Intratracheal Administration of Recombinant Human Keratinocyte Growth Factor Promotes Alveolar Epithelial Cell Proliferation during Compensatory Lung Growth in Rat.

PubMed

Furukawa, Katsuro; Matsumoto, Keitaro; Nagayasu, Takeshi; Yamamoto-Fukuda, Tomomi; Tobinaga, Shuichi; Abo, Takafumi; Yamasaki, Naoya; Tsuchiya, Tomoshi; Miyazaki, Takuro; Kamohara, Ryotaro; Nanashima, Atsushi; Obatake, Masayuki; Koji, Takehiko

2013-12-28

Keratinocyte growth factor (KGF) is considered to be one of the most important mitogens for lung epithelial cells. The objectives of this study were to confirm the effectiveness of intratracheal injection of recombinant human KGF (rhKGF) during compensatory lung growth and to optimize the instillation protocol. Here, trilobectomy in adult rat was performed, followed by intratracheal rhKGF instillation with low (0.4 mg/kg) and high (4 mg/kg) doses at various time-points. The proliferation of alveolar cells was assessed by the immunostaining for proliferating cell nuclear antigen (PCNA) in the residual lung. We also investigated other immunohistochemical parameters such as KGF, KGF receptor and surfactant protein A as well as terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Consequently, intratracheal single injection of rhKGF in high dose group significantly increased PCNA labeling index (LI) of alveolar cells in the remaining lung. Surprisingly, there was no difference in PCNA LI between low and high doses of rhKGF with daily injection, and PCNA LI reached a plateau level with 2 days-consecutive administration (about 60%). Our results indicate that even at low dose, daily intratracheal injection is effective to maintain high proliferative states during the early phase of compensatory lung growth.

Interleukin-35 administration counteracts established murine type 1 diabetes--possible involvement of regulatory T cells.

PubMed

Singh, Kailash; Kadesjö, Erik; Lindroos, Julia; Hjort, Marcus; Lundberg, Marcus; Espes, Daniel; Carlsson, Per-Ola; Sandler, Stellan; Thorvaldson, Lina

2015-07-30

The anti-inflammatory cytokine IL-35 is produced by regulatory T (Treg) cells to suppress autoimmune and inflammatory responses. The role of IL-35 in type 1 diabetes (T1D) remains to be answered. To elucidate this, we investigated the kinetics of Treg cell response in the multiple low dose streptozotocin induced (MLDSTZ) T1D model and measured the levels of IL-35 in human T1D patients. We found that Treg cells were increased in MLDSTZ mice. However, the Treg cells showed a decreased production of anti-inflammatory (IL-10, IL-35, TGF-β) and increased pro-inflammatory (IFN-γ, IL-2, IL-17) cytokines, indicating a phenotypic shift of Treg cells under T1D condition. IL-35 administration effectively both prevented development of, and counteracted established MLDSTZ T1D, seemingly by induction of Eos expression and IL-35 production in Treg cells, thus reversing the phenotypic shift of the Treg cells. IL-35 administration reversed established hyperglycemia in NOD mouse model of T1D. Moreover, circulating IL-35 levels were decreased in human T1D patients compared to healthy controls. These findings suggest that insufficient IL-35 levels play a pivotal role in the development of T1D and that treatment with IL-35 should be investigated in treatment of T1D and other autoimmune diseases.

Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Reveal Bradycardiac Effects Caused by Co-Administration of Sofosbuvir and Amiodarone.

PubMed

Yu, Yankun; Liu, Feng; He, Liuming; Ramakrishna, Seeram; Zheng, Monica; Bu, Lei; Xu, Ying

2018-05-30

Co-administration of sofosbuvir, an anti-hepatitis C virus medication, and antiarrhythmic amiodarone causes symptomatic severe bradycardia in patients and animal models. However, in a few in vitro studies, the combination of sofosbuvir and amiodarone resulted in tachycardiac effects in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). This discrepancy may be attributable to the use of immature hiPSC-CMs in the in vitro studies. To address this, we evaluated the ability of our in-house hiPSC-CMs to assess the interactions between sofosbuvir and amiodarone in vitro. We performed whole-cell patch recordings on hiPSC-CMs to examine the cardiac effect of sofosbuvir and amiodarone, alone or in combination. We found that sofosbuvir and amiodarone caused bradycardiac effects (the beating rate decreased to 75% of the vehicle control, P < 0.001) on our hiPSC-CMs when applied in combination, but they had no significant effect when applied alone. Furthermore, the bradycardiac effect was membrane potential dependent: it increased with depolarization. This raised the possibility that the bradycardiac effects in vivo may originate in nodal cells, which have a more depolarized resting membrane potential compared with ventricular cells. The bradycardiac effects of sofosbuvir plus amiodarone in vitro are consistent with the clinical phenotype and suggest that our hiPSC-CMs may serve as a useful tool in assessing cardiac safety during drug discovery and development process.

Evaluation of Hematopoietic Stem Cell Mobilization Rates with Early Plerixafor Administration for Adult Stem Cell Transplantation.

PubMed

Stover, Jessica T; Shaw, J Ryan; Kuchibhatla, Maragatha; Horwitz, Mitchell E; Engemann, Ashley M

2017-08-01

The addition of plerixafor to high-dose colony-stimulating growth factor has been shown to improve stem cell mobilization rates in autologous transplant patients with multiple myeloma and non-Hodgkin lymphoma. This study evaluates the change in administration time of plerixafor to determine if cell mobilization rates are similar between the US Food and Drug Administration-approved administration time of 11 hours before apheresis and an earlier administration time of 16 hours before apheresis. Medical records of patients age ≥ 18 years undergoing autologous stem cell transplantation requiring the use of plerixafor after at least 4 days of granulocyte colony-stimulating factor therapy to complete stem cell mobilization from January 1, 2010 through September 30, 2014 were retrospectively reviewed. The primary outcome was CD34 + cell mobilization success rates when plerixafor was administered 11 ± 2 hours (standard administration group) compared with 16 ± 2 hours before cell apheresis (early administration group), as defined as collection of  ≥2 × 10 6 CD34 + cells/kg. Secondary outcomes included the number of plerixafor therapy days required to collect a total of ≥2 × 10 6 CD34 + cells/kg, the number of apheresis cycles required to achieve ≥2 × 10 6 CD34 + cells/kg, the median CD34 + cells/kg collected in each apheresis session, and the rates of reported adverse events that occurred in the standard administration time group compared with the early administration time group. Of the 197 patients included, 114 patients received plerixafor 11 ± 2 hours before apheresis and 83 patients received plerixafor 16 hours ± 2 hours before apheresis. Ninety-four percent of patients in the early administration group achieved successful stem cell mobilization compared with 81.6% in the standard administration group (P = .0111). The median number of plerixafor days to reach the collection goal of  ≥2 × 10 6 CD34 + cells/kg was 1 day for

Suppression of Angiogenesis and Therapy of Human Colon Cancer Liver Metastasis by Systemic Administration of Interferon-α1

PubMed Central

Ozawa, Shutaro; Shinohara, Hisashi; Kanayama, Hiro-omi; Bruns, Christiane J; Bucana, Corazon D; Ellis, Lee M; Davis, Darren W; Fidler, Isaiah J

2001-01-01

Abstract The purpose of this study was to determine whether systemic administration of interferon-alpha (IFN-α) can inhibit liver metastasis produced in nude mice by human colon cancer cells. KM12L4 (IFN-α-sensitive) or KM12L4 IFNR (IFN-α-resistant) cells were injected into the spleen of nude mice. Seven days later, the mice were treated with subcutaneous (s.c.) injections of IFN-α (70,000 units/week) at different dosing schedules (1, 2, or 7 times/week). Significant inhibition of tumor growth, vascularization and expression of basic fibroblast growth factor (bFGF) or matrix metalloproteinase-9 (MMP-9) mRNA and protein occurred in mice given daily injections of IFN-α. Kinetic analysis of therapy showed that daily s.c. administrations of 10,000 units of IFN-α induced apoptosis in liver metastasis-associated endothelial cells, followed by inhibition of tumor cell division and apoptosis of tumor cells. These data suggest that the antiangiogenic activity of IFN-α-2a depends on frequent administration of the optimal biologic dose. PMID:11420751

Administrator Bridenstine Speaks at Humans to Mars Summit

NASA Image and Video Library

2018-05-09

NASA Administrator Jim Bridenstine delivered the keynote address at the Humans to Mars Summit 2018, on Wednesday, May 9 at The George Washington University, in Washington. The annual event addresses the technical, scientific and policy challenges of making human exploration of Mars a reality.

Administration of herpes simplex-thymidine kinase-expressing donor T cells with a T-cell-depleted allogeneic marrow graft.

PubMed

Tiberghien, P; Ferrand, C; Lioure, B; Milpied, N; Angonin, R; Deconinck, E; Certoux, J M; Robinet, E; Saas, P; Petracca, B; Juttner, C; Reynolds, C W; Longo, D L; Hervé, P; Cahn, J Y

2001-01-01

Administration of donor T cells expressing the herpes simplex-thymidine kinase (HS-tk) with a hematopoietic stem cell (HSC) transplantation could allow, if graft-versus-host disease (GVHD) was to occur, a selective in vivo depletion of these T cells by the use of ganciclovir (GCV). The study evaluates the feasibility of such an approach. Escalating numbers of donor HS-tk-expressing CD3(+) gene-modified cells (GMCs) are infused with a T-cell-depleted bone marrow transplantation (BMT). Twelve patients with hematological malignancies received 2 x 10(5) (n = 5), 6 x 10(5) (n = 5), or 20 x 10(5) (n = 2) donor CD3(+) GMCs/kg with a BMT from a human leukocyte antigen (HLA)-identical sibling. No acute toxicity was associated with GMC administration. An early increase of circulating GMCs followed by a progressive decrease and long-lasting circulation of GMCs was documented. GCV treatment resulted in significant rapid decrease in circulating GMCs. Three patients developed acute GVHD, with a grade of at least II, while one patient developed chronic GVHD. Treatment with GCV alone was associated with a complete remission (CR) in 2 patients with acute GVHD, while the addition of glucocorticoids was necessary to achieve a CR in the last case. Long-lasting CR occurred with GCV treatment in the patient with chronic GVHD. Unfortunately, Epstein-Barr virus-lymphoproliferative disease occurred in 3 patients. Overall, the administration of low numbers of HS-tk-expressing T cells early following an HLA-identical BMT is associated with no acute toxicity, persistent circulation of the GMCs, and GCV-sensitive GVHD. Such findings open the way to the infusion of higher numbers of gene-modified donor T cells to enhance post-BMT immune competence while preserving GCV-sensitive alloreactivity.

The Human Experience With Ghrelin Administration

PubMed Central

Garin, Margaret C.; Burns, Carrie M.; Kaul, Shailja

2013-01-01

Context: Ghrelin is an endogenous stimulator of GH and is implicated in a number of physiological processes. Clinical trials have been performed in a variety of patient populations, but there is no comprehensive review of the beneficial and adverse consequences of ghrelin administration to humans. Evidence Acquisition: PubMed was utilized, and the reference list of each article was screened. We included 121 published articles in which ghrelin was administered to humans. Evidence Synthesis: Ghrelin has been administered as an infusion or a bolus in a variety of doses to 1850 study participants, including healthy participants and patients with obesity, prior gastrectomy, cancer, pituitary disease, diabetes mellitus, eating disorders, and other conditions. There is strong evidence that ghrelin stimulates appetite and increases circulating GH, ACTH, cortisol, prolactin, and glucose across varied patient populations. There is a paucity of evidence regarding the effects of ghrelin on LH, FSH, TSH, insulin, lipolysis, body composition, cardiac function, pulmonary function, the vasculature, and sleep. Adverse effects occurred in 20% of participants, with a predominance of flushing and gastric rumbles and a mild degree of severity. The few serious adverse events occurred in patients with advanced illness and were not clearly attributable to ghrelin. Route of administration may affect the pattern of adverse effects. Conclusions: Existing literature supports the short-term safety of ghrelin administration and its efficacy as an appetite stimulant in diverse patient populations. There is some evidence to suggest that ghrelin has wider ranging therapeutic effects, although these areas require further investigation. PMID:23533240

Sequentially administrated of pemetrexed with icotinib/erlotinib in lung adenocarcinoma cell lines in vitro

PubMed Central

Feng, Xiuli; Zhang, Yan; Li, Tao; Li, Yu

2017-01-01

Combination of chemotherapy and epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) had been proved to be a potent anti-drug for the treatment of tumors. However, survival time was not extended for the patients with lung adenocarcinoma (AdC) compared with first-line chemotherapy. In the present study, we attempt to assess the optimal schedule of the combined administration of pemetrexed and icotinib/erlotinib in AdC cell lines. Human lung AdC cell lines with wild-type (A549), EGFR T790M (H1975) and activating EGFR mutation (HCC827) were applied in vitro to assess the differential efficacy of various sequential regimens on cell viability, cell apoptosis and cell cycle distribution. The results suggested that the antiproliferative effect of the sequence of pemetrexed followed by icotinib/erlotinib was more effective than that of icotinib/erlotinib followed by pemetrexed. Additionally, a reduction of G1 phase and increased S phase in sequence of pemetrexed followed by icotinib/erlotinib was also observed, promoting cell apoptosis. Thus, the sequential administration of pemetrexed followed by icotinib/erlotinib exerted a synergistic effect on HCC827 and H1975 cell lines compared with the reverse sequence. The sequential treatment of pemetrexed followed by icotinib/erlotinib has been demonstrated promising results. This treatment strategy warrants further confirmation in patients with advanced lung AdC. PMID:29371987

Sequentially administrated of pemetrexed with icotinib/erlotinib in lung adenocarcinoma cell lines in vitro.

