protected intestinal barrier integrity in Caco-2 cells exposed to tumor necrosis factor α under marginal zinc deprivation, with the enriched juice showing higher protection. Multivariate regression analysis indicated that the extent of rescue from stress-induced epithelial dysfunction correlated with the composition in bioactive molecules of the juices and, in particular, with a group of phenolic compounds, including several anthocyanins, quercetin-3-Glc, cryptochlorogenic, neochlorogenic and cinnamic acids. PMID:27529258

  6. Conditioned medium from the stem cells of human dental pulp improves cognitive function in a mouse model of Alzheimer's disease.

    PubMed

    Mita, Tsuneyuki; Furukawa-Hibi, Yoko; Takeuchi, Hideyuki; Hattori, Hisashi; Yamada, Kiyofumi; Hibi, Hideharu; Ueda, Minoru; Yamamoto, Akihito

    2015-10-15

    Alzheimer's disease (AD) is a progressive, neurodegenerative disease characterized by a decline in cognitive abilities and the appearance of β-amyloid plaques in the brain. Although the pathogenic mechanisms associated with AD are not fully understood, activated microglia releasing various neurotoxic factors, including pro-inflammatory cytokines and oxidative stress mediators, appear to play major roles. Here, we investigated the therapeutic benefits of a serum-free conditioned medium (CM) derived from the stem cells of human exfoliated deciduous teeth (SHEDs) in a mouse model of AD. The intranasal administration of SHEDs in these mice resulted in substantially improved cognitive function. SHED-CM contained factors involved in multiple neuroregenerative mechanisms, such as neuroprotection, axonal elongation, neurotransmission, the suppression of inflammation, and microglial regulation. Notably, SHED-CM attenuated the pro-inflammatory responses induced by β-amyloid plaques, and generated an anti-inflammatory/tissue-regenerating environment, which was accompanied by the induction of anti-inflammatory M2-like microglia. Our data suggest that SHED-CM may provide significant therapeutic benefits for AD.

  7. Tropomyosin heterogeneity in human cells

    SciTech Connect

    Giometti, C.S.; Anderson, N.L.

    1984-11-25

    Tropomyosin preparations from human platelets, human peripheral blood leukocytes from normal individuals and from a patient with chronic lymphocytic leukemia, human lymphoblastoid cells (GM607), human epithelial cells, and human skin fibroblasts have all been found to contain more than one protein when analyzed by two-dimensional gel electrophoresis. Although the lymphoid cell preparations consistently contain two proteins of almost identical molecular weight (M/sub r/ = 30,000), the platelet, epithelial cell, and fibroblast preparations contain two or more major proteins with molecular weights between 31,000 and 36,000, in addition to a major protein at 30,000. All of these proteins have characteristics in common with tropomyosin including slightly acidic isoelectric point, stability to heat and organic solvents, association with the cytoskeleton, and reactivity with antibody against skeletal muscle tropomyosin. The nonmuscle tropomyosin-like proteins were compared with tropomyosins from human skeletal, cardiac, and smooth muscle by peptide mapping after partial proteolysis. The results showed one of the nonmuscle proteins to be identical to the major smooth muscle tropomyosin in human uterus (myometrium) and another to be similar but not identical to skeletal muscle ..cap alpha..-tropomyosin. The remainder of the proteins with tropomyosin characteristics was unique to nonmuscle cells. In all, nine distinct human proteins with characteristics of tropomyosin are described. Charge variants of two of these proteins have been described previously. 43 references, 7 figures, 2 tables.

  8. Advanced flow cytometric analysis of nanoparticle targeting to rare leukemic stem cells in peripheral human blood in a defined model system

    NASA Astrophysics Data System (ADS)

    Cooper, Christy L.; Leary, James F.

    2015-03-01

    Leukemia stem cells are both stem-like and leukemic-like. This complicates their detection as rare circulating tumor cells in the peripheral blood of leukemia patients. Since leukemic stem cells are also resistant to standard chemotherapeutic regimens, new therapeutic strategies need to be designed to kill the leukemic stem cells without killing normal stem cells. In these initial targeting studies we utilized a bioinformatics approach to design an antibodyfluorescent nanoparticle conjugate for targeting to these leukemic stem cells and to minimize targeting to normal stemprogenitor cells. Multicolor flow cytometric analyses were performed on a BD FACS Aria III. Human leukemic stem cell-like cell RS4;11 (with putative immunophenotype CD133+/CD24+/-, CD34+/-, CD38+, CD10-/Flt3+) was spiked into normal hematopoietic stem-progenitor cells obtained from a "buffy coat" prep (with putative immunophenotype CD133- /CD34+/CD38-/CD10-/Flt-3-) to be used as a model human leukemia patient. To analyze the model system, digital data mixtures of the two cell types were first created and assigned classifiers in order to create truth sets. ROC (Receiver Operating Characteristic) and multidimensional cluster analyses were used to evaluate the specificity and sensitivity of the immunophenotyping panel and for automated cell population identification, respectively. Costs of misclassification (false targeting) were also accounted for by this analysis scheme. Ultimately, this analysis scheme will be applied to use of nanoparticle-antibody conjugates at therapeutic doses for targeted killing of leukemia stem cells preferentially to normal stem -progenitor cells.

  9. Intrastriatal transplantation of neurotrophic factor-secreting human mesenchymal stem cells improves motor function and extends survival in R6/2 transgenic mouse model for Huntington's disease

    PubMed Central

    Sadan, Ofer; Melamed, Eldad; Offen, Daniel

    2012-01-01

    Stem cell-based treatment for Huntington's disease (HD) is an expanding field of research. Although various stem cells have been shown to be beneficial in vivo, no long standing clinical effect has been demonstrated. To address this issue, we are developing a stem cell-based therapy designed to improve the microenvironment of the diseased tissue via delivery of neurotrophic factors (NTFs). Previously, we established that bone marrow derived human mesenchymal stem cells (MSCs) can be differentiated using medium based cues into NTF-secreting cells (NTF+ cells) that express astrocytic markers. NTF+ cells were shown to alleviate neurodegeneration symptoms in several disease models in vitro and in vivo, including the model for excitotoxicity. In the present study, we explored if the timing of intrastriatal transplantation of hNTF+ cells into the R6/2 transgenic mouse model for HD influences motor function and survival. One hundred thousand cells were transplanted bilaterally into the striatum of immune-suppressed mice at 4.5, 5.5 and 6.5 weeks of age. Contrary to our expectations, early transplantation of NTF+ cells did not improve motor function or overall survival. However, late (6.5 weeks) transplantation resulted in a temporary improvement in motor function and an extension of life span relative to that observed for PBS treated mice. We conclude that late transplantation of NTF+ cells induces a beneficial effect in this transgenic model for HD. Since no transplanted NTF+ cells could be detected in vivo, we suspect that the temporary nature of the beneficial effect is due to poor survival of transplanted cells. In general, we submit that NTF+ cells should be further evaluated for the therapy of HD. PMID:22953237

  10. Glutathione metabolism in the HaCaT cell line as a model for the detoxification of the model sensitisers 2,4-dinitrohalobenzenes in human skin.

    PubMed

    Jacquoilleot, Sandrine; Sheffield, David; Olayanju, Adedamola; Sison-Young, Rowena; Kitteringham, Neil R; Naisbitt, Dean J; Aleksic, Maja

    2015-08-19

    Glutathione (GSH) is the most prominent antioxidant in cells and the co-factor of an important set of enzymes involved in the skin metabolic clearance system, glutathione S-transferases (GST). Here, we describe an LC-MS (liquid chromatography-mass spectroscopy) method to measure GSH and its disulfide form (GSSG) in HaCaT cells and a 3D Reconstructed Human Epidermis (RHE) model. In our assay, the basal level of GSH in both systems was in the low nmol/mg soluble protein range, while the level of GSSG was systematically below our limit of quantification (0.1 μM). We found that 2,4-dinitrohalobenzenes deplete the GSH present in HaCaT cells within the first hour of exposure, in a dose dependent manner. The level of GSH in HaCaT cells treated with a single non-toxic dose of 10 μM of dinitrohalobenzene was also shown to increase after two hours. While cells treated with 1-chloro-2,4-dinitrobenzene (DNCB) and 1-fluoro-2,4-dinitrobenzene (DNFB) repleted GSH to levels similar to untreated control cells within 24h, 1-bromo-2,4-dinitrobenzene (DNBB) seemed to prevent such a repletion and appeared to be the most toxic compound in all assays. A mathematical modelling of experimental results was performed to further rationalise the differences observed between test chemicals. For this purpose the biological phenomena observed were simplified into two sequential events: the initial depletion of the GSH stock after chemical treatment followed by the repletion of the GSH once the chemical was cleared. Activation of the nuclear factor E2-related factor 2 (Nrf2) pathway was observed with all compounds within two hours, and at concentrations less than 10 μM. These data show that GSH depletion and repletion occur rapidly in skin cells and emphasize the importance of conducting kinetic studies when performing in vitro experiments exploring skin sensitization. PMID:26022718

  11. Mesenchymal stem cell like (MSCl) cells generated from human embryonic stem cells support pluripotent cell growth

    SciTech Connect

    Varga, Nora; Vereb, Zoltan; Rajnavoelgyi, Eva; Nemet, Katalin; Uher, Ferenc; Sarkadi, Balazs; Apati, Agota

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer MSC like cells were derived from hESC by a simple and reproducible method. Black-Right-Pointing-Pointer Differentiation and immunosuppressive features of MSCl cells were similar to bmMSC. Black-Right-Pointing-Pointer MSCl cells as feeder cells support the undifferentiated growth of hESC. -- Abstract: Mesenchymal stem cell like (MSCl) cells were generated from human embryonic stem cells (hESC) through embryoid body formation, and isolated by adherence to plastic surface. MSCl cell lines could be propagated without changes in morphological or functional characteristics for more than 15 passages. These cells, as well as their fluorescent protein expressing stable derivatives, efficiently supported the growth of undifferentiated human embryonic stem cells as feeder cells. The MSCl cells did not express the embryonic (Oct4, Nanog, ABCG2, PODXL, or SSEA4), or hematopoietic (CD34, CD45, CD14, CD133, HLA-DR) stem cell markers, while were positive for the characteristic cell surface markers of MSCs (CD44, CD73, CD90, CD105). MSCl cells could be differentiated toward osteogenic, chondrogenic or adipogenic directions and exhibited significant inhibition of mitogen-activated lymphocyte proliferation, and thus presented immunosuppressive features. We suggest that cultured MSCl cells can properly model human MSCs and be applied as efficient feeders in hESC cultures.

  12. Assessing the Cytotoxicity of Black Carbon As A Model for Ultrafine Anthropogenic Aerosol Across Human and Murine Cells: A Chronic Exposure Model of Nanosized Particulate Matter

    NASA Astrophysics Data System (ADS)

    Salinas, E.

    2015-12-01

    Combustion-derived nanomaterials or ultrafine (<1 μm) atmospheric aerosols are primarily products of anthropogenic activities, such as the burning of fossil fuels. Ultrafine particles (UFPs) can absorb other noxious pollutants including volatile organic compounds (VOCs), polycyclic aromatic hydrocarbons (PAHs), toxic organic compounds, and heavy metals. The combination of high population density, meteorological conditions, and industrial productivity brings high levels of air pollution to the metropolitan area of El Paso, Texas, USA/ Ciudad Juarez, Chihuahua, Mexico, comprising the Paso del Norte air basin. A study conducted by scientists from the Research Triangle Park in North Carolina, analyzed sites adjacent to heavy-traffic highways in El Paso and elucidated higher UFP concentrations in comparison to previously published work exploring pollution and adverse health effects in the basin. UFPs can penetrate deep into the alveolar sacs of the lung, reaching distant alveolar sacs and inducing a series of immune responses that are detrimental to the body: evidence suggests that UFPs can also cross the alveolar-blood barrier and potentially endanger the body's immune response. The physical properties of UFPs and the dynamics of local atmospheric and topographical conditions indicate that emissions of nanosized carbonaceous aerosols could pose significant threats to biological tissues upon inhalation by local residents of the Paso del Norte. This study utilizes Black Carbon (BC) as a model for environmental UFPs and its effects on the immunological response. An in vitro approach is used to measure the ability of BC to promote cell death upon long-term exposure. Human epithelial lung cells (A549), human peripheral-blood monocytes (THP-1), murine macrophages (RAW264.7), and murine epithelial lung cells (LA-4) were treated with BC and assessed for metabolic activity after chronic exposure utilizing three distinct and independent cell viability assays. The cell viability

  13. SV40 replication in human mesothelial cells induces HGF/Met receptor activation: A model for viral-related carcinogenesis of human malignant mesothelioma

    PubMed Central

    Cacciotti, Paola; Libener, Roberta; Betta, Piergiacomo; Martini, Fernanda; Porta, Camillo; Procopio, Antonio; Strizzi, Luigi; Penengo, Lorenza; Tognon, Mauro; Mutti, Luciano; Gaudino, Giovanni

    2001-01-01

    Recent studies suggested that simian virus 40 (SV40) may cause malignant mesothelioma, although the pathogenic mechanism is unclear. We found that in SV40-positive malignant mesothelioma cells, the hepatocyte growth factor (HGF) receptor (Met) was activated. In human mesothelial cells (HMC) transfected with full-length SV40 DNA (SV40-HMC), Met receptor activation was associated with S-phase entry, acquisition of a fibroblastoid morphology, and the assembly of viral particles. Coculture experiments revealed the ability of SV40-HMC to infect permissive monkey cells (CV-1), HMC, and murine BNL CL cells. Cocultured human and murine SV40-positive cells expressed HGF, showed Met tyrosine phosphorylation and S-phase entry, and acquired a spindle-shaped morphology (spBNL), whereas CV-1 cells were lysed. Cocultured HMC inherited from SV40-HMC the infectivity, as they induced lysis in cocultured CV-1 cells. Treatment with suramin or HGF-blocking antibodies inhibited Met tyrosine phosphorylation in all large T antigen (Tag)-positive cells and reverted the spindle-shaped morphology of spBNL. This finding indicated that Met activation and subsequent biological effects were mediated by an autocrine HGF circuit. This, in turn, was causally related to Tag expression, being induced by transfection with the SV40 early region alone. Our findings suggest that when SV40 infects HMC it causes Met activation via an autocrine loop. Furthermore, SV40 replicates in HMC and infects the adjacent HMC, inducing an HGF-dependent Met activation and cell-cycle progression into S phase. This may explain how a limited number of SV40-positive cells may be sufficient to direct noninfected HMC toward malignant transformation. PMID:11572935

  14. Thymic and Postthymic Regulation of Naïve CD4+ T-Cell Lineage Fates in Humans and Mice Models

    PubMed Central

    Belizário, José E.; Brandão, Wesley; Rossato, Cristiano; Peron, Jean Pierre

    2016-01-01

    Our understanding of how thymocytes differentiate into many subtypes has been increased progressively in its complexity. At early life, the thymus provides a suitable microenvironment with specific combination of stromal cells, growth factors, cytokines, and chemokines to induce the bone marrow lymphoid progenitor T-cell precursors into single-positive CD4+ and CD8+ T effectors and CD4+CD25+ T-regulatory cells (Tregs). At postthymic compartments, the CD4+ T-cells acquire distinct phenotypes which include the classical T-helper 1 (Th1), T-helper 2 (Th2), T-helper 9 (Th9), T-helper 17 (Th17), follicular helper T-cell (Tfh), and induced T-regulatory cells (iTregs), such as the regulatory type 1 cells (Tr1) and transforming growth factor-β- (TGF-β-) producing CD4+ T-cells (Th3). Tregs represent only a small fraction, 5–10% in mice and 1-2% in humans, of the overall CD4+ T-cells in lymphoid tissues but are essential for immunoregulatory circuits mediating the inhibition and expansion of all lineages of T-cells. In this paper, we first provide an overview of the major cell-intrinsic developmental programs that regulate T-cell lineage fates in thymus and periphery. Next, we introduce the SV40 immortomouse as a relevant mice model for implementation of new approaches to investigate thymus organogenesis, CD4 and CD8 development, and thymus cells tumorogenesis. PMID:27313405

  15. Myeloid Dysregulation in a Human Induced Pluripotent Stem Cell Model of PTPN11-Associated Juvenile Myelomonocytic Leukemia.

    PubMed

    Mulero-Navarro, Sonia; Sevilla, Ana; Roman, Angel C; Lee, Dung-Fang; D'Souza, Sunita L; Pardo, Sherly; Riess, Ilan; Su, Jie; Cohen, Ninette; Schaniel, Christoph; Rodriguez, Nelson A; Baccarini, Alessia; Brown, Brian D; Cavé, Hélène; Caye, Aurélie; Strullu, Marion; Yalcin, Safak; Park, Christopher Y; Dhandapany, Perundurai S; Yongchao, Ge; Edelmann, Lisa; Bahieg, Sawsan; Raynal, Patrick; Flex, Elisabetta; Tartaglia, Marco; Moore, Kateri A; Lemischka, Ihor R; Gelb, Bruce D

    2015-10-20

    Somatic PTPN11 mutations cause juvenile myelomonocytic leukemia (JMML). Germline PTPN11 defects cause Noonan syndrome (NS), and specific inherited mutations cause NS/JMML. Here, we report that hematopoietic cells differentiated from human induced pluripotent stem cells (hiPSCs) harboring NS/JMML-causing PTPN11 mutations recapitulated JMML features. hiPSC-derived NS/JMML myeloid cells exhibited increased signaling through STAT5 and upregulation of miR-223 and miR-15a. Similarly, miR-223 and miR-15a were upregulated in 11/19 JMML bone marrow mononuclear cells harboring PTPN11 mutations, but not those without PTPN11 defects. Reducing miR-223's function in NS/JMML hiPSCs normalized myelogenesis. MicroRNA target gene expression levels were reduced in hiPSC-derived myeloid cells as well as in JMML cells with PTPN11 mutations. Thus, studying an inherited human cancer syndrome with hiPSCs illuminated early oncogenesis prior to the accumulation of secondary genomic alterations, enabling us to discover microRNA dysregulation, establishing a genotype-phenotype association for JMML and providing therapeutic targets. PMID:26456833

  16. Mancozeb, a fungicide routinely used in agriculture, worsens nonalcoholic fatty liver disease in the human HepG2 cell model.