PubMed

Feng, Xiuli; Zhang, Yan; Li, Tao; Li, Yu

2017-12-26

Combination of chemotherapy and epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) had been proved to be a potent anti-drug for the treatment of tumors. However, survival time was not extended for the patients with lung adenocarcinoma (AdC) compared with first-line chemotherapy. In the present study, we attempt to assess the optimal schedule of the combined administration of pemetrexed and icotinib/erlotinib in AdC cell lines. Human lung AdC cell lines with wild-type (A549), EGFR T790M (H1975) and activating EGFR mutation (HCC827) were applied in vitro to assess the differential efficacy of various sequential regimens on cell viability, cell apoptosis and cell cycle distribution. The results suggested that the antiproliferative effect of the sequence of pemetrexed followed by icotinib/erlotinib was more effective than that of icotinib/erlotinib followed by pemetrexed. Additionally, a reduction of G1 phase and increased S phase in sequence of pemetrexed followed by icotinib/erlotinib was also observed, promoting cell apoptosis. Thus, the sequential administration of pemetrexed followed by icotinib/erlotinib exerted a synergistic effect on HCC827 and H1975 cell lines compared with the reverse sequence. The sequential treatment of pemetrexed followed by icotinib/erlotinib has been demonstrated promising results. This treatment strategy warrants further confirmation in patients with advanced lung AdC.

Potential therapeutic effect of the secretome from human uterine cervical stem cells against both cancer and stromal cells compared with adipose tissue stem cells.

PubMed

Eiró, Noemí; Sendon-Lago, Juan; Seoane, Samuel; Bermúdez, María A; Lamelas, Maria Luz; Garcia-Caballero, Tomás; Schneider, José; Perez-Fernandez, Roman; Vizoso, Francisco J

2014-11-15

Evidences indicate that tumor development and progression towards a malignant phenotype depend not only on cancer cells themselves, but are also deeply influenced by tumor stroma reactivity. The present study uses mesenchymal stem cells from normal human uterine cervix (hUCESCs), isolated by the minimally invasive method of routine Pap cervical smear, to study their effect on the three main cell types in a tumor: cancer cells, fibroblasts and macrophages. Administration of hUCESCs-conditioned medium (CM) to a highly invasive breast cancer MDA-MB-231 cell line and to human breast tumors with high cell proliferation rates had the effect of reducing cell proliferation, modifying the cell cycle, inducing apoptosis, and decreasing invasion. In a xenograft mouse tumor model, hUCESCs-CM reduced tumor growth and increased overall survival. In cancer-associated fibroblasts, administration of hUCESCs-CM resulted in reduced cell proliferation, greater apoptosis and decreased invasion. In addition, hUCESCs-CM inhibited and reverted macrophage differentiation. The analysis of hUCESCs-CM (fresh and lyophilized) suggests that a complex paracrine signaling network could be implicated in the anti-tumor potential of hUCESCs. In light of their anti-tumor potential, the easy cell isolation method, and the fact that lyophilization of their CM conserves original properties make hUCESCs good candidates for experimental or clinical applications in anticancer therapy.

Potential therapeutic effect of the secretome from human uterine cervical stem cells against both cancer and stromal cells compared with adipose tissue stem cells

PubMed Central

Seoane, Samuel; Bermúdez, María A.; Lamelas, Maria Luz; Garcia-Caballero, Tomás; Schneider, José; Perez-Fernandez, Roman; Vizoso, Francisco J.

2014-01-01

Evidences indicate that tumor development and progression towards a malignant phenotype depend not only on cancer cells themselves, but are also deeply influenced by tumor stroma reactivity. The present study uses mesenchymal stem cells from normal human uterine cervix (hUCESCs), isolated by the minimally invasive method of routine Pap cervical smear, to study their effect on the three main cell types in a tumor: cancer cells, fibroblasts and macrophages. Administration of hUCESCs-conditioned medium (CM) to a highly invasive breast cancer MDA-MB-231 cell line and to human breast tumors with high cell proliferation rates had the effect of reducing cell proliferation, modifying the cell cycle, inducing apoptosis, and decreasing invasion. In a xenograft mouse tumor model, hUCESCs-CM reduced tumor growth and increased overall survival. In cancer-associated fibroblasts, administration of hUCESCs-CM resulted in reduced cell proliferation, greater apoptosis and decreased invasion. In addition, hUCESCs-CM inhibited and reverted macrophage differentiation. The analysis of hUCESCs-CM (fresh and lyophilized) suggests that a complex paracrine signaling network could be implicated in the anti-tumor potential of hUCESCs. In light of their anti-tumor potential, the easy cell isolation method, and the fact that lyophilization of their CM conserves original properties make hUCESCs good candidates for experimental or clinical applications in anticancer therapy. PMID:25296979

Neuroblast survival depends on mature vascular network formation after mouse stroke: role of endothelial and smooth muscle progenitor cell co-administration.

PubMed

Nih, Lina R; Deroide, Nicolas; Leré-Déan, Carole; Lerouet, Dominique; Soustrat, Mathieu; Levy, Bernard I; Silvestre, Jean-Sébastien; Merkulova-Rainon, Tatiana; Pocard, Marc; Margaill, Isabelle; Kubis, Nathalie

2012-04-01

Pro-angiogenic cell-based therapies constitute an interesting and attractive approach to enhancing post-stroke neurogenesis and decreasing neurological deficit. However, most new stroke-induced neurons die during the first few weeks after ischemia, thus impairing total recovery. Although the neovascularization process involves different cell types and various growth factors, most cell therapy protocols are based on the biological effects of single-cell-type populations or on the administration of heterogeneous populations of progenitors, namely human cord blood-derived CD34(+) cells, with scarce vascular progenitor cells. Tight cooperation between endothelial cells and smooth muscle cells/pericytes is critical for the development of functional neovessels. We hypothesized that neuroblast survival in stroke brain depends on mature vascular network formation. In this study, we injected a combination of endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SMPCs), isolated from human umbilical cord blood, into a murine model of permanent focal ischemia induced by middle cerebral artery occlusion. The co-administration of SMPCs and EPCs induced enhanced angiogenesis and vascular remodeling in the peri-infarct and infarct areas, where vessels exhibited a more mature phenotype. This activation of vessel growth resulted in the maintenance of neurogenesis and neuroblast migration to the peri-ischemic cortex. Our data suggest that a mature vascular network is essential for neuroblast survival after cerebral ischemia, and that co-administration of EPCs and SMPCs may constitute a novel therapeutic strategy for improving the treatment of stroke. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

Long-term administration of inulin-type fructans has no significant lipid-lowering effect in normolipidemic humans.

PubMed

Forcheron, Fabien; Beylot, Michel

2007-08-01

Short-term studies have shown that the addition to diet of inulin-type fructans, a nondigestible carbohydrate, may have a plasma lipid-lowering effect in humans. Whether this beneficial effect persists during long-term administration has not been determined. The study was aimed at determining whether a prolonged (6 months) administration of inulin-type fructans to healthy subjects has a lipid-lowering action. In a double-blind, randomized, placebo-controlled study, 17 healthy subjects were studied before and after 6 months of daily administration of placebo (8 subjects) or 10 g of a mix of inulin and oligofructose (9 subjects). During this 6-month period, they consumed their usual diet and did not modify their everyday way of life. We measured plasma lipid concentrations; cholesterol synthesis and hepatic lipogenesis; and adipose tissue and circulating mononuclear cell messenger RNA concentrations of key regulatory genes of cholesterol metabolism. Compared with the administration of placebo, the administration of inulin-type fructans had no effect on plasma triacylglycerol concentrations and hepatic lipogenesis and induced only a nonsignificant trend for decreased plasma total and low-density lipoprotein cholesterol levels and increased high-density lipoprotein cholesterol concentration. Cholesterol synthesis was not significantly modified. Of all the messenger RNA concentrations measured, none was significantly modified by the administration of inulin-type fructans. In conclusion, contrary to what was observed in short-term studies, we observed no significant beneficial effect of a long-term (6-month) administration of inulin-type fructans on plasma lipids in healthy human subjects.

Cell Permeability, Migration, and Reactive Oxygen Species Induced by Multi-Walled Carbon Nanotubes in Human Microvascular Endothelial Cells

PubMed Central

Pacurari, M; Qian, Y; Fu, W; Schwegler-Berry, D; Ding, M; Castranova, V; Guo, NL

2011-01-01

Multi-walled carbon nanotubes (MWCNT) have elicited great interest in biomedical applications due to their extraordinary physical, chemical, and optical properties. Intravenous administration of MWCNT-based medical imaging agents and drugs in animal models was utilized. However, the potential harmful health effects of MWCNT administration in humans have not yet been elucidated. Furthermore, to date, there are no apparent reports regarding the precise mechanisms of translocation of MWCNT into target tissues and organs from blood circulation. This study demonstrates that exposure to MWCNT leads to an increase in cell permeability in human microvascular endothelial cells (HMVEC). The results obtained from this study also showed that the MWCNT-induced rise in endothelial permeability is mediated by reactive oxygen species (ROS) production and actin filament remodeling. In addition, it was found that MWCNT promoted cell migration in HMVEC. Mechanistically, MWCNT exposure elevated the levels of monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule 1 (ICAM-1) in HMVEC. Taken together, these results provide new insights into the bioreactivity of MWCNT, which may have implications in the biomedical application of MWCNT in vascular targeting, imaging, and drug delivery. The results generated from this study also elucidate the potential adverse effects of MWCNT exposure on humans at the cellular level. PMID:22129238

Lipid-core nanocapsules as a nanomedicine for parenteral administration of tretinoin: development and in vitro antitumor activity on human myeloid leukaemia cells.

PubMed

Ourique, A F; Azoubel, S; Ferreira, C V; Silva, C B; Marchiori, M C L; Pohlmann, A R; Guterres, S S; Beck, R C R

2010-06-01

Tretinoin-loaded conventional nanocapsules have showed a significant protection of this drug against UVC radiation. However, this formulation presents a limited stability on storage. We hypothesized that the association of tretinoin to lipid-core nanocapsules could increase the physicochemical stability of such formulations, focusing on the development of a reliable nanomedicine for parenteral administration. However, this advantage should still be accompanied by the known photoprotective effect of conventional polymeric nanocapsules against the exposure of tretinoin to UV radiation. Results showed that tretinoin-loaded lipid-core nanocapsules improved the physicochemical stability of formulations under storage, without changing their ability to protect tretinoin either against UVA or UVC radiation. In addition, the effect of nanoencapsulation on the antiproliferative and differentiation properties of tretinoin was studied on human myeloid leukemia cells (HL60 cells) showing that tretinoin-loaded lipid-core nanocapsules presents a longer antitumor efficiency compared to the free tretinoin. These results allow us to propose the current formulation (tretinoin-loaded lipid-core nanocapsules) as a promising parenteral nanomedicine for the treatment of acute promyelocytic leukaemia.

The Human Cell Atlas.