    PubMed

    Pirozzi, Anna Virginia Adriana; Stellavato, Antonietta; La Gatta, Annalisa; Lamberti, Monica; Schiraldi, Chiara

    2016-05-13

    Mancozeb, a manganese/zinc ethylene-bis-dithiocarbamate, is a fungicide routinely used in pest control programs. However, it has been found to have deleterious effects on human health and on the environment. Indeed, its massive use has raised the issue of possible health risks for agrarian communities; the molecule can also reach human cells via the food chain and alter metabolism, endocrine activity and cell survival. In particular, mancozeb induces many toxic effects on hepatic cell metabolism. For this reason, we investigated its effect in an in vitro model of hepatic damage, namely fatty acid-induced nonalcoholic fatty liver disease in the HepG2 cell line. We found that the hepatic toxicity of the fungicide exacerbated fatty acid-induced steatosis, as manifested by an increase in intracellular lipid droplet accumulation. Furthermore, mancozeb altered cell metabolism and induced cell death through upregulation of lactate dehydrogenase and cytochrome c, respectively, in dose-dependent manners. Therefore, mancozeb may play an important role in the pathogenesis and progression of chronic disease in humans and represents a danger for human health in high doses. PMID:27016407

  17. C/EBP-β Regulates Endoplasmic Reticulum Stress–Triggered Cell Death in Mouse and Human Models

    PubMed Central

    Meir, Ofir; Dvash, Efrat; Werman, Ariel; Rubinstein, Menachem

    2010-01-01

    Endoplasmic reticulum (ER) stress elicits the unfolded protein response (UPR), initially aimed at coping with the stress, but triggering cell death upon further stress. ER stress induces the C/EBP-® variant Liver-enriched Activating Protein (LAP), followed by the dominant-negative variant, Liver Inhibitory Protein (LIP). However, the distinct role of LAP and LIP in ER stress is unknown. We found that the kinetics of the ER stress-induced expression of LIP overlapped with that of the cell death in mouse B16 melanoma cells. Furthermore, inducible over-expression of LIP augmented ER stress-triggered cell death whereas over-expression of LAP attenuated cell death. Similar results were obtained in human 293T cells. Limited vasculature in tumors triggers hypoxia, nutrient shortage and accumulation of toxic metabolites, all of which eliciting continuous ER stress. We found that LAP promoted and LIP inhibited B16 melanoma tumor progression without affecting angiogenesis or accelerating the cell cycle. Rather, LAP attenuated, whereas LIP augmented tumor ER stress. We therefore suggest that C/EBP-® regulates the transition from the protective to the death–promoting phase of the UPR. We further suggest that the over-expression of LAP observed in many solid tumors promotes tumor progression by attenuating ER stress–triggered tumor cell death. PMID:20209087

  18. Mesenchymal stromal cells from the human placenta promote neovascularization in a mouse model in vivo.

    PubMed

    Kinzer, M; Hingerl, K; König, J; Reinisch, A; Strunk, D; Huppertz, B; Lang, I

    2014-07-01

    Cell transplantation is a promising strategy in regenerative medicine for revascularization of ischemic tissues. Based on our observation that placental mesenchymal stromal cells (PMSC) enhance endothelial cell viability in vitro via secretion of angiogenic factors, we asked whether PMSC support vascular growth in vivo. PMSC were isolated from amnion and placental endothelial cells (PLEC) from chorion and either separately or co-transplanted subcutaneously into immune-deficient mice. Co-transplantation resulted in a higher number of perfused human vessels (CD31+/vimentin+) containing mouse glycophorin A+ erythrocytes. Results indicate positive effects of PMSC on neovascularization in vivo, making them attractive candidates to create autologous PMSC/PLEC pairs for research and transplantation.

  19. Electrophoretic separation and analysis of living cells from solid tissues by several methods - Human embryonic kidney cell cultures as a model

    NASA Technical Reports Server (NTRS)

    Todd, Paul; Plank, Lindsay D.; Kunze, M. Elaine; Lewis, Marian L.; Morrison, Dennis R.

    1986-01-01

    The use of free-fluid electrophoresis methods to separate tissue cells having a specific function is discussed. It is shown that cells suspended by trypsinization from cultures of human embryonic kidney are electrophoretically heterogeneous and tolerate a wide range of electrophoresis buffers and conditions without significant attenuation of function. Moreover, these cells do not separate electrophoretically on the basis of size or cell position alone and can be separated according to their ability to give rise to progeny that produce specific plasminogen activators.

  20. In vitro study of biodegradation of a Co-Cr alloy using a human cell culture model.

    PubMed

    Harmand, M F

    1995-01-01

    The evaluation of a potential biomaterial is based on two approaches: firstly, the study of the local and systemic effects of the biomaterial implanted in the host; and secondly the study of the behaviour of the biomaterial itself with increasing time. The progress achieved in human cell culturing allows in vitro evaluation of a new biomaterial using the human cell(s) system(s) characteristic of the tissue which it will be exposed to in vivo. This kind of approach permits the assessment of the biodegradation of a biomaterial whatever it is: metal; alloy; ceramic; glass; polymer; with or without specialized coating.... The experimental approach is as follows: discs representative of the biomaterial (surface state, cleaning, sterilization process) are manufactured in order to cover the bottom of the culture wells. Thereafter, they are either brought in the presence of complete culture medium alone, or in the presence of a subconfluent cell layer. A kinetic analysis is performed using various incubation periods at 37 degrees C. Released biodegradation products are identified and quantified, in both the medium and cell compartment, and on the other hand cytotoxicity is assessed. A Co-Cr alloy was studied over a 9-day period according to the experimental schedule, and showed a higher corrosion rate in the presence of osteoblasts in the range of 25-30%. Moreover, an intracellular uptake of both Cr and Co was detected, which will have physiological importance. PMID:7772567

  1. Intravenous Administration of Human Umbilical Cord Blood-Derived AC133+ Endothelial Progenitor Cells in Rat Stroke Model Reduces Infarct Volume: Magnetic Resonance Imaging and Histological Findings

    PubMed Central

    Iskander, Asm; Knight, Robert A.; Zhang, Zheng Gang; Ewing, James R.; Shankar, Adarsh; Varma, Nadimpalli Ravi S.; Bagher-Ebadian, Hassan; Ali, Meser M.; Arbab, Ali S.

    2013-01-01

    Abstract Endothelial progenitor cells (EPCs) hold enormous therapeutic potential for ischemic vascular diseases. Previous studies have indicated that stem/progenitor cells derived from human umbilical cord blood (hUCB) improve functional recovery in stroke models. Here, we examined the effect of hUCB AC133+ EPCs on stroke development and resolution in a middle cerebral artery occlusion (MCAo) rat model. Since the success of cell therapies strongly depends on the ability to monitor in vivo the migration of transplanted cells, we also assessed the capacity of magnetic resonance imaging (MRI) to track in vivo the magnetically labeled cells that were administered. Animals were subjected to transient MCAo and 24 hours later injected intravenously with 107 hUCB AC133+ EPCs. MRI performed at days 1, 7, and 14 after the insult showed accumulation of transplanted cells in stroke-affected hemispheres and revealed that stroke volume decreased at a significantly higher rate in cell-treated animals. Immunohistochemistry analysis of brain tissues localized the administered cells in the stroke-affected hemispheres only and indicated that these cells may have significantly affected the magnitude of endogenous proliferation, angiogenesis, and neurogenesis. We conclude that transplanted cells selectively migrated to the ischemic brain parenchyma, where they exerted a therapeutic effect on the extent of tissue damage, regeneration, and time course of stroke resolution. PMID:23934909

  2. Sensory neurons do not induce motor neuron loss in a human stem cell model of spinal muscular atrophy.

    PubMed

    Schwab, Andrew J; Ebert, Allison D

    2014-01-01

    Spinal muscular atrophy (SMA) is an autosomal recessive disorder leading to paralysis and early death due to reduced SMN protein. It is unclear why there is such a profound motor neuron loss, but recent evidence from fly and mouse studies indicate that cells comprising the whole sensory-motor circuit may contribute to motor neuron dysfunction and loss. Here, we used induced pluripotent stem cells derived from SMA patients to test whether sensory neurons directly contribute to motor neuron loss. We generated sensory neurons from SMA induced pluripotent stem cells and found no difference in neuron generation or survival, although there was a reduced calcium response to depolarizing stimuli. Using co-culture of SMA induced pluripotent stem cell derived sensory neurons with control induced pluripotent stem cell derived motor neurons, we found no significant reduction in motor neuron number or glutamate transporter boutons on motor neuron cell bodies or neurites. We conclude that SMA sensory neurons do not overtly contribute to motor neuron loss in this human stem cell system.

  3. The bovine model for elucidating the role of γδ T cells in controlling infectious diseases of importance to cattle and humans.

    PubMed

    Baldwin, Cynthia L; Telfer, Janice C

    2015-07-01

    There are several instances of co-investigation and related discoveries and achievements in bovine and human immunology; perhaps most interesting is the development of the BCG vaccine, the tuberculin skin test and the more recent interferon-gamma test that were developed first in cattle to prevent and diagnosis bovine tuberculosis and then applied to humans. There are also a number of immune-physiological traits that ruminant share with humans including the development of their immune systems in utero which increases the utility of cattle as a model for human immunology. These are reviewed here with a particular focus on the use of cattle to unravel γδ T cell biology. Based on the sheer number of γδ T cells in this γδ T cell high species, it is reasonable to expect γδ T cells to play an important role in protective immune responses. For that reason alone cattle may provide good models for elucidating at least some of the roles γδ T cells play in protective immunity in all species. This includes fundamental research on γδ T cells as well as the responses of ruminant γδ T cells to a variety of infectious disease situations including to protozoan and bacterial pathogens. The role that pattern recognition receptors (PRR) play in the activation of γδ T cells may be unique relative to αβ T cells. Here we focus on that of the γδ T cell specific family of molecules known as WC1 or T19 in ruminants, which are part of the CD163 scavenger receptor cysteine rich (SRCR) family that includes SCART1 and SCART2 expressed on murine γδ T cells. We review the evidence for WC1 being a PRR as well as an activating co-receptor and the role that γδ T cells bearing these receptors play in immunity to leptospirosis and tuberculosis. This includes the generation of memory responses to vaccines, thereby continuing the tradition of co-discovery between cattle and humans.

  4. Co-transplantation of human hematopoietic stem cells and human breast cancer cells in NSG mice

    PubMed Central

    Wege, Anja K; Schmidt, Marcus; Ueberham, Elke; Ponnath, Marvin; Ortmann, Olaf; Brockhoff, Gero; Lehmann, Jörg

    2014-01-01

    Humanized tumor mice (HTM) were generated by the co-transplantation of human hematopoietic stem cells and human breast cancer cells overexpressing HER2 into neonatal NOD-scid IL2Rγnull (NSG) mice. These mice are characterized by the development of a human immune system in combination with human breast cancer growth. Due to concurrent transplantation into newborn mice, transfer of MHC-mismatched tumor cells resulted in solid coexistence and immune cell activation (CD4+ T cells, natural killer cells, and myeloid cells), but without evidence for rejection. Histological staining of the spleen of HTM revealed co-localization of human antigen-presenting cells together with human T and B cells allowing MHC-dependent interaction, and thereby the generation of T cell-dependent antibody production. Here, we investigated the capability of these mice to generate human tumor-specific antibodies and correlated immunoglobulin titers with tumor outgrowth. We found detectable IgM and also IgG amounts in the serum of HTM, which apparently controlled tumor development when IgG serum concentrations were above 10 µg/ml. Western blot analyses revealed that the tumor-specific antibodies generated in HTM did not recognize HER2/neu antigens, but different, possibly relevant antigens for breast cancer therapy. In conclusion, HTM offer a novel approach to generate complete human monoclonal antibodies that do not require further genetic manipulation (e. g., humanization) for a potential application in humans. In addition, efficacy and safety of the generated antibodies can be tested in the same mouse model under human-like conditions. This might be of particular interest for cancer subtypes with no currently available antibody therapy. PMID:24870377

  5. Species-specific vesicular monoamine transporter 2 (VMAT2) expression in mammalian pancreatic beta cells: implications for optimising radioligand-based human beta cell mass (BCM) imaging in animal models

    PubMed Central

    Hartwig, N. R.; Kalmbach, N.; Klietz, M.; Anlauf, M.; Eiden, L. E.; Weihe, E.

    2014-01-01

    Aims/hypothesis Imaging of beta cell mass (BCM) is a major challenge in diabetes research. The vesicular monoamine transporter 2 (VMAT2) is abundantly expressed in human beta cells. Radiolabelled analogues of tetrabenazine (TBZ; a low-molecular-weight, cell-permeant VMAT2-selective ligand) have been employed for pancreatic islet imaging in humans. Since reports on TBZ-based VMAT2 imaging in rodent pancreas have been fraught with confusion, we compared VMAT2 gene expression patterns in the mouse, rat, pig and human pancreas, to identify appropriate animal models with which to further validate and optimise TBZ imaging in humans. Methods We used a panel of highly sensitive VMAT2 antibodies developed against equivalently antigenic regions of the transporter from each species in combination with immunostaining for insulin and species-specific in situ hybridisation probes. Individual pancreatic islets were obtained by laser-capture microdissection and subjected to analysis of mRNA expression of VMAT2. Results The VMAT2 protein was not expressed in beta cells in the adult pancreas of common mouse or rat laboratory strains, in contrast to its expression in beta cells (but not other pancreatic endocrine cell types) in the pancreas of pigs and humans. VMAT2- and tyrosine hydroxylase co-positive (catecholaminergic) innervation was less abundant in humans than in rodents. VMAT2-positive mast cells were identified in the pancreas of all species. Conclusions/interpretation Primates and pigs are suitable models for TBZ imaging of beta cells. Rodents, because of a complete lack of VMAT2 expression in the endocrine pancreas, are a ‘null’ model for assessing interference with BCM measurements by VMAT2-positive mast cells and sympathetic innervation in the pancreas. PMID:23404442

  6. Chitosan-poly(butylene succinate) scaffolds and human bone marrow stromal cells induce bone repair in a mouse calvaria model.

    PubMed

    Costa-Pinto, A R; Correlo, V M; Sol, P C; Bhattacharya, M; Srouji, S; Livne, E; Reis, R L; Neves, N M

    2012-01-01

    Tissue engineering sustains the need of a three-dimensional (3D) scaffold to promote the regeneration of tissues in volume. Usually, scaffolds are seeded with an adequate cell population, allowing their growth and maturation upon implantation in vivo. Previous studies obtained by our group evidenced significant growth patterns and osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) when seeded and cultured on melt-based porous chitosan fibre mesh scaffolds (cell constructs). Therefore, it is crucial to test the in vivo performance of these in vitro 3D cell constructs. In this study, chitosan-based scaffolds were seeded and cultured in vitro with hBMSCs for 3 weeks under osteogenic stimulation conditions and analysed for cell adhesion, proliferation and differentiation. Implantation of 2 weeks precultured cell constructs in osteogenic culture conditions was performed into critical cranial size defects in nude mice. The objective of this study was to verify the scaffold integration and new bone formation. At 8 weeks of implantation, scaffolds were harvested and prepared for micro-computed tomography (µCT) analysis. Retrieved implants showed good integration with the surrounding tissue and significant bone formation, more evident for the scaffolds cultured and implanted with human cells. The results of this work demonstrated that chitosan-based scaffolds, besides supporting in vitro proliferation and osteogenic differentiation of hBMSCs, induced bone formation in vivo. Thus, their osteogenic potential in orthotopic location in immunodeficient mice was validated, evidencing good prospects for their use in bone tissue-engineering therapies.

  7. Human Urine-derived Stem Cells Seeded Surface Modified Composite Scaffold Grafts for Bladder Reconstruction in a Rat Model.

    PubMed

    Lee, Jun Nyung; Chun, So Young; Lee, Hyo-Jung; Jang, Yu-Jin; Choi, Seock Hwan; Kim, Dae Hwan; Oh, Se Heang; Song, Phil Hyun; Lee, Jin Ho; Kim, Jong Kun; Kwon, Tae Gyun

    2015-12-01

    We conducted this study to investigate the synergistic effect of human urine-derived stem cells (USCs) and surface modified composite scaffold for bladder reconstruction in a rat model. The composite scaffold (Polycaprolactone/Pluronic F127/3 wt% bladder submucosa matrix) was fabricated using an immersion precipitation method, and heparin was immobilized on the surface via covalent conjugation. Basic fibroblast growth factor (bFGF) was loaded onto the heparin-immobilized scaffold by a simple dipping method. In maximal bladder capacity and compliance analysis at 8 weeks post operation, the USCs-scaffold(heparin-bFGF) group showed significant functional improvement (2.34 ± 0.25 mL and 55.09 ± 11.81 µL/cm H2O) compared to the other groups (2.60 ± 0.23 mL and 56.14 ± 9.00 µL/cm H2O for the control group, 1.46 ± 0.18 mL and 34.27 ± 4.42 µL/cm H2O for the partial cystectomy group, 1.76 ± 0.22 mL and 35.62 ± 6.69 µL/cm H2O for the scaffold group, and 1.92 ± 0.29 mL and 40.74 ± 7.88 µL/cm H2O for the scaffold(heparin-bFGF) group, respectively). In histological and immunohistochemical analysis, the USC-scaffold(heparin-bFGF) group showed pronounced, well-differentiated, and organized smooth muscle bundle formation, a multi-layered and pan-cytokeratin-positive urothelium, and high condensation of submucosal area. The USCs seeded scaffold(heparin-bFGF) exhibits significantly increased bladder capacity, compliance, regeneration of smooth muscle tissue, multi-layered urothelium, and condensed submucosa layers at the in vivo study.

  8. Modeling structural and functional deficiencies of RBM20 familial dilated cardiomyopathy using human induced pluripotent stem cells.

    PubMed

    Wyles, Saranya P; Li, Xing; Hrstka, Sybil C; Reyes, Santiago; Oommen, Saji; Beraldi, Rosanna; Edwards, Jessica; Terzic, Andre; Olson, Timothy M; Nelson, Timothy J

    2016-01-15

    Dilated cardiomyopathy (DCM) is a leading cause of heart failure. In families with autosomal-dominant DCM, heterozygous missense mutations were identified in RNA-binding motif protein 20 (RBM20), a spliceosome protein induced during early cardiogenesis. Dermal fibroblasts from two unrelated patients harboring an RBM20 R636S missense mutation were reprogrammed to human induced pluripotent stem cells (hiPSCs) and differentiated to beating cardiomyocytes (CMs). Stage-specific transcriptome profiling identified differentially expressed genes ranging from angiogenesis regulator to embryonic heart transcription factor as initial molecular aberrations. Furthermore, gene expression analysis for RBM20-dependent splice variants affected sarcomeric (TTN and LDB3) and calcium (Ca(2+)) handling (CAMK2D and CACNA1C) genes. Indeed, RBM20 hiPSC-CMs exhibited increased sarcomeric length (RBM20: 1.747 ± 0.238 µm versus control: 1.404 ± 0.194 µm; P < 0.0001) and decreased sarcomeric width (RBM20: 0.791 ± 0.609 µm versus control: 0.943 ± 0.166 µm; P < 0.0001). Additionally, CMs showed defective Ca(2+) handling machinery with prolonged Ca(2+) levels in the cytoplasm as measured by greater area under the curve (RBM20: 814.718 ± 94.343 AU versus control: 206.941 ± 22.417 AU; P < 0.05) and higher Ca(2+) spike amplitude (RBM20: 35.281 ± 4.060 AU versus control:18.484 ± 1.518 AU; P < 0.05). β-adrenergic stress induced with 10 µm norepinephrine demonstrated increased susceptibility to sarcomeric disorganization (RBM20: 86 ± 10.5% versus control: 40 ± 7%; P < 0.001). This study features the first hiPSC model of RBM20 familial DCM. By monitoring human cardiac disease according to stage-specific cardiogenesis, this study demonstrates RBM20 familial DCM is a developmental disorder initiated by molecular defects that pattern maladaptive cellular mechanisms of pathological cardiac remodeling. Indeed, hiPSC-CMs recapitulate RBM20 familial DCM phenotype in a dish and establish a tool