PubMed

Regev, Aviv; Teichmann, Sarah A; Lander, Eric S; Amit, Ido; Benoist, Christophe; Birney, Ewan; Bodenmiller, Bernd; Campbell, Peter; Carninci, Piero; Clatworthy, Menna; Clevers, Hans; Deplancke, Bart; Dunham, Ian; Eberwine, James; Eils, Roland; Enard, Wolfgang; Farmer, Andrew; Fugger, Lars; Göttgens, Berthold; Hacohen, Nir; Haniffa, Muzlifah; Hemberg, Martin; Kim, Seung; Klenerman, Paul; Kriegstein, Arnold; Lein, Ed; Linnarsson, Sten; Lundberg, Emma; Lundeberg, Joakim; Majumder, Partha; Marioni, John C; Merad, Miriam; Mhlanga, Musa; Nawijn, Martijn; Netea, Mihai; Nolan, Garry; Pe'er, Dana; Phillipakis, Anthony; Ponting, Chris P; Quake, Stephen; Reik, Wolf; Rozenblatt-Rosen, Orit; Sanes, Joshua; Satija, Rahul; Schumacher, Ton N; Shalek, Alex; Shapiro, Ehud; Sharma, Padmanee; Shin, Jay W; Stegle, Oliver; Stratton, Michael; Stubbington, Michael J T; Theis, Fabian J; Uhlen, Matthias; van Oudenaarden, Alexander; Wagner, Allon; Watt, Fiona; Weissman, Jonathan; Wold, Barbara; Xavier, Ramnik; Yosef, Nir

2017-12-05

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

The Human Cell Atlas

PubMed Central

Amit, Ido; Benoist, Christophe; Birney, Ewan; Bodenmiller, Bernd; Campbell, Peter; Carninci, Piero; Clatworthy, Menna; Clevers, Hans; Deplancke, Bart; Dunham, Ian; Eberwine, James; Eils, Roland; Enard, Wolfgang; Farmer, Andrew; Fugger, Lars; Göttgens, Berthold; Hacohen, Nir; Haniffa, Muzlifah; Hemberg, Martin; Kim, Seung; Klenerman, Paul; Kriegstein, Arnold; Lein, Ed; Linnarsson, Sten; Lundberg, Emma; Lundeberg, Joakim; Majumder, Partha; Marioni, John C; Merad, Miriam; Mhlanga, Musa; Nawijn, Martijn; Netea, Mihai; Nolan, Garry; Pe'er, Dana; Phillipakis, Anthony; Ponting, Chris P; Quake, Stephen; Reik, Wolf; Rozenblatt-Rosen, Orit; Sanes, Joshua; Satija, Rahul; Schumacher, Ton N; Shalek, Alex; Shapiro, Ehud; Sharma, Padmanee; Shin, Jay W; Stegle, Oliver; Stratton, Michael; Stubbington, Michael J T; Theis, Fabian J; Uhlen, Matthias; van Oudenaarden, Alexander; Wagner, Allon; Watt, Fiona; Weissman, Jonathan; Wold, Barbara; Xavier, Ramnik; Yosef, Nir

2017-01-01

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community. PMID:29206104

Human mesenchymal stem cells suppress donor CD4(+) T cell proliferation and reduce pathology in a humanized mouse model of acute graft-versus-host disease.

PubMed

Tobin, L M; Healy, M E; English, K; Mahon, B P

2013-05-01

Acute graft-versus-host disease (aGVHD) is a life-threatening complication following allogeneic haematopoietic stem cell transplantation (HSCT), occurring in up to 30-50% of patients who receive human leucocyte antigen (HLA)-matched sibling transplants. Current therapies for steroid refractory aGVHD are limited, with the prognosis of patients suboptimal. Mesenchymal stem or stromal cells (MSC), a heterogeneous cell population present in many tissues, display potent immunomodulatory abilities. Autologous and allogeneic ex-vivo expanded human MSC have been utilized to treat aGVHD with promising results, but the mechanisms of therapeutic action remain unclear. Here a robust humanized mouse model of aGVHD based on delivery of human peripheral blood mononuclear cells (PBMC) to non-obese diabetic (NOD)-severe combined immunodeficient (SCID) interleukin (IL)-2rγ(null) (NSG) mice was developed that allowed the exploration of the role of MSC in cell therapy. MSC therapy resulted in the reduction of liver and gut pathology and significantly increased survival. Protection was dependent upon the timing of MSC therapy, with conventional MSC proving effective only after delayed administration. In contrast, interferon (IFN)-γ-stimulated MSC were effective when delivered with PBMC. The beneficial effect of MSC therapy in this model was not due to the inhibition of donor PBMC chimerism, as CD45(+) and T cells engrafted successfully in this model. MSC therapy did not induce donor T cell anergy, FoxP3(+) T regulatory cells or cause PBMC apoptosis in this model; however, it was associated with the direct inhibition of donor CD4(+) T cell proliferation and reduction of human tumour necrosis factor-α in serum. © 2012 British Society for Immunology.

Intranasal Neuropeptide Administration To Target the Human Brain in Health and Disease.

PubMed

Spetter, Maartje S; Hallschmid, Manfred

2015-08-03

Central nervous system control of metabolic function relies on the input of endocrine messengers from the periphery, including the pancreatic hormone insulin and the adipokine leptin. This concept primarily derives from experiments in animals where substances can be directly applied to the brain. A feasible approach to study the impact of peptidergic messengers on brain function in humans is the intranasal (IN) route of administration, which bypasses the blood-brain barrier and delivers neuropeptides to the brain compartment, but induces considerably less, if any, peripheral uptake than other administration modes. Experimental IN insulin administration has been extensively used to delineate the role of brain insulin signaling in the control of energy homeostasis, but also cognitive function in healthy humans. Clinical pilot studies have found beneficial effects of IN insulin in patients with memory deficits, suggesting that the IN delivery of this and other peptides bears some promise for new, selectively brain-targeted pharmaceutical approaches in the treatment of metabolic and cognitive disorders. More recently, experiments relying on the IN delivery of the hypothalamic hormone oxytocin, which is primarily known for its involvement in psychosocial processes, have provided evidence that oxytocin influences metabolic control in humans. The IN administration of leptin has been successfully tested in animal models but remains to be investigated in the human setting. We briefly summarize the literature on the IN administration of insulin, leptin, and oxytocin, with a particular focus on metabolic effects, and address limitations and perspectives of IN neuropeptide administration.

Fluorescent nanodiamonds enable quantitative tracking of human mesenchymal stem cells in miniature pigs

NASA Astrophysics Data System (ADS)

Su, Long-Jyun; Wu, Meng-Shiue; Hui, Yuen Yung; Chang, Be-Ming; Pan, Lei; Hsu, Pei-Chen; Chen, Yit-Tsong; Ho, Hong-Nerng; Huang, Yen-Hua; Ling, Thai-Yen; Hsu, Hsao-Hsun; Chang, Huan-Cheng

2017-03-01

Cell therapy is a promising strategy for the treatment of human diseases. While the first use of cells for therapeutic purposes can be traced to the 19th century, there has been a lack of general and reliable methods to study the biodistribution and associated pharmacokinetics of transplanted cells in various animal models for preclinical evaluation. Here, we present a new platform using albumin-conjugated fluorescent nanodiamonds (FNDs) as biocompatible and photostable labels for quantitative tracking of human placenta choriodecidual membrane-derived mesenchymal stem cells (pcMSCs) in miniature pigs by magnetic modulation. With this background-free detection technique and time-gated fluorescence imaging, we have been able to precisely determine the numbers as well as positions of the transplanted FND-labeled pcMSCs in organs and tissues of the miniature pigs after intravenous administration. The method is applicable to single-cell imaging and quantitative tracking of human stem/progenitor cells in rodents and other animal models as well.

Morphofunctional study of the therapeutic efficacy of human mesenchymal and neural stem cells in rats with diffuse brain injury.

PubMed

Tsyb, A F; Yuzhakov, V V; Roshal', L M; Sukhikh, G T; Konoplyannikov, A G; Sushkevich, G N; Yakovleva, N D; Ingel', I E; Bandurko, L N; Sevan'kaeva, L E; Mikhina, L N; Fomina, N K; Marei, M V; Semenova, Zh B; Konoplyannikova, O A; Kal'sina, S Sh; Lepekhina, L A; Semenkova, I V; Agaeva, E V; Shevchuk, A S; Pavlova, L N; Tokarev, O Yu; Karaseva, O V; Chernyshova, T A

2009-01-01

We studied the effect of transplantation of human stem cells from various tissues on reparative processes in the brain of rats with closed craniocerebral injury. Combined treatment with standard drugs and systemic administration of xenogeneic stem cells had a neuroprotective effect. The morphology of neurons rapidly returned to normal after administration of fetal neural stem cells. Fetal mesenchymal stem cells produced a prolonged effect on proliferative activity of progenitor cells in the subventricular zone of neurogenesis. Adult mesenchymal stem cells had a strong effect on recovery of the vascular bed in ischemic regions.

Human Dental Pulp Stem Cells Are More Effective Than Human Bone Marrow-Derived Mesenchymal Stem Cells in Cerebral Ischemic Injury.

PubMed

Song, Miyeoun; Lee, Jae-Hyung; Bae, Jinhyun; Bu, Youngmin; Kim, Eun-Cheol

2017-06-09

We compared the therapeutic effects and mechanism of transplanted human dental pulp stem cells (hDPSCs) and human bone marrow-derived mesenchymal stem cells (hBM-MSCs) in a rat stroke model and an in vitro model of ischemia. Rats were intravenously injected with hDPSCs or hBM-MSCs 24 h after middle cerebral artery occlusion (MCAo), and both groups showed improved functional recovery and reduced infarct volume versus control rats, but the hDPSC group showed greater reduction in infarct volume than the hBM-MSC group. The positive area for the endothelial cell marker was greater in the lesion boundary areas in the hDPSC group than in the hBM-MSC group. Administration of hDPSCs to rats with stroke significantly decreased reactive gliosis, as evidenced by the attenuation of MCAo-induced GFAP+/nestin+ and GFAP+/Musashi-1+ cells, compared with hBM-MSCs. In vivo findings were confirmed by in vitro data illustrating that hDPSCs showed superior neuroprotective, migratory, and in vitro angiogenic effects in oxygen-glucose deprivation (OGD)-injured human astrocytes (hAs) versus hBM-MSCs. Comprehensive comparative bioinformatics analyses from hDPSC- and hBM-MSC-treated in vitro OGD-injured hAs were examined by RNA sequencing technology. In gene ontology and KEGG pathway analyses, significant pathways in the hDPSC-treated group were the MAPK and TGF-β signaling pathways. Thus, hDPSCs may be a better cell therapy source for ischemic stroke than hBM-MSCs.

Improved memory for reward cues following acute buprenorphine administration in humans.

PubMed

Syal, Supriya; Ipser, Jonathan; Terburg, David; Solms, Mark; Panksepp, Jaak; Malcolm-Smith, Susan; Bos, Peter A; Montoya, Estrella R; Stein, Dan J; van Honk, Jack

2015-03-01

In rodents, there is abundant evidence for the involvement of the opioid system in the processing of reward cues, but this system has remained understudied in humans. In humans, the happy facial expression is a pivotal reward cue. Happy facial expressions activate the brain's reward system and are disregarded by subjects scoring high on depressive mood who are low in reward drive. We investigated whether a single 0.2mg administration of the mixed mu-opioid agonist/kappa-antagonist, buprenorphine, would influence short-term memory for happy, angry or fearful expressions relative to neutral faces. Healthy human subjects (n38) participated in a randomized placebo-controlled within-subject design, and performed an emotional face relocation task after administration of buprenorphine and placebo. We show that, compared to placebo, buprenorphine administration results in a significant improvement of memory for happy faces. Our data demonstrate that acute manipulation of the opioid system by buprenorphine increases short-term memory for social reward cues. Copyright © 2015. Published by Elsevier Ltd.

Redistribution, Hyperproliferation, Activation of Natural Killer Cells and CD8 T Cells, and Cytokine Production During First-in-Human Clinical Trial of Recombinant Human Interleukin-15 in Patients With Cancer

PubMed Central

Conlon, Kevin C.; Lugli, Enrico; Welles, Hugh C.; Rosenberg, Steven A.; Fojo, Antonio Tito; Morris, John C.; Fleisher, Thomas A.; Dubois, Sigrid P.; Perera, Liyanage P.; Stewart, Donn M.; Goldman, Carolyn K.; Bryant, Bonita R.; Decker, Jean M.; Chen, Jing; Worthy, Tat'Yana A.; Figg, William D.; Peer, Cody J.; Sneller, Michael C.; Lane, H. Clifford; Yovandich, Jason L.; Creekmore, Stephen P.; Roederer, Mario; Waldmann, Thomas A.

2015-01-01

Purpose Interleukin-15 (IL-15) has significant potential in cancer immunotherapy as an activator of antitumor CD8 T and natural killer (NK) cells. The primary objectives of this trial were to determine safety, adverse event profile, dose-limiting toxicity, and maximum-tolerated dose of recombinant human IL-15 (rhIL-15) administered as a daily intravenous bolus infusion for 12 consecutive days in patients with metastatic malignancy. Patients and Methods We performed a first in-human trial of Escherichia coli–produced rhIL-15. Bolus infusions of 3.0, 1.0, and 0.3 μg/kg per day of IL-15 were administered for 12 consecutive days to patients with metastatic malignant melanoma or metastatic renal cell cancer. Results Flow cytometry of peripheral blood lymphocytes revealed dramatic efflux of NK and memory CD8 T cells from the circulating blood within minutes of IL-15 administration, followed by influx and hyperproliferation yielding 10-fold expansions of NK cells that ultimately returned to baseline. Up to 50-fold increases of serum levels of multiple inflammatory cytokines were observed. Dose-limiting toxicities observed in patients receiving 3.0 and 1.0 μg/kg per day were grade 3 hypotension, thrombocytopenia, and elevations of ALT and AST, resulting in 0.3 μg/kg per day being determined the maximum-tolerated dose. Indications of activity included clearance of lung lesions in two patients. Conclusion IL-15 could be safely administered to patients with metastatic malignancy. IL-15 administration markedly altered homeostasis of lymphocyte subsets in blood, with NK cells and γδ cells most dramatically affected, followed by CD8 memory T cells. To reduce toxicity and increase efficacy, alternative dosing strategies have been initiated, including continuous intravenous infusions and subcutaneous IL-15 administration. PMID:25403209

Development of an orally-administrative MELK-targeting inhibitor that suppresses the growth of various types of human cancer.

PubMed

Chung, Suyoun; Suzuki, Hanae; Miyamoto, Takashi; Takamatsu, Naofumi; Tatsuguchi, Ayako; Ueda, Koji; Kijima, Kyoko; Nakamura, Yusuke; Matsuo, Yo

2012-12-01

We previously reported MELK (maternal embryonic leucine zipper kinase) as a novel therapeutic target for breast cancer. MELK was also reported to be highly upregulated in multiple types of human cancer. It was implied to play indispensable roles in cancer cell survival and indicated its involvement in the maintenance of tumor-initiating cells. We conducted a high-throughput screening of a compound library followed by structure-activity relationship studies, and successfully obtained a highly potent MELK inhibitor OTSSP167 with IC₅₀ of 0.41 nM. OTSSP167 inhibited the phosphorylation of PSMA1 (proteasome subunit alpha type 1) and DBNL (drebrin-like), which we identified as novel MELK substrates and are important for stem-cell characteristics and invasiveness. The compound suppressed mammosphere formation of breast cancer cells and exhibited significant tumor growth suppression in xenograft studies using breast, lung, prostate, and pancreas cancer cell lines in mice by both intravenous and oral administration. This MELK inhibitor should be a promising compound possibly to suppress the growth of tumor-initiating cells and be applied for treatment of a wide range of human cancer.

Effect of human umbilical cord blood stem cell transplantation on oval cell response in 2-AAF/CCL4 liver injury model: experimental immunohistochemical study.

PubMed

Abdellatif, Hussein; Shiha, Gamal; Saleh, Dalia M; Eltahry, Huda; Botros, Kamal G

2017-01-01

Oval cells, specific liver progenitors, are activated in response to injury. The human umbilical cord blood (hUCB) is a possible source of transplantable hepatic progenitors and can be used in cases of severe liver injury. We detected the effect of hUCB stem cell transplantation on natural response of oval cells to injury. Twenty-four female albino rats were randomly divided into three groups: (A) control, (B) liver injury with hepatocyte block, and (C) hUCB transplanted group. Hepatocyte block was performed by administration of 2-acetylaminofluorene (2-AAF) for 12 days. CCL4 was administrated at day 5 from experiment start. Animals were sacrificed at 9 days post CCL4 administration, and samples were collected for biochemical and histopathological analysis. Oval cell response to injury was evaluated by the percentage of oval cells in the liver tissue and frequency of cells incorporated into new ducts. Immunohistochemical analysis of oval cell response to injury was performed. There was significant deviation in the hUCB-transplanted (4.9 ± 1.4) and liver injury groups (2.4 ± 0.9) as compared to control (0.89 ± 0.4) 9 days post injury. Detection of oval cell response was dependant on OV-6 immunoreactivity. For mere localization of cells with human origin, CD34 antihuman immunoreactivity was performed. There was no significant difference in endogenous OV-6 immunoreactivity following stem cell transplantation as compared to the liver injury group. In vivo transplantation of cord blood stem cells (hUCB) does not interfere with natural oval cell response to liver injury.

Homing of human B cells to lymphoid organs and B-cell lymphoma engraftment are controlled by cell adhesion molecule JAM-C.

PubMed

Doñate, Carmen; Ody, Christiane; McKee, Thomas; Ruault-Jungblut, Sylvie; Fischer, Nicolas; Ropraz, Patricia; Imhof, Beat A; Matthes, Thomas

2013-01-15

Junctional adhesion molecule C (JAM-C) is expressed by vascular endothelium and human but not mouse B lymphocytes. The level of JAM-C expression defines B-cell differentiation stages and allows the classification of marginal zone-derived (JAM-C-positive) and germinal center-derived (JAM-C-negative) B-cell lymphomas. In the present study, we investigated the role of JAM-C in homing of human B cells, using a xenogeneic nonobese diabetic/severe combined immunodeficient mouse model. Treatment with anti-JAM-C antibodies in short-term experiments reduced migration of normal and malignant JAM-C-expressing B cells to bone marrow, lymph nodes, and spleen. Blocking homing to the spleen is remarkable, as most other antiadhesion antibodies reduce homing of B cells only to bone marrow and lymph nodes. Long-term administration of anti-JAM-C antibodies prevented engraftment of JAM-Cpos lymphoma cells in bone marrow, spleen, and lymph nodes of mice. Plasmon resonance studies identified JAM-B as the major ligand for JAM-C, whereas homotypic JAM-C interactions remained at background levels. Accordingly, anti-JAM-C antibodies blocked adhesion of JAM-C-expressing B cells to their ligand JAM-B, and immunofluorescence analysis showed the expression of JAM-B on murine and human lymphatic endothelial cells. Targeting JAM-C could thus constitute a new therapeutic strategy to prevent lymphoma cells from reaching supportive microenvironments not only in the bone marrow and lymph nodes but also in the spleen.

Relevance Revisited: Curriculum Development in the Humanities. No. II: Administrative Strategies for Curriculum Change.

ERIC Educational Resources Information Center

Mondale, Clarence C., Ed.

Papers presented in advance of a workshop on "administrative strategies" for curricular change in the humanities and brief summaries of discussions taking place at the workshop are provided. Background papers include: "Curricular Change and the Humanities," by Edward A. Lindell; "Developing Administrative Strategies for…

Targeting eradication of malignant cells derived from human bone marrow mesenchymal stromal cells

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

Yang, Yingbin; School of Life Science, Southwest University, Chongqing 400715; Cai, Shaoxi, E-mail: sxcai@cqu.edu.cn

2010-12-10

Human bone marrow mesenchymal stromal cells (hBMSC) have been shown to participate in malignant transformation. However, hampered by the low frequency of malignant transformation of hBMSC, we do not yet know how to prevent malignant transformation of implanted hBMSC. In this study, in order to establish a model for the eradication of hBMSC-derived malignant cells, a gene fusion consisting of a human telomerase (hTERT) promoter modified with both c-Myc and myeloid zinc finger protein2 (MZF-2) binding elements and followed by the E. coli cytosine deaminase (CD) and luciferase genes was stably transferred into hBMSC via lentiviral transduction; n-phosphonacelyl-L-aspartic acid (PALA)more » selection was used to generate malignant cell colonies derived from transduced hBMSC after treatment with the carcinogenic reagent BPDE. Cells that were amplified after PALA selection were used for transplantation and 5-FC pro-drug cytotoxicity tests. The results showed that PALA-resistant malignant cells could be generated from hBMSC co-induced with lentiviral transduction and treatment with Benzo(a)pyrene Diol Epoxide (BPDE); the modification of c-Myc and MZF-2 binding elements could remarkably enhance the transcriptional activities of the hTERT promoter in malignant cells, whereas transcriptional activity was depressed in normal hBMSC; malignant cells stably expressing CD under the control of the modified hTERT promoter could be eliminated by 5-FC administration. This study has provided a method for targeted eradication of malignant cells derived from hBMSC.« less

Human innate lymphoid cells.

PubMed

Montaldo, Elisa; Vacca, Paola; Vitale, Chiara; Moretta, Francesca; Locatelli, Franco; Mingari, Maria Cristina; Moretta, Lorenzo

2016-11-01

The interest in innate lymphoid cells (ILC) has rapidly grown during the last decade. ILC include distinct cell types that are collectively involved in host protection against pathogens and tumor cells and in the regulation of tissue homeostasis. Studies in mice enabled a broad characterization of ILC function and of their developmental requirements. In humans all mature ILC subsets have been characterized and their role in the pathogenesis of certain disease is emerging. Nonetheless, still limited information is available on human ILC development. Indeed, only the cell precursors committed toward NK cells or ILC3 have been described. Here, we review the most recent finding on human mature ILC, discussing their tissue localization and function. Moreover, we summarize the available data regarding human ILC development. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

PubMed

Unger, Christian; Felldin, Ulrika; Rodin, Sergey; Nordenskjöld, Agneta; Dilber, Sirac; Hovatta, Outi

2016-02-03

After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts. Copyright © 2016 John Wiley & Sons, Inc.

Engineering human T cells for resistance to methotrexate and mycophenolate mofetil as an in vivo cell selection strategy.

PubMed

Jonnalagadda, Mahesh; Brown, Christine E; Chang, Wen-Chung; Ostberg, Julie R; Forman, Stephen J; Jensen, Michael C

2013-01-01

Gene transfer and drug selection systems that enforce ongoing transgene expression in vitro and in vivo which are compatible with human pharmaceutical drugs are currently underdeveloped. Here, we report on the utility of incorporating human enzyme muteins that confer resistance to the lymphotoxic/immunosuppressive drugs methotrexate (MTX) and mycophenolate mofetil (MMF) in a multicistronic lentiviral vector for in vivo T lymphocyte selection. We found that co-expression of human dihydrofolate reductase (DHFR(FS); L22F, F31S) and inosine monophosphate dehydrogenase II (IMPDH2(IY); T333I, S351Y) conferred T cell resistance to the cytocidal and anti-proliferative effects of these drugs at concentrations that can be achieved clinically (up to 0.1 µM MTX and 1.0 µM MPA). Furthermore, using a immunodeficient mouse model that supports the engraftment of central memory derived human T cells, in vivo selection studies demonstrate that huEGFRt(+)DHFR(FS+)IMPDH2(IY+) T cells could be enriched following adoptive transfer either by systemic administration of MTX alone (4.4 -fold), MMF alone (2.9-fold), or combined MTX and MMF (4.9-fold). These findings demonstrate the utility of both DHFR(FS)/MTX and IMPDH2(IY)/MMF for in vivo selection of lentivirally transduced human T cells. Vectors incorporating these muteins in combination with other therapeutic transgenes may facilitate the selective engraftment of therapeutically active cells in recipients.