  9. Human Stem Cells for Craniomaxillofacial Reconstruction

    PubMed Central

    Kirkpatrick, William Niall Alexander; Cameron, Malcolm Gregor

    2014-01-01

    Human stem cell research represents an exceptional opportunity for regenerative medicine and the surgical reconstruction of the craniomaxillofacial complex. The correct architecture and function of the vastly diverse tissues of this important anatomical region are critical for life supportive processes, the delivery of senses, social interaction, and aesthetics. Craniomaxillofacial tissue loss is commonly associated with inflammatory responses of the surrounding tissue, significant scarring, disfigurement, and psychological sequelae as an inevitable consequence. The in vitro production of fully functional cells for skin, muscle, cartilage, bone, and neurovascular tissue formation from human stem cells, may one day provide novel materials for the reconstructive surgeon operating on patients with both hard and soft tissue deficit due to cancer, congenital disease, or trauma. However, the clinical translation of human stem cell technology, including the application of human pluripotent stem cells (hPSCs) in novel regenerative therapies, faces several hurdles that must be solved to permit safe and effective use in patients. The basic biology of hPSCs remains to be fully elucidated and concerns of tumorigenicity need to be addressed, prior to the development of cell transplantation treatments. Furthermore, functional comparison of in vitro generated tissue to their in vivo counterparts will be necessary for confirmation of maturity and suitability for application in reconstructive surgery. Here, we provide an overview of human stem cells in disease modeling, drug screening, and therapeutics, while also discussing the application of regenerative medicine for craniomaxillofacial tissue deficit and surgical reconstruction. PMID:24564584

  10. Protective effects of citrus and rosemary extracts on UV-induced damage in skin cell model and human volunteers.

    PubMed

    Pérez-Sánchez, A; Barrajón-Catalán, E; Caturla, N; Castillo, J; Benavente-García, O; Alcaraz, M; Micol, V

    2014-07-01

    Ultraviolet radiation absorbed by the epidermis is the major cause of various cutaneous disorders, including photoaging and skin cancers. Although topical sunscreens may offer proper skin protection, dietary plant compounds may significantly contribute to lifelong protection of skin health, especially when unconsciously sun UV exposed. A combination of rosemary and citrus bioflavonoids extracts was used to inhibit UV harmful effects on human HaCaT keratinocytes and in human volunteers after oral intake. Survival of HaCaT cells after UVB radiation was higher in treatments using the combination of extracts than in those performed with individual extracts, indicating potential synergic effects. The combination of extracts also decreased UVB-induced intracellular radical oxygen species (ROS) and prevented DNA damage in HaCaT cells by comet assay and decreased chromosomal aberrations in X-irradiated human lymphocytes. The oral daily consumption of 250 mg of the combination by human volunteers revealed a significant minimal erythema dose (MED) increase after eight weeks (34%, p<0.05). Stronger protection was achieved after 12 weeks (56%, p<0.01). The combination of citrus flavonoids and rosemary polyphenols and diterpenes may be considered as an ingredient for oral photoprotection. Their mechanism of action may deserve further attention.

  11. Intramyocardial transplantation and tracking of human mesenchymal stem cells in a novel intra-uterine pre-immune fetal sheep myocardial infarction model: a proof of concept study.

    PubMed

    Emmert, Maximilian Y; Weber, Benedikt; Wolint, Petra; Frauenfelder, Thomas; Zeisberger, Steffen M; Behr, Luc; Sammut, Sebastien; Scherman, Jacques; Brokopp, Chad E; Schwartländer, Ruth; Vogel, Viola; Vogt, Peter; Grünenfelder, Jürg; Alkadhi, Hatem; Falk, Volkmar; Boss, Andreas; Hoerstrup, Simon P

    2013-01-01

    Although stem-cell therapies have been suggested for cardiac-regeneration after myocardial-infarction (MI), key-questions regarding the in-vivo cell-fate remain unknown. While most available animal-models require immunosuppressive-therapy when applying human cells, the fetal-sheep being pre-immune until day 75 of gestation has been proposed for the in-vivo tracking of human cells after intra-peritoneal transplantation. We introduce a novel intra-uterine myocardial-infarction model to track human mesenchymal stem cells after direct intra-myocardial transplantation into the pre-immune fetal-sheep. Thirteen fetal-sheep (gestation age: 70-75 days) were included. Ten animals either received an intra-uterine induction of MI only (n = 4) or MI+intra-myocardial injection (IMI;n = 6) using micron-sized, iron-oxide (MPIO) labeled human mesenchymal stem cells either derived from the adipose-tissue (ATMSCs;n = 3) or the bone-marrow (BMMSCs;n = 3). Three animals received an intra-peritoneal injection (IPI;n = 3; ATMSCs;n = 2/BMMSCs;n = 1). All procedures were performed successfully and follow-up was 7-9 days. To assess human cell-fate, multimodal cell-tracking was performed via MRI and/or Micro-CT, Flow-Cytometry, PCR and immunohistochemistry. After IMI, MRI displayed an estimated amount of 1×10(5)-5×10(5) human cells within ventricular-wall corresponding to the injection-sites which was further confirmed on Micro-CT. PCR and IHC verified intra-myocardial presence via detection of human-specific β-2-microglobulin, MHC-1, ALU-Sequence and anti-FITC targeting the fluorochrome-labeled part of the MPIOs. The cells appeared viable, integrated and were found in clusters or in the interstitial-spaces. Flow-Cytometry confirmed intra-myocardial presence, and showed further distribution within the spleen, lungs, kidneys and brain. Following IPI, MRI indicated the cells within the intra-peritoneal-cavity involving the liver and kidneys. Flow

  12. Investigating the utility of human embryonic stem cell-derived neurons to model ageing and neurodegenerative disease using whole-genome gene expression and splicing analysis

    PubMed Central

    Patani, Rickie; Lewis, Patrick A; Trabzuni, Daniah; Puddifoot, Clare A; Wyllie, David J A; Walker, Robert; Smith, Colin; Hardingham, Giles E; Weale, Michael; Hardy, John; Chandran, Siddharthan; Ryten, Mina

    2012-01-01

    A major goal in regenerative medicine is the predictable manipulation of human embryonic stem cells (hESCs) to defined cell fates that faithfully represent their somatic counterparts. Directed differentiation of hESCs into neuronal populations has galvanized much interest into their potential application in modelling neurodegenerative disease. However, neurodegenerative diseases are age-related, and therefore establishing the maturational comparability of hESC-derived neural derivatives is critical to generating accurate in vitro model systems. We address this issue by comparing genome-wide, exon-specific expression analyses of pluripotent hESCs, multipotent neural precursor cells and a terminally differentiated enriched neuronal population to expression data from post-mortem foetal and adult human brain samples. We show that hESC-derived neuronal cultures (using a midbrain differentiation protocol as a prototypic example of lineage restriction), while successful in generating physiologically functional neurons, are closer to foetal than adult human brain in terms of molecular maturation. These findings suggest that developmental stage has a more dominant influence on the cellular transcriptome than regional identity. In addition, we demonstrate that developmentally regulated gene splicing is common, and potentially a more sensitive measure of maturational state than gene expression profiling alone. In summary, this study highlights the value of genomic indices in refining and validating optimal cell populations appropriate for modelling ageing and neurodegeneration. PMID:22681703

  13. In vitro and in vivo activity of 4-thio-uridylate against JY cells, a model for human acute lymphoid leukemia

    SciTech Connect

    Berenyi, Erika; Benko, Ilona; Vamosi, Gyoergy; Geresi, Krisztina; Tarkanyi, Ilona; Szegedi, Istvan; Lukacs, Levente; Juhasz, Istvan; Kiss, Csongor; Fesues, Laszlo; and others

    2011-07-08

    Highlights: {yields} s{sup 4}UMP a naturally occurring thiolated nucleotide, effectively inhibited the proliferation of JY cells in vitro and in vivo. {yields} s{sup 4}UMP decreased the cell number and colony forming activity of leukemia cells in SCID mice. {yields} The effect of s{sup 4}UMP was undetectable on the bone marrow of healthy mice. {yields} The biochemical changes of the treated cells suggested that s{sup 4}UMP induced apoptosis. -- Abstract: We have previously reported the in vitro anti-proliferative effect of 4-thio-uridylate (s{sup 4}UMP) on OCM-1 uveal melanoma cells. Here, we assessed the efficacy of s{sup 4}UMP on JY cells. Treatment of JY cells with s{sup 4}UMP suppressed their colony forming activity and induced apoptosis; healthy human bone marrow granulocyte-macrophage progenitor cells were 14-fold less sensitive to the nucleotide. In vivo effectiveness of s{sup 4}UMP was determined using xenograft SCID mouse model. s{sup 4}UMP decreased the cell number and colony forming activity of the total cell content of the femur of SCID mice transplanted with JY cells without affecting the bone marrow of healthy mice. These results suggest that s{sup 4}UMP alone or in combination with other clinically approved anti-leukemic remedies should be further explored as a potential novel therapeutic agent.

  14. Survival, Differentiation, and Neuroprotective Mechanisms of Human Stem Cells Complexed With Neurotrophin-3-Releasing Pharmacologically Active Microcarriers in an Ex Vivo Model of Parkinson’s Disease

    PubMed Central

    Daviaud, Nicolas; Garbayo, Elisa; Sindji, Laurence; Martínez-Serrano, Alberto; Schiller, Paul C.

    2015-01-01

    Stem cell-based regenerative therapies hold great potential for the treatment of degenerative disorders such as Parkinson’s disease (PD). We recently reported the repair and functional recovery after treatment with human marrow-isolated adult multilineage inducible (MIAMI) cells adhered to neurotrophin-3 (NT3) releasing pharmacologically active microcarriers (PAMs) in hemiparkinsonian rats. In order to comprehend this effect, the goal of the present work was to elucidate the survival, differentiation, and neuroprotective mechanisms of MIAMI cells and human neural stem cells (NSCs), both adhering to NT3-releasing PAMs in an ex vivo organotypic model of nigrostriatal degeneration made from brain sagittal slices. It was shown that PAMs led to a marked increase in MIAMI cell survival and neuronal differentiation when releasing NT3. A significant neuroprotective effect of MIAMI cells adhering to PAMs was also demonstrated. NSCs barely had a neuroprotective effect and differentiated mostly into dopaminergic neuronal cells when adhering to PAM-NT3. Moreover, those cells were able to release dopamine in a sufficient amount to induce a return to baseline levels. Reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay analyses identified vascular endothelial growth factor (VEGF) and stanniocalcin-1 as potential mediators of the neuroprotective effect of MIAMI cells and NSCs, respectively. It was also shown that VEGF locally stimulated tissue vascularization, which might improve graft survival, without excluding a direct neuroprotective effect of VEGF on dopaminergic neurons. These results indicate a prospective interest of human NSC/PAM and MIAMI cell/PAM complexes in tissue engineering for PD. Significance Stem cell-based regenerative therapies hold great potential for the treatment of degenerative disorders such as Parkinson’s disease (PD). The present work elucidates and compares the survival, differentiation, and

  15. Process-Based Expansion and Neural Differentiation of Human Pluripotent Stem Cells for Transplantation and Disease Modeling

    PubMed Central

    Stover, Alexander E.; Brick, David J.; Nethercott, Hubert E.; Banuelos, Maria G.; Sun, Lei; O’Dowd, Diane K.; Schwartz, Philip H.

    2014-01-01

    Robust strategies for developing patient-specific, human, induced pluripotent stem cell (iPSC)-based therapies of the brain require an ability to derive large numbers of highly defined neural cells. Recent progress in iPSC culture techniques includes partial-to-complete elimination of feeder layers, use of defined media, and single-cell passaging. However, these techniques still require embryoid body formation or coculture for differentiation into neural stem cells (NSCs). In addition, none of the published methodologies has employed all of the advances in a single culture system. Here we describe a reliable method for long-term, single-cell passaging of PSCs using a feeder-free, defined culture system that produces confluent, adherent PSCs that can be differentiated into NSCs. To provide a basis for robust quality control, we have devised a system of cellular nomenclature that describes an accurate genotype and phenotype of the cells at specific stages in the process. We demonstrate that this protocol allows for the efficient, large-scale, cGMP-compliant production of transplantable NSCs from all lines tested. We also show that NSCs generated from iPSCs produced with the process described are capable of forming both glia defined by their expression of S100β and neurons that fire repetitive action potentials. PMID:23893392

  16. Transplantation of Human Neural Stem Cells in a Parkinsonian Model Exerts Neuroprotection via Regulation of the Host Microenvironment.

    PubMed

    Zuo, Fu-Xing; Bao, Xin-Jie; Sun, Xi-Cai; Wu, Jun; Bai, Qing-Ran; Chen, Guo; Li, Xue-Yuan; Zhou, Qiang-Yi; Yang, Yuan-Fan; Shen, Qin; Wang, Ren-Zhi

    2015-11-05

    Parkinson's disease (PD) is characterized by a progressive loss of dopaminergic neurons and consequent dopamine (DA) deficit, and current treatment still remains a challenge. Although neural stem cells (NSCs) have been evaluated as appealing graft sources, mechanisms underlying the beneficial phenomena are not well understood. Here, we investigate whether human NSCs (hNSCs) transplantation could provide neuroprotection against DA depletion by recruiting endogenous cells to establish a favorable niche. Adult mice subjected to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were transplanted with hNSCs or vehicle into the striatum. Behavioral and histological analyses demonstrated significant neurorescue response observed in hNSCs-treated animals compared with the control mice. In transplanted animals, grafted cells survived, proliferated, and migrated within the astrocytic scaffold. Notably, more local astrocytes underwent de-differentiation, acquiring the properties of NSCs or neural precursor cells (NPCs) in mice given hNSCs. Additionally, we also detected significantly higher expression of host-derived growth factors in hNSCs-transplanted mice compared with the control animals, together with inhibition of local microglia and proinflammatory cytokines. Overall, our results indicate that hNSCs transplantation exerts neuroprotection in MPTP-insulted mice via regulating the host niche. Harnessing synergistic interaction between the grafts and host cells may help optimize cell-based therapies for PD.

  17. Subpopulations of MCF7 cells separate by Percoll gradient centrifugation: a model to analyze the heterogeneity of human breast cancer

    SciTech Connect

    Resnicoff, M.; Medrano, E.E.; Podhajcer, O.L.; Bravo, A.I.; Bover, L.; Mordoh, J.

    1987-10-01

    Exponentially growing MCF7 human breast cancer cells were separated in Percoll gradients into six different fractions of increasing density (A to F). These fractions could be subcultured and were found to contain different cellular subpopulations as defined by the following criteria: ability to generate other cellular subpopulations; growth rate; DNA synthesis; and expression of estrogen receptors, ras oncogene-encoded protein p21, and carcinoembryonic antigen. One of the minor fractions (E), which contained about 5% of the total cell number, appeared to contain the stem cells, on the basis of the following criteria: (i) its ability to reproduce the other cellular subpopulations, (ii) its high rate of growth and DNA synthesis, and (iii) the inability of the other subpopulations to generate it. The most differentiated subpopulation appeared to be the densest one (F), since it was the slowest growing and appeared to be the end point of the other subpopulations.

  18. Hexachlorobenzene promotes angiogenesis in vivo, in a breast cancer model and neovasculogenesis in vitro, in the human microvascular endothelial cell line HMEC-1.

    PubMed

    Pontillo, Carolina; Español, Alejandro; Chiappini, Florencia; Miret, Noelia; Cocca, Claudia; Alvarez, Laura; Kleiman de Pisarev, Diana; Sales, María Elena; Randi, Andrea Silvana

    2015-11-19

    Exposure to environmental pollutants may alter proangiogenic ability and promotes tumor growth. Hexachlorobenzene (HCB) is an organochlorine pesticide found in maternal milk and in lipid foods, and a weak ligand of the aryl hydrocarbon receptor (AhR). HCB induces migration and invasion in human breast cancer cells, as well as tumor growth and metastasis in vivo. In this study, we examined HCB action on angiogenesis in mammary carcinogenesis. HCB stimulates angiogenesis and increases vascular endothelial growth factor (VEGF) expression in a xenograft model with the human breast cancer cell line MDA-MB-231. Human microvascular endothelial cells HMEC-1 exposed to HCB (0.005, 0.05, 0.5 and 5μM) showed an increase in cyclooxygenase-2 (COX-2) and VEGF protein expression involving AhR. In addition, we found that HCB enhances VEGF-Receptor 2 (VEGFR2) expression, and activates its downstream pathways p38 and ERK1/2. HCB induces cell migration and neovasculogenesis in a dose-dependent manner. Cells pretreatment with AhR, COX-2 and VEGFR2 selective inhibitors, suppressed these effects. In conclusion, our results show that HCB promotes angiogenesis in vivo and in vitro. HCB-induced cell migration and tubulogenesis are mediated by AhR, COX-2 and VEGFR2 in HMEC-1. These findings may help to understand the association among HCB exposure, angiogenesis and mammary carcinogenesis. PMID:26358519

  19. IL22 Regulates Human Urothelial Cell Sensory and Innate Functions through Modulation of the Acetylcholine Response, Immunoregulatory Cytokines and Antimicrobial Peptides: Assessment of an In Vitro Model

    PubMed Central

    Le, Phong T.; Pearce, Meghan M.; Zhang, Shubin; Campbell, Edward M.; Fok, Cynthia S.; Mueller, Elizabeth R.; Brincat, Cynthia A.; Wolfe, Alan J.; Brubaker, Linda

    2014-01-01

    Human urinary disorders are generally studied in rodent models due to limitations of functional in vitro culture models of primary human urothelial cells (HUCs). Current HUC culture models are often derived from immortalized cancer cell lines, which likely have functional characteristics differ from healthy human urothelium. Here, we described a simple explant culture technique to generate HUCs and assessed their in vitro functions. Using transmission electron microscopy, we assessed morphology and heterogeneity of the generated HUCs and characterized their intercellular membrane structural proteins relative to ex vivo urothelium tissue. We demonstrated that our cultured HUCs are free of fibroblasts. They are also heterogeneous, containing cells characteristic of both immature basal cells and mature superficial urothelial cells. The cultured HUCs expressed muscarinic receptors (MR1 and MR2), carnitine acetyltransferase (CarAT), immunoregulatory cytokines IL7, IL15, and IL23, as well as the chemokine CCL20. HUCs also expressed epithelial cell-specific molecules essential for forming intercellular structures that maintain the functional capacity to form the physiological barrier of the human bladder urothelium. A subset of HUCs, identified by the high expression of CD44, expressed the Toll-like receptor 4 (TLR4) along with its co-receptor CD14. We demonstrated that HUCs express, at the mRNA level, both forms of the IL22 receptor, the membrane-associated (IL22RA1) and the secreted soluble (IL22RA2) forms; in turn, IL22 inhibited expression of MR1 and induced expression of CarAT and two antimicrobial peptides (S100A9 and lipocalin-2). While the cellular sources of IL22 have yet to be identified, the HUC cytokine and chemokine profiles support the concept that IL22-producing cells are present in the human bladder mucosa tissue and that IL22 plays a regulatory role in HUC functions. Thus, the described explant technique is clearly capable of generating functional HUCs

  20. IL22 regulates human urothelial cell sensory and innate functions through modulation of the acetylcholine response, immunoregulatory cytokines and antimicrobial peptides: assessment of an in vitro model.