Administration of CD34+ cells after stroke enhances neurogenesis via angiogenesisin a mouse model

PubMed Central

Taguchi, Akihiko; Soma, Toshihiro; Tanaka, Hidekazu; Kanda, Takayoshi; Nishimura, Hiroyuki; Yoshikawa, Hiroo; Tsukamoto, Yoshitane; Iso, Hiroyuki; Fujimori, Yoshihiro; Stern, David M.; Naritomi, Hiroaki; Matsuyama, Tomohiro

2004-01-01

Thrombo-occlusive cerebrovascular disease resulting in stroke and permanent neuronal loss is an important cause of morbidity and mortality. Because of the unique properties of cerebral vasculature and the limited reparative capability of neuronal tissue, it has been difficult to devise effective neuroprotective therapies in cerebral ischemia. Our results demonstrate that systemic administration of human cord blood–derived CD34+ cells to immunocompromised mice subjected to stroke 48 hours earlier induces neovascularization in the ischemic zone and provides a favorable environment for neuronal regeneration. Endogenous neurogenesis, suppressed by an antiangiogenic agent, is accelerated as a result of enhanced migration of neuronal progenitor cells to the damaged area, followed by their maturation and functional recovery. Our data suggest an essential role for CD34+ cells in promoting directly or indirectly an environment conducive to neovascularization of ischemic brain so that neuronal regeneration can proceed. PMID:15286799

Administration of interleukin-12 enhances the therapeutic efficacy of dendritic cell-based tumor vaccines in mouse hepatocellular carcinoma.

PubMed

Tatsumi, T; Takehara, T; Kanto, T; Miyagi, T; Kuzushita, N; Sugimoto, Y; Jinushi, M; Kasahara, A; Sasaki, Y; Hori, M; Hayashi, N

2001-10-15

Dendritic cells (DCs) are potent antigen-presenting cells that are capable of priming systemic antitumor immune response. Here, we evaluated the combined effectiveness of tumor lysate-pulsed DC immunization and interleukin (IL)-12 administration on the induction of antitumor immunity in a mouse hepatocellular carcinoma (HCC) model. Mouse DCs were pulsed with lysate of BNL 1ME A.7R.1 (BNL), a BALB/c-derived HCC cell line, and then injected into syngeneic mice in combination with systemic administration of IL-12. Lymphocytes from mice treated with BNL lysate-pulsed DCs and IL-12 showed stronger cytolytic activity and produced higher amounts of IFN-gamma than those from mice treated with BNL lysate-pulsed DCs alone. Although immunization with BNL lysate-pulsed DCs alone did not lead to complete regression of established tumors, it significantly inhibited tumor growth compared with vehicle injection. Importantly, the combined therapy of BNL lysate-pulsed DCs and IL-12 resulted in tumor rejection or significant inhibition of tumor growth compared with mice treated with BNL lysate-pulsed DCs alone. In vivo lymphocyte depletion experiments demonstrated that this combination was dependent on both CD8+ and CD4+ T cells, but not natural killer cells. These results demonstrated that IL-12 administration enhanced the therapeutic effect of immunization of tumor lysate-pulsed DCs against HCC in mice. This combination of IL-12 and DCs may be useful for suppressing the growth of residual tumor after primary therapy of human HCC.

Genome engineering in human cells.

PubMed

Song, Minjung; Kim, Young-Hoon; Kim, Jin-Soo; Kim, Hyongbum

2014-01-01

Genome editing in human cells is of great value in research, medicine, and biotechnology. Programmable nucleases including zinc-finger nucleases, transcription activator-like effector nucleases, and RNA-guided engineered nucleases recognize a specific target sequence and make a double-strand break at that site, which can result in gene disruption, gene insertion, gene correction, or chromosomal rearrangements. The target sequence complexities of these programmable nucleases are higher than 3.2 mega base pairs, the size of the haploid human genome. Here, we briefly introduce the structure of the human genome and the characteristics of each programmable nuclease, and review their applications in human cells including pluripotent stem cells. In addition, we discuss various delivery methods for nucleases, programmable nickases, and enrichment of gene-edited human cells, all of which facilitate efficient and precise genome editing in human cells.

AHR Activation Is Protective against Colitis Driven by T Cells in Humanized Mice.

PubMed

Goettel, Jeremy A; Gandhi, Roopali; Kenison, Jessica E; Yeste, Ada; Murugaiyan, Gopal; Sambanthamoorthy, Sharmila; Griffith, Alexandra E; Patel, Bonny; Shouval, Dror S; Weiner, Howard L; Snapper, Scott B; Quintana, Francisco J

2016-10-25

Existing therapies for inflammatory bowel disease that are based on broad suppression of inflammation result in variable clinical benefit and unwanted side effects. A potential therapeutic approach for promoting immune tolerance is the in vivo induction of regulatory T cells (Tregs). Here we report that activation of the aryl hydrocarbon receptor using the non-toxic agonist 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) induces human Tregs in vitro that suppress effector T cells through a mechanism mediated by CD39 and Granzyme B. We then developed a humanized murine system whereby human CD4 + T cells drive colitis upon exposure to 2,4,6-trinitrobenzenesulfonic acid and assessed ITE as a potential therapeutic. ITE administration ameliorated colitis in humanized mice with increased CD39, Granzyme B, and IL10-secreting human Tregs. These results develop an experimental model to investigate human CD4 + T responses in vivo and identify the non-toxic AHR agonist ITE as a potential therapy for promoting immune tolerance in the intestine. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Tolerability, usability and acceptability of dissolving microneedle patch administration in human subjects

PubMed Central

Arya, Jaya; Henry, Sebastien; Kalluri, Haripriya; McAllister, Devin V.; Pewin, Winston P.; Prausnitz, Mark R.

2017-01-01

To support translation of microneedle patches from pre-clinical development into clinical trials, this study examined the effect of microneedle patch application on local skin reactions, reliability of use and acceptability to patients. Placebo patches containing dissolving microneedles were administered to fifteen human participants. Microneedle patches were well tolerated in the skin with no pain or swelling and only mild erythema localized to the site of patch administration that resolved fully within seven days. Microneedle patches could be administered by hand without the need of an applicator and delivery efficiencies were similar for investigator-administration and self-administration. Microneedle patch administration was not considered painful and the large majority of subjects were somewhat or fully confident that they self-administered patches correctly. Microneedle patches were overwhelmingly preferred over conventional needle and syringe injection. Altogether, these results demonstrate that dissolving microneedle patches were well tolerated, easily usable and strongly accepted by human subjects, which will facilitate further clinical translation of this technology. PMID:28285193

Emodin induces apoptosis of human osteosarcoma cells via mitochondria- and endoplasmic reticulum stress-related pathways.

PubMed

Ying, Jinhe; Xu, Huan; Wu, Dhua; Wu, Xiaoguang

2015-01-01

Emodin showed anti-cancer activity against multiple human malignant tumors by inducing apoptosis. However, the apoptotic inducing effect against human osteosarcoma and related mechanism are still not studied. This study was aimed to investigate them. Emodin was used to incubate human OS cell U2OS cells at serially diluted concentrations. Hoechst staining was used to evaluate apoptosis; flow cytometry was applied to assess the collapse of mitochondrial membrane potential (MMP); intracellular ROS generation was detected by DCFH-DA staining; endoplasmic reticulum stress activation was examined by western blotting. Cell apoptosis of U2OS cells was induced by emodin incubation in a concentration-dependent manner; MMP collapse and ROS generation were identified at starting concentration of 80 μmol/L of emodin in a concentration-dependent manner. ER stress activation was found at beginning concentration of 40 μmol/L of emodin. The MMP collapse was inhibited while the ER stress was not inhibited by NAC administration. Emodin induces death of human osteosarcoma cells by initiating ROS-dependent mitochondria-induced and ROS-independent ER stress-induced apoptosis.

The role of human drug self-administration procedures in the development of medications

PubMed Central

Comer, SD; Ashworth, JB; Foltin, RW; Johanson, CE; Zacny, JP; Walsh, SL

2008-01-01

The purpose of this review is to illustrate the utility and value of employing human self-administration procedures in medication development, including abuse liability assessments of novel medications and evaluation of potential pharmacotherapies for substance use disorders. Traditionally, human abuse liability testing has relied primarily on subjective reports describing drug action by use of questionnaires; similarly, drug interactions between putative treatment agents and the drugs of abuse have relied on these measures. Subjective reports are highly valued because they provide qualitative and quantitative information about the characteristics of central and peripheral pharmacodynamic effects as well as safety and tolerability. However, self-administration procedures directly examine the behavior of interest – that is, drug taking. The present paper 1) reviews the most commonly used human self-administration procedures, 2) discusses the concordance of subjective reports and self-administration within the context of medications development for substance use disorders, focusing primarily on illustrative examples from development efforts with opioid and cocaine dependence, and 3) explores the utility of applying self-administration procedures to assess the abuse liability of novel compounds, including “abuse deterrent” formulations (ADFs). The review will focus on opioid and cocaine dependence because a rich database from both clinical laboratory and clinical trial research exists for these two drug classes. The data reviewed suggest that drug-induced changes in self-administration and subjective effects are not always concordant. Therefore, assessment of self-administration in combination with subjective effects provides a more comprehensive picture that may have improved predictive validity for translating to the clinical setting. PMID:18436394

76 FR 36543 - Draft Guidance for Industry and Food and Drug Administration Staff: Applying Human Factors and...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-06-22

... DEPARTMENT OF HEALTH AND HUMAN SERVICES Food and Drug Administration [Docket No. FDA-2011-D-0469] Draft Guidance for Industry and Food and Drug Administration Staff: Applying Human Factors and Usability Engineering To Optimize Medical Device Design; Availability AGENCY: Food and Drug Administration, HHS. ACTION...

Glycolysis in Panc-1 human pancreatic cancer cells is inhibited by everolimus.

PubMed

Liu, Ling; Gong, Liansheng; Zhang, Yangde; Li, Nianfeng

2013-01-01

The aim of this study was to evaluate the effects and molecular mechanisms of everolimus on Panc-1 human pancreatic cancer cells. Panc-1 human pancreatic cancer cells were treated with everolimus (10 μg/ml) at selected time points (6, 12 and 24 h). Cell proliferation and apoptosis were evaluated by MTT and flow cytometric analyses. The glycolytic activity was determined by measuring the activity of the key enzyme lactate dehydrogenase (LDH) and lactate production. The activity of mammalian target of rapamycin (mTOR) signaling was measured by western blotting. The expression of genes, including hexokinase 2 (HK2) and microRNA-143 (miR-143), was evaluated by real-time polymerase chain reaction (PCR). The administration of everolimus time-dependently inhibited proliferation and glycolysis and induced apoptosis in the Panc-1 human pancreatic cancer cells. As the time of treatment with everolimus increased, the mTOR signaling activity decreased, indicated by lower phosphorylation levels of S6 kinase; however, the phosphorylation levels of mTOR barely changed. Moreover, our data showed an everolimus-induced increase in miR-143 and decrease in HK2 in Panc-1 cells in a time-dependent manner. In conclusion, the current study indicates a novel role of everolimus in its antitumor effect as an inhibitor of glycolysis in Panc-1 human pancreatic cancer cells. Furthermore, our data highlights the significance of exploring the mechanisms of everolimus and miR-143 in malignant tumors.

Intraperitoneal administration of tumor-targeting Salmonella typhimurium A1-R inhibits disseminated human ovarian cancer and extends survival in nude mice

PubMed Central

Zhang, Yong; Zhao, Ming; Yano, Shuya; Uehara, Fuminari; Yamamoto, Mako; Hiroshima, Yukihiko; Toneri, Makoto; Bouvet, Michael; Matsubara, Hisahiro; Tsuchiya, Hiroyuki; Hoffman, Robert M.

2015-01-01

Peritoneal disseminated cancer is highly treatment resistant. We here report the efficacy of intraperitoneal (i.p.) administration of tumor-targeting Salmonella typhimurium A1-R in a nude mouse model of disseminated human ovarian cancer. The mouse model was established by intraperitoneal injection of the human ovarian cancer cell line SKOV3-GFP. Seven days after implantation, mice were treated with S. typhimurium A1-R via intravenous (i.v.) or i.p. administration at the same dose, 5×107 CFU, once per week. Both i.v. and i.p. treatments effected prolonged survival compared with the untreated control group (P=0.025 and P<0.001, respectively). However, i.p. treatment was less toxic than i.v. treatment. Tumor-specific targeting of S. typhimurium A1-R was confirmed with bacterial culture from tumors and various organs and tumor or organ colony formation after i.v. or i.p. injection. Selective tumor targeting was most effective with i.p. administration. The results of the present study show S. typhimurium A1-R has promising clinical potential for disseminated ovarian cancer, especially via i.p. administration. PMID:25957417

Round window administration of gentamicin: a new method for the study of ototoxicity of cochlear hair cells.