    PubMed

    Le, Phong T; Pearce, Meghan M; Zhang, Shubin; Campbell, Edward M; Fok, Cynthia S; Mueller, Elizabeth R; Brincat, Cynthia A; Wolfe, Alan J; Brubaker, Linda

    2014-01-01

    Human urinary disorders are generally studied in rodent models due to limitations of functional in vitro culture models of primary human urothelial cells (HUCs). Current HUC culture models are often derived from immortalized cancer cell lines, which likely have functional characteristics differ from healthy human urothelium. Here, we described a simple explant culture technique to generate HUCs and assessed their in vitro functions. Using transmission electron microscopy, we assessed morphology and heterogeneity of the generated HUCs and characterized their intercellular membrane structural proteins relative to ex vivo urothelium tissue. We demonstrated that our cultured HUCs are free of fibroblasts. They are also heterogeneous, containing cells characteristic of both immature basal cells and mature superficial urothelial cells. The cultured HUCs expressed muscarinic receptors (MR1 and MR2), carnitine acetyltransferase (CarAT), immunoregulatory cytokines IL7, IL15, and IL23, as well as the chemokine CCL20. HUCs also expressed epithelial cell-specific molecules essential for forming intercellular structures that maintain the functional capacity to form the physiological barrier of the human bladder urothelium. A subset of HUCs, identified by the high expression of CD44, expressed the Toll-like receptor 4 (TLR4) along with its co-receptor CD14. We demonstrated that HUCs express, at the mRNA level, both forms of the IL22 receptor, the membrane-associated (IL22RA1) and the secreted soluble (IL22RA2) forms; in turn, IL22 inhibited expression of MR1 and induced expression of CarAT and two antimicrobial peptides (S100A9 and lipocalin-2). While the cellular sources of IL22 have yet to be identified, the HUC cytokine and chemokine profiles support the concept that IL22-producing cells are present in the human bladder mucosa tissue and that IL22 plays a regulatory role in HUC functions. Thus, the described explant technique is clearly capable of generating functional HUCs

  1. Human Adipose Stem Cells Improve Mechanical Allodynia and Enhance Functional Recovery in a Rat Model of Neuropathic Pain.

    PubMed

    Lee, Hye Yeong; Lee, Hye-Lan; Yun, Yeomin; Kim, Jin-Su; Ha, Yoon; Yoon, Do Heum; Lee, Soo-Hong; Shin, Dong Ah

    2015-07-01

    Stem cells are a promising source of tissue engineering due to their differentiation potential. Today, direct transplantation of stem cells for cell therapy is commonly performed. However, in cases of nerve injury, direct transplantation of cells could lead to secondary nerve damage. Male Sprague-Dawley rats were randomized into four groups: the phosphate-buffered saline epineural transplantation (PBS-ENT) group, the PBS intraneural transplantation (PBS-INT) group, the human adipose-derived stem cells epineural transplantation (hASCs-ENT) group, and human adipose-derived stem cells intraneural transplantation (hASCs-INT) group. Transplantation was conducted 1 week later after inflicting a crush injury with subsequent observation for 5 weeks. To evaluate pain, each group was examined with regard to paw withdrawal latency and evoked potentials. The sciatic functional index (SFI) was calculated to estimate functional recovery. The sciatic nerve was also examined histologically. The hASCs-ENT group showed a more rapid paw withdrawal threshold and SFI recovery than the other groups (p<0.05). The hASCs-ENT group also showed shorter initial latencies in both somatosensory evoked potential (SSEP) and motor evoked potential (MEP) than the PBS-INT group (p<0.05). In addition, the N1 latency of the MEP and the N1 and P1 latencies of the SSEP were significantly shorter than those of the PBS-INT group (p<0.05). Histological examination revealed that the transplanted groups showed better neural recovery and remyelination than the groups injected with PBS. These results show that the transplantation of hASCs into the injured sciatic nerve improved mechanical allodynia and functional recovery as determined by the paw withdrawal test, SFI analysis, and electrophysiological studies. ENT is superior to INT in terms of invasiveness and better outcomes.

  2. Human Adipose Stem Cells Improve Mechanical Allodynia and Enhance Functional Recovery in a Rat Model of Neuropathic Pain.

    PubMed

    Lee, Hye Yeong; Lee, Hye-Lan; Yun, Yeomin; Kim, Jin-Su; Ha, Yoon; Yoon, Do Heum; Lee, Soo-Hong; Shin, Dong Ah

    2015-07-01

    Stem cells are a promising source of tissue engineering due to their differentiation potential. Today, direct transplantation of stem cells for cell therapy is commonly performed. However, in cases of nerve injury, direct transplantation of cells could lead to secondary nerve damage. Male Sprague-Dawley rats were randomized into four groups: the phosphate-buffered saline epineural transplantation (PBS-ENT) group, the PBS intraneural transplantation (PBS-INT) group, the human adipose-derived stem cells epineural transplantation (hASCs-ENT) group, and human adipose-derived stem cells intraneural transplantation (hASCs-INT) group. Transplantation was conducted 1 week later after inflicting a crush injury with subsequent observation for 5 weeks. To evaluate pain, each group was examined with regard to paw withdrawal latency and evoked potentials. The sciatic functional index (SFI) was calculated to estimate functional recovery. The sciatic nerve was also examined histologically. The hASCs-ENT group showed a more rapid paw withdrawal threshold and SFI recovery than the other groups (p<0.05). The hASCs-ENT group also showed shorter initial latencies in both somatosensory evoked potential (SSEP) and motor evoked potential (MEP) than the PBS-INT group (p<0.05). In addition, the N1 latency of the MEP and the N1 and P1 latencies of the SSEP were significantly shorter than those of the PBS-INT group (p<0.05). Histological examination revealed that the transplanted groups showed better neural recovery and remyelination than the groups injected with PBS. These results show that the transplantation of hASCs into the injured sciatic nerve improved mechanical allodynia and functional recovery as determined by the paw withdrawal test, SFI analysis, and electrophysiological studies. ENT is superior to INT in terms of invasiveness and better outcomes. PMID:25857679

  3. Human platelets produced in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice upon transplantation of human cord blood CD34(+) cells are functionally active in an ex vivo flow model of thrombosis.

    PubMed

    Salles, Isabelle I; Thijs, Tim; Brunaud, Christine; De Meyer, Simon F; Thys, Johan; Vanhoorelbeke, Karen; Deckmyn, Hans

    2009-12-01

    Xenotransplantation systems have been used with increasing success to better understand human hematopoiesis and thrombopoiesis. In this study, we demonstrate that production of human platelets in nonobese diabetic/severe combined immunodeficient mice after transplantation of unexpanded cord-blood CD34(+) cells was detected within 10 days after transplantation, with the number of circulating human platelets peaking at 2 weeks (up to 87 x 10(3)/microL). This rapid human platelet production was followed by a second wave of platelet formation 5 weeks after transplantation, with a population of 5% still detected after 8 weeks, attesting for long-term engraftment. Platelets issued from human hematopoietic stem cell progenitors are functional, as assessed by increased CD62P expression and PAC1 binding in response to collagen-related peptide and thrombin receptor-activating peptide activation and their ability to incorporate into thrombi formed on a collagen-coated surface in an ex vivo flow model of thrombosis. This interaction was abrogated by addition of inhibitory monoclonal antibodies against human glycoprotein Ibalpha (GPIbalpha) and GPIIb/IIIa. Thus, our mouse model with production of human platelets may be further explored to study the function of genetically modified platelets, but also to investigate the effect of stimulators or inhibitors of human thrombopoiesis in vivo.

  4. Modeling collective cell motility

    NASA Astrophysics Data System (ADS)

    Rappel, Wouter-Jan

    Eukaryotic cells often move in groups, a critical aspect of many biological and medical processes including wound healing, morphogenesis and cancer metastasis. Modeling can provide useful insights into the fundamental mechanisms of collective cell motility. Constructing models that incorporate the physical properties of the cells, however, is challenging. Here, I discuss our efforts to build a comprehensive cell motility model that includes cell membrane properties, cell-substrate interactions, cell polarity, and cell-cell interaction. The model will be applied to a variety of systems, including motion on micropatterned substrates and the migration of border cells in Drosophila. This work was supported by NIH Grant No. P01 GM078586 and NSF Grant No. 1068869.

  5. Multidrug resistance-associated protein (MRP1, 2, 4 and 5) expression in human corneal cell culture models and animal corneal tissue.

    PubMed

    Verstraelen, Jessica; Reichl, Stephan

    2014-07-01

    Preclinical studies addressing the transcorneal absorption of ophthalmic drugs are mainly performed using ex vivo animal corneas and in vitro corneal cell culture models, leaving open the question of transferability to humans in an in vivo situation. While passive drug absorption through corneal tissue is well understood, little is known about the expression of transporter proteins and active drug transport in human and animal corneas as well as corneal cell culture models. Therefore, the aim of this study was to conduct an expression analysis of four multidrug resistance-associated proteins (MRP1, 2, 4 and 5) in various in vitro and ex vivo corneal models, leading to a better understanding of the comparability of different corneal models regarding drug absorption and transferability to humans. Two well-established in vitro human corneal models, the HCE-T epithelial model and the more organotypic Hemicornea construct, both of which are based on the SV40 immortalized human corneal epithelial cell line HCE-T, were analyzed, as were excised rabbit and porcine cornea. Specimens of abraded epithelia from human donor corneas were also tested. MRP mRNA expression was determined via reverse transcriptase polymerase chain reaction. Protein expression was examined using Western blot experiments and immunohistochemistry. The functional activity of the MRP efflux transporter was detected in transport assays using specific marker and inhibitor substances. The functional expression of all of the tested MRP transporters was detected in the HCE-T epithelial model. Hemicornea constructs displayed a similar expression pattern for MRP1, 4 and 5, whereas no MRP2 protein expression or activity was detected. However, excised animal corneas exhibited different expression profiles. In porcine cornea, no functional expression of MRP1, 2, or 5 was observed, and we failed to detect MRP4 expression in rabbit cornea. The results suggest that MRP1, 2, 4, and 5 are expressed in the human corneal

  6. Regulation of IL-17A Production Is Distinct from IL-17F in a Primary Human Cell Co-culture Model of T Cell-Mediated B Cell Activation

    PubMed Central

    Melton, Andrew C.; Melrose, Jennifer; Alajoki, Liisa; Privat, Sylvie; Cho, Hannah; Brown, Naomi; Plavec, Ana Marija; Nguyen, Dat; Johnston, Elijah D.; Yang, Jian; Polokoff, Mark A.; Plavec, Ivan; Berg, Ellen L.; O'Mahony, Alison

    2013-01-01

    Improper regulation of B cell responses leads to excessive production of antibodies and contributes to the development of autoimmune disease. T helper 17 (Th17) cells also drive the development of autoimmune disease, but the role of B cells in shaping Th17 cell-mediated immune responses, as well as the reciprocal regulation of B cell responses by IL-17 family cytokines, remains unclear. The aim of this study was to characterize the regulation of IL-17A and IL-17F in a model of T cell-dependent B cell activation. Stimulation of primary human B cell and peripheral blood mononuclear cell (BT) co-cultures with α-IgM and a non-mitogenic concentration of superantigens for three days promoted a Th17 cell response as evidenced by increased expression of Th17-related gene transcripts, including Il17f, Il21, Il22, and Il23r, in CD4 T cells, as well as the secretion of IL-17A and IL-17F protein. We tested the ability of 144 pharmacologic modulators representing 91 different targets or pathways to regulate IL-17A and IL-17F production in these stimulated BT co-cultures. IL-17A production was found to be preferentially sensitive to inhibition of the PI3K/mTOR pathway, while prostaglandin EP receptor agonists, including PGE2, increased IL-17A concentrations. In contrast, the production of IL-17F was inhibited by PGE2, but selectively increased by TLR2 and TLR5 agonists. These results indicate that IL-17A regulation is distinct from IL-17F in stimulated BT co-cultures and that this co-culture approach can be used to identify pathway mechanisms and novel agents that selectively inhibit production of IL-17A or IL-17F. PMID:23505568

  7. Simulation of DNA Damage in Human Cells from Space Radiation Using a Physical Model of Stochastic Particle Tracks and Chromosomes

    NASA Technical Reports Server (NTRS)

    Ponomarev, Artem; Plante, Ianik; Hada, Megumi; George, Kerry; Wu, Honglu

    2015-01-01

    The formation of double-strand breaks (DSBs) and chromosomal aberrations (CAs) is of great importance in radiation research and, specifically, in space applications. We are presenting a recently developed model, in which chromosomes simulated by NASARTI (NASA Radiation Tracks Image) is combined with nanoscopic dose calculations performed with the Monte-Carlo simulation by RITRACKS (Relativistic Ion Tracks) in a voxelized space. The model produces the number of DSBs, as a function of dose for high-energy iron, oxygen, and carbon ions, and He ions. The combined model calculates yields of radiation-induced CAs and unrejoined chromosome breaks in normal and repair deficient cells. The merged computational model is calibrated using the relative frequencies and distributions of chromosomal aberrations reported in the literature. The model considers fractionated deposition of energy to approximate dose rates of the space flight environment. The merged model also predicts of the yields and sizes of translocations, dicentrics, rings, and more complex-type aberrations formed in the G0/G1 cell cycle phase during the first cell division after irradiation.

  8. Spatio-temporal Model of Endogenous ROS and Raft-Dependent WNT/Beta-Catenin Signaling Driving Cell Fate Commitment in Human Neural Progenitor Cells

    PubMed Central

    Haack, Fiete; Lemcke, Heiko; Ewald, Roland; Rharass, Tareck; Uhrmacher, Adelinde M.

    2015-01-01

    Canonical WNT/β-catenin signaling is a central pathway in embryonic development, but it is also connected to a number of cancers and developmental disorders. Here we apply a combined in-vitro and in-silico approach to investigate the spatio-temporal regulation of WNT/β-catenin signaling during the early neural differentiation process of human neural progenitors cells (hNPCs), which form a new prospect for replacement therapies in the context of neurodegenerative diseases. Experimental measurements indicate a second signal mechanism, in addition to canonical WNT signaling, being involved in the regulation of nuclear β-catenin levels during the cell fate commitment phase of neural differentiation. We find that the biphasic activation of β-catenin signaling observed experimentally can only be explained through a model that combines Reactive Oxygen Species (ROS) and raft dependent WNT/β-catenin signaling. Accordingly after initiation of differentiation endogenous ROS activates DVL in a redox-dependent manner leading to a transient activation of down-stream β-catenin signaling, followed by continuous auto/paracrine WNT signaling, which crucially depends on lipid rafts. Our simulation studies further illustrate the elaborate spatio-temporal regulation of DVL, which, depending on its concentration and localization, may either act as direct inducer of the transient ROS/β-catenin signal or as amplifier during continuous auto-/parcrine WNT/β-catenin signaling. In addition we provide the first stochastic computational model of WNT/β-catenin signaling that combines membrane-related and intracellular processes, including lipid rafts/receptor dynamics as well as WNT- and ROS-dependent β-catenin activation. The model’s predictive ability is demonstrated under a wide range of varying conditions for in-vitro and in-silico reference data sets. Our in-silico approach is realized in a multi-level rule-based language, that facilitates the extension and modification of the

  9. Humanized Mouse Models of HIV Infection

    PubMed Central

    Denton, Paul W.; Garcia, J. Victor

    2013-01-01

    Because of the limited tropism of HIV, in vivo modeling of this virus has been almost exclusively limited to other lentiviruses such as SIV that reproduce many important characteristics of HIV infection. However, there are significant genetic and biological differences among lentiviruses and some HIV-specific interventions are not effective against other lentiviruses in non-human hosts. For these reasons much emphasis has recently been placed on developing alternative animal models that support HIV replication and recapitulate key aspects of HIV infection and pathogenesis in humans. Humanized mice, CD34+ hematopoietic progenitor cell transplanted immunodeficient mice and in particular mice also implanted with human thymic/liver tissue (BLT mice) that develop a functional human immune system, have been the focus of a great deal of attention as possible models to study virtually all aspects of HIV biology and pathogenesis. Humanized mice are systemically reconstituted with human lymphoid cells offering rapid, reliable and reproducible experimental systems for HIV research. Peripheral blood of humanized mice can be readily sampled longitudinally to assess reconstitution with human cells and to monitor HIV replication permitting the evaluation of multiple parameters of HIV infection such as viral load levels, CD4+ T cell depletion, immune activation, as well as the effects of therapeutic interventions. Of high relevance to HIV transmission is the extensive characterization and validation of the reconstitution with human lymphoid cells of the female reproductive tract and of the gastrointestinal tract of humanized BLT mice that renders them susceptible to both vaginal and rectal HIV infection. Other important attributes of all types of humanized mice include: 1) their small size and cost that make them broadly accessible; 2) multiple cohorts of humanized mice can be made from multiple human donors and each cohort has identical human cells, permitting control of

  10. The Use of Mesenchymal Stem Cells for the Treatment of Autoimmunity: From Animals Models to Human Disease.

    PubMed

    Fierabracci, Alessandra; Del Fattore, Andrea; Muraca, Marta; Delfino, Domenico Vittorio; Muraca, Maurizio

    2016-01-01

    Mesenchymal stem cells are multipotent progenitors able to differentiate into osteoblasts, chondrocytes and adipocytes. These cells also exhibit remarkable immune regulatory properties, which stimulated both in vitro and in vivo experimental studies to unravel the underlying mechanisms as well as extensive clinical applications. Here, we describe the effects of MSCs on immune cells and their application in animal models as well as in clinical trials of autoimmune diseases. It should be pointed out that, while the number of clinical applications is increasing steadily, results should be interpreted with caution, in order to avoid rising false expectations. Major issues conditioning clinical application are the heterogeneity of MSCs and their unpredictable behavior following therapeutic administration. However, increasing knowledge on the interaction between exogenous cell and host tissue, as well as some encouraging clinical observations suggest that the therapeutic applications of MSCs will be further expanded on firmer grounds in the near future. PMID:26201487

  11. Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models

    PubMed Central

    Wen, Li; Voronina, Svetlana; Javed, Muhammad A.; Awais, Muhammad; Szatmary, Peter; Latawiec, Diane; Chvanov, Michael; Collier, David; Huang, Wei; Barrett, John; Begg, Malcolm; Stauderman, Ken; Roos, Jack; Grigoryev, Sergey; Ramos, Stephanie; Rogers, Evan; Whitten, Jeff; Velicelebi, Gonul; Dunn, Michael; Tepikin, Alexei V.; Criddle, David N.; Sutton, Robert

    2015-01-01

    Background & Aims Sustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release–activated calcium modulator ORAI1 is the most abundant Ca2+ entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice. Methods Mouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then