PubMed

Husmann, K R; Morgan, A S; Girod, D A; Durham, D

1998-11-01

Damage to inner ear sensory hair cells after systemic administration of ototoxic drugs has been documented in humans and animals. Birds have the ability to regenerate new hair cells to replace those damaged by drugs or noise. Unfortunately, the systemic administration of gentamicin damages both ears in a variable fashion with potentially confounding systemic drug effects. We developed a method of direct application of gentamicin to one cochlea of hatchling chickens, allowing the other ear to serve as a within-animal control. We tested variables including the vehicle for application, location of application, dosage, and duration of gentamicin exposure. After 5 or 28 days survival, the percent length damage to the cochlea and regeneration of hair cells was evaluated using scanning electron microscopy. Controls consisted of the opposite unexposed cochlea and additional animals which received saline instead of gentamicin. Excellent damage was achieved using gentamicin-soaked Gelfoam pledgets applied to the round window membrane. The percent length damage could be varied from 15 to 100% by changing the dosage of gentamicin, with exposures as short as 30 min. No damage was observed in control animals. Regeneration of hair cells was observed in both the base and apex by 28 days survival.

Human alpha-lactalbumin made lethal to tumor cells (HAMLET) kills human glioblastoma cells in brain xenografts by an apoptosis-like mechanism and prolongs survival.

PubMed

Fischer, Walter; Gustafsson, Lotta; Mossberg, Ann-Kristin; Gronli, Janne; Mork, Sverre; Bjerkvig, Rolf; Svanborg, Catharina

2004-03-15

Malignant brain tumors present a major therapeutic challenge because no selective or efficient treatment is available. Here, we demonstrate that intratumoral administration of human alpha-lactalbumin made lethal to tumor cells (HAMLET) prolongs survival in a human glioblastoma (GBM) xenograft model, by selective induction of tumor cell apoptosis. HAMLET is a protein-lipid complex that is formed from alpha-lactalbumin when the protein changes its tertiary conformation and binds oleic acid as a cofactor. HAMLET induces apoptosis in a wide range of tumor cells in vitro, but the therapeutic effect in vivo has not been examined. In this study, invasively growing human GBM tumors were established in nude rats (Han:rnu/rnu Rowett, n = 20) by transplantation of human GBM biopsy spheroids. After 7 days, HAMLET was administered by intracerebral convection-enhanced delivery for 24 h into the tumor area; and alpha-lactalbumin, the native, folded variant of the same protein, was used as a control. HAMLET reduced the intracranial tumor volume and delayed the onset of pressure symptoms in the tumor-bearing rats. After 8 weeks, all alpha-lactalbumin-treated rats had developed pressure symptoms, but the HAMLET-treated rats remained asymptomatic. Magnetic resonance imaging scans revealed large differences in tumor volume (456 versus 63 mm(3)). HAMLET caused apoptosis in vivo in the tumor but not in adjacent intact brain tissue or in nontransformed human astrocytes, and no toxic side effects were observed. The results identify HAMLET as a new candidate in cancer therapy and suggest that HAMLET should be additionally explored as a novel approach to controlling GBM progression.

Cell-based interventions for neurologic conditions: ethical challenges for early human trials.

PubMed

Mathews, D J H; Sugarman, J; Bok, H; Blass, D M; Coyle, J T; Duggan, P; Finkel, J; Greely, H T; Hillis, A; Hoke, A; Johnson, R; Johnston, M; Kahn, J; Kerr, D; Kurtzberg, J; Liao, S M; McDonald, J W; McKhann, G; Nelson, K B; Rao, M; Regenberg, A; Siegel, A W; Smith, K; Solter, D; Song, H; Vescovi, A; Young, W; Gearhart, J D; Faden, R

2008-07-22

Attempts to translate basic stem cell research into treatments for neurologic diseases and injury are well under way. With a clinical trial for one such treatment approved and in progress in the United States, and additional proposals under review, we must begin to address the ethical issues raised by such early forays into human clinical trials for cell-based interventions for neurologic conditions. An interdisciplinary working group composed of experts in neuroscience, cell biology, bioethics, law, and transplantation, along with leading disease researchers, was convened twice over 2 years to identify and deliberate on the scientific and ethical issues raised by the transition from preclinical to clinical research of cell-based interventions for neurologic conditions. While the relevant ethical issues are in many respects standard challenges of human subjects research, they are heightened in complexity by the novelty of the science, the focus on the CNS, and the political climate in which the science is proceeding. Distinctive challenges confronting US scientists, administrators, institutional review boards, stem cell research oversight committees, and others who will need to make decisions about work involving stem cells and their derivatives and evaluate the ethics of early human trials include evaluating the risks, safety, and benefits of these trials, determining and evaluating cell line provenance, and determining inclusion criteria, informed consent, and the ethics of conducting early human trials in the public spotlight. Further study and deliberation by stakeholders is required to move toward professional and institutional policies and practices governing this research.

Immunosuppressive Mesenchymal Stromal Cells Derived from Human-Induced Pluripotent Stem Cells Induce Human Regulatory T Cells In Vitro and In Vivo

PubMed Central

Roux, Clémence; Saviane, Gaëlle; Pini, Jonathan; Belaïd, Nourhène; Dhib, Gihen; Voha, Christine; Ibáñez, Lidia; Boutin, Antoine; Mazure, Nathalie M.; Wakkach, Abdelilah; Blin-Wakkach, Claudine; Rouleau, Matthieu

2018-01-01

Despite mesenchymal stromal cells (MSCs) are considered as a promising source of cells to modulate immune functions on cells from innate and adaptive immune systems, their clinical use remains restricted (few number, limited in vitro expansion, absence of a full phenotypic characterization, few insights on their in vivo fate). Standardized MSCs derived in vitro from human-induced pluripotent stem (huIPS) cells, remediating part of these issues, are considered as well as a valuable tool for therapeutic approaches, but their functions remained to be fully characterized. We generated multipotent MSCs derived from huiPS cells (huiPS-MSCs), and focusing on their immunosuppressive activity, we showed that human T-cell activation in coculture with huiPS-MSCs was significantly reduced. We also observed the generation of functional CD4+ FoxP3+ regulatory T (Treg) cells. Further tested in vivo in a model of human T-cell expansion in immune-deficient NSG mice, huiPS-MSCs immunosuppressive activity prevented the circulation and the accumulation of activated human T cells. Intracytoplasmic labeling of cytokines produced by the recovered T cells showed reduced percentages of human-differentiated T cells producing Th1 inflammatory cytokines. By contrast, T cells producing IL-10 and FoxP3+-Treg cells, absent in non-treated animals, were detected in huiPS-MSCs treated mice. For the first time, these results highlight the immunosuppressive activity of the huiPS-MSCs on human T-cell stimulation with a concomitant generation of human Treg cells in vivo. They may favor the development of new tools and strategies based on the use of huiPS cells and their derivatives for the induction of immune tolerance. PMID:29422893

Immunosuppressive Mesenchymal Stromal Cells Derived from Human-Induced Pluripotent Stem Cells Induce Human Regulatory T Cells In Vitro and In Vivo.

PubMed

Roux, Clémence; Saviane, Gaëlle; Pini, Jonathan; Belaïd, Nourhène; Dhib, Gihen; Voha, Christine; Ibáñez, Lidia; Boutin, Antoine; Mazure, Nathalie M; Wakkach, Abdelilah; Blin-Wakkach, Claudine; Rouleau, Matthieu

2017-01-01

Despite mesenchymal stromal cells (MSCs) are considered as a promising source of cells to modulate immune functions on cells from innate and adaptive immune systems, their clinical use remains restricted (few number, limited in vitro expansion, absence of a full phenotypic characterization, few insights on their in vivo fate). Standardized MSCs derived in vitro from human-induced pluripotent stem (huIPS) cells, remediating part of these issues, are considered as well as a valuable tool for therapeutic approaches, but their functions remained to be fully characterized. We generated multipotent MSCs derived from huiPS cells (huiPS-MSCs), and focusing on their immunosuppressive activity, we showed that human T-cell activation in coculture with huiPS-MSCs was significantly reduced. We also observed the generation of functional CD4 + FoxP3 + regulatory T (Treg) cells. Further tested in vivo in a model of human T-cell expansion in immune-deficient NSG mice, huiPS-MSCs immunosuppressive activity prevented the circulation and the accumulation of activated human T cells. Intracytoplasmic labeling of cytokines produced by the recovered T cells showed reduced percentages of human-differentiated T cells producing Th1 inflammatory cytokines. By contrast, T cells producing IL-10 and FoxP3 + -Treg cells, absent in non-treated animals, were detected in huiPS-MSCs treated mice. For the first time, these results highlight the immunosuppressive activity of the huiPS-MSCs on human T-cell stimulation with a concomitant generation of human Treg cells in vivo . They may favor the development of new tools and strategies based on the use of huiPS cells and their derivatives for the induction of immune tolerance.

Formation of human hepatocyte-like cells with different cellular phenotypes by human umbilical cord blood-derived cells in the human-rat chimeras

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

Sun, Yan; Xiao, Dong; Zhang, Ruo-Shuang

2007-06-15

We took advantage of the proliferative and permissive environment of the developing pre-immune fetus to develop a noninjury human-rat xenograft small animal model, in which the in utero transplantation of low-density mononuclear cells (MNCs) from human umbilical cord blood (hUCB) into fetal rats at 9-11 days of gestation led to the formation of human hepatocyte-like cells (hHLCs) with different cellular phenotypes, as revealed by positive immunostaining for human-specific alpha-fetoprotein (AFP), cytokeratin 19 (CK19), cytokeratin 8 (CK8), cytokeratin 18 (CK18), and albumin (Alb), and with some animals exhibiting levels as high as 10.7% of donor-derived human cells in the recipient liver.more » More interestingly, donor-derived human cells stained positively for CD34 and CD45 in the liver of 2-month-old rat. Human hepatic differentiation appeared to partially follow the process of hepatic ontogeny, as evidenced by the expression of AFP gene at an early stage and albumin gene at a later stage. Human hepatocytes generated in this model retained functional properties of normal hepatocytes. In this xenogeneic system, the engrafted donor-derived human cells persisted in the recipient liver for at least 6 months after birth. Taken together, these findings suggest that the donor-derived human cells with different cellular phenotypes are found in the recipient liver and hHLCs hold biological activity. This humanized small animal model, which offers an in vivo environment more closely resembling the situations in human, provides an invaluable approach for in vivo investigating human stem cell behaviors, and further in vivo examining fundamental mechanisms controlling human stem cell fates in the future.« less

Administration of recombinant human granulocyte-colony-stimulating factor does not induce long-lasting detectable epigenetic alterations in healthy donors.

PubMed

Leitner, Gerda C; Faschingbauer, Martin; Wenda, Sabine; Weigel, Günter; Fischer, Gottfried

2014-12-01

The short-term safety profile of recombinant human granulocyte-colony-stimulating factor (rHuG-CSF) in the allogeneic stem cell setting seems acceptable; only few data on long-term safety are available. To further study possible epigenetic alterations, we investigated prospectively the influence of rHuG-CSF on DNA methyltransferase (DNMT) activity and on changes in DNA methylation of candidate genes in peripheral blood cells of healthy unrelated stem cell donors within an observation period of 1 year. In this study, 20 stem cell donors (14 male/six female; median age, 40 years; range, 22-54 years) and 20 sex- and age-matched blood component donors (controls) were included. Sampling was performed before rHuG-CSF administration; at the time of donation; and on Days (+1), 7, 30, 100, 180, and 360 in both groups. Analysis of DNMT activity in nuclear extracts was performed using a modified radionuclide assay. We performed methylation-specific polymerase chain reaction to detect the methylation status of promoter CpG islands of the genes of the retinoic acid receptor beta (RAR-B) and the Ras association domain family 1A (RASSF1A). DNMT activity increased significantly on the day of donation and 1 day after (p < 0.05). By Day +7 baseline values were reached. No further significant alterations of DNMT activity in the treated group compared to the controls were observed. We could not detect any differences in the gene methylation of RAR-B and RASSF1A between both groups. In our prospective study no evidence of long-lasting increased DNMT activity or enhanced DNA methylation in a limited panel of target genes after recombinant human G-CSF administration was observed in healthy stem cell donors. © 2014 AABB.

Alloimmune Responses of Humanized Mice to Human Pluripotent Stem Cell Therapeutics.

PubMed

Kooreman, Nigel G; de Almeida, Patricia E; Stack, Jonathan P; Nelakanti, Raman V; Diecke, Sebastian; Shao, Ning-Yi; Swijnenburg, Rutger-Jan; Sanchez-Freire, Veronica; Matsa, Elena; Liu, Chun; Connolly, Andrew J; Hamming, Jaap F; Quax, Paul H A; Brehm, Michael A; Greiner, Dale L; Shultz, Leonard D; Wu, Joseph C

2017-08-22

There is growing interest in using embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) derivatives for tissue regeneration. However, an increased understanding of human immune responses to stem cell-derived allografts is necessary for maintaining long-term graft persistence. To model this alloimmunity, humanized mice engrafted with human hematopoietic and immune cells could prove to be useful. In this study, an in-depth analysis of graft-infiltrating human lymphocytes and splenocytes revealed that humanized mice incompletely model human immune responses toward allogeneic stem cells and their derivatives. Furthermore, using an "allogenized" mouse model, we show the feasibility of reconstituting immunodeficient mice with a functional mouse immune system and describe a key role of innate immune cells in the rejection of mouse stem cell allografts. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

c-Myc-Dependent Cell Competition in Human Cancer Cells.

PubMed

Patel, Manish S; Shah, Heta S; Shrivastava, Neeta

2017-07-01

Cell Competition is an interaction between cells for existence in heterogeneous cell populations of multicellular organisms. This phenomenon is involved in initiation and progression of cancer where heterogeneous cell populations compete directly or indirectly for the survival of the fittest based on differential gene expression. In Drosophila, cells having lower dMyc expression are eliminated by cell competition through apoptosis when present in the milieu of cells having higher dMyc expression. Thus, we designed a study to develop c-Myc (human homolog) dependent in vitro cell competition model of human cancer cells. Cells with higher c-Myc were transfected with c-myc shRNA to prepare cells with lower c-Myc and then co-cultured with the same type of cells having a higher c-Myc in equal ratio. Cells with lower c-Myc showed a significant decrease in numbers when compared with higher c-Myc cells, suggesting "loser" and "winner" status of cells, respectively. During microscopy, engulfment of loser cells by winner cells was observed with higher expression of JNK in loser cells. Furthermore, elimination of loser cells was prevented significantly, when co-cultured cells were treated with the JNK (apoptosis) inhibitor. Above results indicate elimination of loser cells in the presence of winner cells by c-Myc-dependent mechanisms of cell competition in human cancer cells. This could be an important mechanism in human tumors where normal cells are eliminated by c-Myc-overexpressed tumor cells. J. Cell. Biochem. 118: 1782-1791, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Generation of Beta Cells from Human Pluripotent Stem Cells: Potential for Regenerative Medicine

PubMed Central

Nostro, Maria Cristina; Keller, Gordon

2015-01-01

The loss of beta cells in Type I Diabetes ultimately leads to insulin dependence and major complications that are difficult to manage by insulin injections. Given the complications associated with long-term administration of insulin, cell-replacement therapy is now under consideration as an alternative treatment that may someday provide a cure for this disease. Over the past 10 years, islet transplantation trials have demonstrated that it is possible to replenish beta cell function in Type I Diabetes patients and, at least temporarily, eliminate their dependency on insulin. While not yet optimal, the success of these trials has provided proof-of-principle that cell replacement therapy is a viable option for treating diabetes. Limited access to donor islets has launched a search for alternative source of beta cells for cell therapy purposes and focused the efforts of many investigators on the challenge of deriving such cells from human embryonic and induced pluripotent stem cells. Over the past five years, significant advances have been made in understanding the signaling pathways that control lineage development from hPSCs and as a consequence, it is now possible to routinely generate human insulin producing cells from both hESCs and hiPSCs. While these achievements are impressive, significant challenges do still exist, as the majority of insulin producing cells generated under these conditions are polyhormonal and non functional, likely reflecting the emergence of the polyhormonal population that is known to arise in the early embryo during the phase of pancreatic development known as the ‘first transition’. Functional beta cells, which arise during the second phase or transition of pancreatic development have been generated from hPSCs, however they are detected only following transplantation of progenitor stage cells into immunocompromised mice. With this success, our challenge now is to define the pathways that control the development and maturation of this

Emodin induces apoptosis of human osteosarcoma cells via mitochondria- and endoplasmic reticulum stress-related pathways

PubMed Central

Ying, Jinhe; Xu, Huan; Wu, Dhua; Wu, Xiaoguang

2015-01-01

Aim: Emodin showed anti-cancer activity against multiple human malignant tumors by inducing apoptosis. However, the apoptotic inducing effect against human osteosarcoma and related mechanism are still not studied. This study was aimed to investigate them. Methods: Emodin was used to incubate human OS cell U2OS cells at serially diluted concentrations. Hoechst staining was used to evaluate apoptosis; flow cytometry was applied to assess the collapse of mitochondrial membrane potential (MMP); intracellular ROS generation was detected by DCFH-DA staining; endoplasmic reticulum stress activation was examined by western blotting. Results: Cell apoptosis of U2OS cells was induced by emodin incubation in a concentration-dependent manner; MMP collapse and ROS generation were identified at starting concentration of 80 μmol/L of emodin in a concentration-dependent manner. ER stress activation was found at beginning concentration of 40 μmol/L of emodin. The MMP collapse was inhibited while the ER stress was not inhibited by NAC administration. Conclusions: Emodin induces death of human osteosarcoma cells by initiating ROS-dependent mitochondria-induced and ROS-independent ER stress-induced apoptosis. PMID:26722474

[Efficacy of HSV-tk/GCV system on human laryngeal squamous cell cancer in vitro].

PubMed

Ding, Xiu-yong; Qin, Yong; Li, Fu-ying; Cong, Tie-chuan

2006-05-01

Efficacy of HSV-tk/GCV system antitumor effects was assessed on human laryngeal cancer cell line Hep-2 in vitro. To assess the HSV-tk/CGV system whether has an antitumour effect on human laryngeal squamous cell cancer Hep-2 in vitro. The mechanisms of cytotoxity were also assessed. Hep-2 cells were transfected with HSV-tk gene by lipofection. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the HSV-tk gene expression. MTT was utilized to test for the cytotoxicity of this system. The cell-circle arrest and apoptosis were analyzed by flowcytometry assay. HSV-tk gene transfected cells demonstrated obvious cytoreductivity followed by ganciclovir (GCV) administration and this cytoreductivity showed partial GCV dose-independent. HSV-tk gene transfected cells demonstrated obvious s-phase arrest, no apoptosis and necrosis occurred. The HSV-tk/GCV system can inhabit the growth of Hep-2 cells effectively. S-phase arrest perhaps is the main reason that leads to the cell inhibition in our study. HSV-tk/GCV system has potential antitumor effects for the future clinical practice.

Human Neutrophil Peptides Mediate Endothelial-Monocyte Interaction, Foam Cell Formation, and Platelet Activation

PubMed Central

Quinn, Kieran L.; Henriques, Melanie; Tabuchi, Arata; Han, Bing; Yang, Hong; Cheng, Wei-Erh; Tole, Soumitra; Yu, Hanpo; Luo, Alice; Charbonney, Emmanuel; Tullis, Elizabeth; Lazarus, Alan; Robinson, Lisa A.; Ni, Heyu; Peterson, Blake R.; Kuebler, Wolfgang M.; Slutsky, Arthur S.; Zhang, Haibo

2016-01-01

Objective Neutrophils are involved in the inflammatory responses during atherosclerosis. Human neutrophil peptides (HNPs) released from activated neutrophils exert immune modulating properties. We hypothesized that HNPs play an important role in neutrophil-mediated inflammatory cardiovascular responses in atherosclerosis. Methods and Results We examined the role of HNPs in endothelial-leukocyte interaction, platelet activation, and foam cell formation in vitro and in vivo. We demonstrated that stimulation of human coronary artery endothelial cells with clinically relevant concentrations of HNPs resulted in monocyte adhesion and transmigration; induction of oxidative stress in human macrophages, which accelerates foam cell formation; and activation and aggregation of human platelets. The administration of superoxide dismutase or anti-CD36 antibody reduced foam cell formation and cholesterol efflux. Mice deficient in double genes of low-density lipoprotein receptor and low-density lipoprotein receptor–related protein (LRP), and mice deficient in a single gene of LRP8, the only LRP phenotype expressed in platelets, showed reduced leukocyte rolling and decreased platelet aggregation and thrombus formation in response to HNP stimulation. Conclusion HNPs exert proatherosclerotic properties that appear to be mediated through LRP8 signaling pathways, suggesting an important role for HNPs in the development of inflammatory cardiovascular diseases. PMID:21817096

Activation of human T cells in hypertension: Studies of Humanized Mice and Hypertensive Humans

PubMed Central

Itani, Hana A.; McMaster, William G.; Saleh, Mohamed A.; Nazarewicz, Rafal R.; Mikolajczyk, Tomasz P.; Kaszuba, Anna; 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.

2016-01-01

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 employed a humanized mouse model in which the murine immune system is replaced by the human immune system. Angiotensin II increased systolic pressure to 162 mm Hg vs. 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 IL-17A producing CD4+ T cells and both CD4+ and CD8+ T cells that produce IFN-γ in hypertensive compared to 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

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. © 2016 American Heart Association, Inc.

Versatile graphene biosensors for enhancing human cell therapy.

PubMed

Vlăsceanu, George M; Amărandi, Roxana-Maria; Ioniță, Mariana; Tite, Teddy; Iovu, Horia; Pilan, Luisa; Burns, Jorge S

2018-05-01

Technological advances in engineering and cell biology stimulate novel approaches for medical treatment, in particular cell-based therapy. The first cell-based gene therapy against cancer was recently approved by the US Food and Drug Administration. Progress in cancer diagnosis includes a blood test detecting five cancer types. Numerous stem cell phase I/II clinical trials showing safety and efficacy will soon pursue qualifying criteria for advanced therapy medicinal products (ATMP), aspiring to join the first stem-cell therapy approved by the European Medicines Agency. Cell based therapy requires extensive preclinical characterisation of biomarkers indicating mechanisms of action crucial to the desired therapeutic effect. Quantitative analyses monitoring critical functions for the manufacture of optimal cell and tissue-based clinical products include successful potency assays for implementation. The challenge to achieve high quality measurement is increasingly met by progress in biosensor design. We adopt a cell therapy perspective to highlight recent examples of graphene-enhanced biointerfaces for measurement of biomarkers relevant to cancer treatment, diagnosis and tissue regeneration. Graphene based biosensor design problems can thwart their use for health care transformative point of care testing and real-time applications. We discuss concerns to be addressed and emerging solutions for establishing clinical grade biosensors to accelerate human cell therapy. Copyright © 2018 Elsevier B.V. All rights reserved.

Reconstituted normal human breast in nude mice: effect of host pregnancy environment and human chorionic gonadotropin on proliferation.

PubMed

Popnikolov, N; Yang, J; Liu, A; Guzman, R; Nandi, S

2001-03-01

The proliferation of normal human breast epithelial cells in women is highest during the first trimester of pregnancy. In an attempt to analyze this hormonal environment in a model system, the effect of host mouse pregnancy and the administration of human chorionic gonadotropin (hCG) were assessed in normal human breast epithelial cells transplanted into athymic nude mice. Human breast epithelial cells, dissociated from reduction mammoplasty specimens and embedded inside the extracellular matrices comprised of collagen gel and Matrigel, were transplanted into nude mice. Proliferation was measured in vivo by BrdU labeling followed by immunostaining of sections from recovered gels in response to an altered hormonal environment of the host animal. The host animal was mated to undergo pregnancy and the complex hormonal environment of the host animal pregnancy stimulated growth of transplanted human cells. This effect increased with progression of pregnancy and reached the maximum during late pregnancy prior to parturition. In order to determine whether additional stimulation could be achieved, the transplanted human cells were exposed to a second cycle of host mouse pregnancy by immediately mating the animal after parturition. This additional exposure of host mouse pregnancy did not result in further increase of proliferation. The effect of hCG administration on transplanted human cells was also tested, since hCG level is highest during the first trimester of human pregnancy and coincides with the maximal breast cell proliferation. Administration of hCG alone stimulated proliferation of human cells in a dose-dependent manner, and could further enhance stimulation achieved with estrogen. The host mouse mammary gland also responded to hCG treatment resulting in increased branching and lobulo-alveolar development. However, the hCG effect on both human and mouse cells was dependent on intact ovary since the stimulation did not occur in ovariectomized animals. Although h

Human umbilical cord blood-derived mesenchymal stem cells attenuate hyperoxia-induced lung injury in neonatal rats.

PubMed

Chang, Yun Sil; Oh, Wonil; Choi, Soo Jin; Sung, Dong Kyung; Kim, Soo Yoon; Choi, Eun Yang; Kang, Saem; Jin, Hye Jin; Yang, Yoon Sun; Park, Won Soon

2009-01-01

Recent evidence suggests mesenchymal stem cells (MSCs) can downmodulate bleomycin-induced lung injury, and umbilical cord blood (UCB) is a promising source for human MSCs. This study examined whether intratracheal or intraperitoneal transplantation of human UCB-derived MSCs can attenuate hyperoxia-induced lung injury in immunocompetent newborn rats. Wild-type Sprague-Dawley rats were randomly exposed to 95% oxygen or air from birth. In the transplantation groups, a single dose of PKH26-labeled human UCB-derived MSCs was administered either intratracheally (2 x 10(6) cells) or intraperitoneally (5 x 10(5) cells) at postnatal day (P) 5. At P14, the harvested lungs were examined for morphometric analyses of alveolarization and TUNEL staining, as well as the myeoloperoxidase activity, the level of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and transforming growth factor (TGF)-beta mRNA, alpha-smooth muscle actin (SMA) protein, and collagen levels. Differentiation of MSCs to the respiratory epithelium was also evaluated both in vitro before transplantation and in vivo after transplantation. Despite one fourth dosage of MSCs, significantly more PKH26-labeled donor cells were recovered with intratracheal administration than with intraperitoneal administration both during normoxia and hyperoxia. The hyperoxia-induced increase in the number of TUNEL-positive cells, myeloperoixdase activity, and the level of IL-6 mRNA were significantly attenuated with both intratracheal and intraperitoneal MSCs transplantation. However, the hyperoxia-induced impaired alveolarization and increased the level of TNF-alpha and TGF-beta mRNA, alpha-SMA protein, and collagen were significantly attenuated only with intratracheal MSCs transplantation. MSCs differentiated into respiratory epithelium in vitro and a few PKH26-positive donor cells were colocalized with pro surfactant protein C in the damaged lungs. In conclusion, intratracheal transplantation of human UCB-derived MSCs is

Topical administration of orbital fat-derived stem cells promotes corneal tissue regeneration.

PubMed

Lin, Ko-Jo; Loi, Mei-Xue; Lien, Gi-Shih; Cheng, Chieh-Feng; Pao, Hsiang-Yin; Chang, Yun-Chuang; Ji, Andrea Tung-Qian; Ho, Jennifer Hui-Chun

2013-06-14

Topical administration of eye drops is the major route for drug delivery to the cornea. Orbital fat-derived stem cells (OFSCs) possess an in vitro corneal epithelial differentiation capacity. Both the safety and immunomodulatory ability of systemic OFSC transplantation were demonstrated in our previous work. In this study, we investigated the safety, therapeutic effect, and mechanism(s) of topical OFSC administration in an extensive alkali-induced corneal wound. Corneal injury was created by contact of a piece of 0.5 N NaOH-containing filter paper on the corneal surface of a male Balb/c mouse for 30 s. The area of the filter paper covered the central 70% or 100% of the corneal surface. OFSCs (2 × 10(5)) in 5 μl phosphate-buffered saline (PBS) were given by topical administration (T) twice a day or by two intralimbal (IL) injections in the right cornea, while 5 μl of PBS in the left cornea served as the control. Topical OFSCs promoted corneal re-epithelialization of both the limbal-sparing and limbal-involved corneal wounds. In the first three days, topical OFSCs significantly reduced alkali-induced corneal edema and stromal infiltration according to a histopathological examination. Immunohistochemistry and immunofluorescence staining revealed that transplanted cells were easily detectable in the corneal epithelium, limbal epithelium and stroma, but only some of transplanted cells at the limbal epithelium had differentiated into cytokeratin 3-expressing cells. OFSCs did not alter neutrophil (Ly6G) levels in the cornea, but significantly reduced macrophage (CD68) infiltration and inducible nitrous oxide synthetase (iNOS) production during acute corneal injury as quantified by a Western blot analysis. Continuous topical administration of OFSCs for seven days improved corneal transparency, and this was accompanied by diffuse stromal engraftment of transplanted cells and differentiation into p63-expressing cells at the limbal area. The therapeutic effect of the

Topical administration of orbital fat-derived stem cells promotes corneal tissue regeneration

PubMed Central

2013-01-01

Introduction Topical administration of eye drops is the major route for drug delivery to the cornea. Orbital fat-derived stem cells (OFSCs) possess an in vitro corneal epithelial differentiation capacity. Both the safety and immunomodulatory ability of systemic OFSC transplantation were demonstrated in our previous work. In this study, we investigated the safety, therapeutic effect, and mechanism(s) of topical OFSC administration in an extensive alkali-induced corneal wound. Methods Corneal injury was created by contact of a piece of 0.5 N NaOH-containing filter paper on the corneal surface of a male Balb/c mouse for 30 s. The area of the filter paper covered the central 70% or 100% of the corneal surface. OFSCs (2 × 105) in 5 μl phosphate-buffered saline (PBS) were given by topical administration (T) twice a day or by two intralimbal (IL) injections in the right cornea, while 5 μl of PBS in the left cornea served as the control. Results Topical OFSCs promoted corneal re-epithelialization of both the limbal-sparing and limbal-involved corneal wounds. In the first three days, topical OFSCs significantly reduced alkali-induced corneal edema and stromal infiltration according to a histopathological examination. Immunohistochemistry and immunofluorescence staining revealed that transplanted cells were easily detectable in the corneal epithelium, limbal epithelium and stroma, but only some of transplanted cells at the limbal epithelium had differentiated into cytokeratin 3-expressing cells. OFSCs did not alter neutrophil (Ly6G) levels in the cornea, but significantly reduced macrophage (CD68) infiltration and inducible nitrous oxide synthetase (iNOS) production during acute corneal injury as quantified by a Western blot analysis. Continuous topical administration of OFSCs for seven days improved corneal transparency, and this was accompanied by diffuse stromal engraftment of transplanted cells and differentiation into p63-expressing cells at the limbal area. The

Induction of insulin-producing cells from human pancreatic progenitor cells.

PubMed

Noguchi, H; Naziruddin, B; Shimoda, M; Fujita, Y; Chujo, D; Takita, M; Peng, H; Sugimoto, K; Itoh, T; Tamura, Y; Olsen, G S; Kobayashi, N; Onaca, N; Hayashi, S; Levy, M F; Matsumoto, S

2010-01-01

We previously established a mouse pancreatic stem cell line without genetic manipulation. In this study, we sought to identify and isolate human pancreatic stem/progenitor cells. We also tested whether growth factors and protein transduction of pancreatic and duodenal homeobox factor-1 (PDX-1) and BETA2/NeuroD into human pancreatic stem/progenitor cells induced insulin or pancreas-related gene expressions. Human pancreata from brain-dead donors were used for islet isolation with the standard Ricordi technique modified by the Edmonton protocol. The cells from a duct-rich population were cultured in several media, based on those designed for mouse pancreatic or for human embryonic stem cells. To induce cell differentiation, cells were cultured for 2 weeks with exendin-4, nicotinamide, keratinocyte growth factor, PDX-1 protein, or BETA2/NeuroD protein. The cells in serum-free media showed morphologies similar to a mouse pancreatic stem cell line, while the cells in the medium for human embryonic stem cells formed fibroblast-like morphologies. The nucleus/cytoplasm ratios of the cells in each culture medium decreased during the culture. The cells stopped dividing after 30 days, suggesting that they had entered senescence. The cells treated with induction medium differentiated into insulin-producing cells, expressing pancreas-related genes. Duplications of cells from a duct-rich population were limited. Induction therapy with several growth factors and transduction proteins might provide a potential new strategy for induction of transplantable insulin-producing cells. Copyright 2010 Elsevier Inc. All rights reserved.

Autophagy mediates cytotoxicity of human colorectal cancer cells treated with garcinielliptone FC.

PubMed

Won, Shen-Jeu; Yen, Cheng-Hsin; Lin, Ting-Yu; Jiang-Shieh, Ya-Fen; Lin, Chun-Nan; Chen, Jyun-Ti; Su, Chun-Li

2018-01-01

The tautomeric pair of garcinielliptone FC (GFC) is a novel tautomeric pair of polyprenyl benzophenonoid isolated from the pericarps of Garcinia subelliptica Merr. (G. subelliptica, Clusiaceae), a tree with abundant sources of polyphenols. Our previous report demonstrated that GFC induced apoptosis on various types of human cancer cell lines including chemoresistant human colorectal cancer HT-29 cells. In the present study, we observed that many autophagy-related genes in GFC-treated HT-29 cells were up- and down-regulated using a cDNA microarray containing oncogenes and kinase genes. GFC-induced autophagy of HT-29 cells was confirmed by observing the formation of acidic vesicular organelles, LC3 puncta, and double-membrane autophagic vesicles using flow cytometry, confocal microscopy, and transmission electron microscopy, respectively. Inhibition of AKT/mTOR/P70S6K signaling as well as formation of Atg5-Atg12 and PI3K/Beclin-1 complexes were observed using Western blot. Administration of autophagy inhibitor (3-methyladenine and shRNA Atg5) and apoptosis inhibitor Z-VAD showed that the GFC-induced autophagy was cytotoxic form and GFC-induced apoptosis enhanced GFC-induced autophagy. Our data suggest the involvement of autophagy and apoptosis in GFC-induced anticancer mechanisms of human colorectal cancer. © 2017 Wiley Periodicals, Inc.

The Human Airway Epithelial Basal Cell Transcriptome

PubMed Central

Wang, Rui; Zwick, Rachel K.; Ferris, Barbara; Witover, Bradley; Salit, Jacqueline; Crystal, Ronald G.

2011-01-01

Background The human airway epithelium consists of 4 major cell types: ciliated, secretory, columnar and basal cells. During natural turnover and in response to injury, the airway basal cells function as stem/progenitor cells for the other airway cell types. The objective of this study is to better understand human airway epithelial basal cell biology by defining the gene expression signature of this cell population. Methodology/Principal Findings Bronchial brushing was used to obtain airway epithelium from healthy nonsmokers. Microarrays were used to assess the transcriptome of basal cells purified from the airway epithelium in comparison to the transcriptome of the differentiated airway epithelium. This analysis identified the “human airway basal cell signature” as 1,161 unique genes with >5-fold higher expression level in basal cells compared to differentiated epithelium. The basal cell signature was suppressed when the basal cells differentiated into a ciliated airway epithelium in vitro. The basal cell signature displayed overlap with genes expressed in basal-like cells from other human tissues and with that of murine airway basal cells. Consistent with self-modulation as well as signaling to other airway cell types, the human airway basal cell signature was characterized by genes encoding extracellular matrix components, growth factors and growth factor receptors, including genes related to the EGF and VEGF pathways. Interestingly, while the basal cell signature overlaps that of basal-like cells of other organs, the human airway basal cell signature has features not previously associated with this cell type, including a unique pattern of genes encoding extracellular matrix components, G protein-coupled receptors, neuroactive ligands and receptors, and ion channels. Conclusion/Significance The human airway epithelial basal cell signature identified in the present study provides novel insights into the molecular phenotype and biology of the stem

Human errors and violations in computer and information security: the viewpoint of network administrators and security specialists.

PubMed

Kraemer, Sara; Carayon, Pascale

2007-03-01

This paper describes human errors and violations of end users and network administration in computer and information security. This information is summarized in a conceptual framework for examining the human and organizational factors contributing to computer and information security. This framework includes human error taxonomies to describe the work conditions that contribute adversely to computer and information security, i.e. to security vulnerabilities and breaches. The issue of human error and violation in computer and information security was explored through a series of 16 interviews with network administrators and security specialists. The interviews were audio taped, transcribed, and analyzed by coding specific themes in a node structure. The result is an expanded framework that classifies types of human error and identifies specific human and organizational factors that contribute to computer and information security. Network administrators tended to view errors created by end users as more intentional than unintentional, while errors created by network administrators as more unintentional than intentional. Organizational factors, such as communication, security culture, policy, and organizational structure, were the most frequently cited factors associated with computer and information security.

A potential oral anticancer drug candidate, Moringa oleifera leaf extract, induces the apoptosis of human hepatocellular carcinoma cells

PubMed Central

JUNG, IL LAE; LEE, JU HYE; KANG, SE CHAN

2015-01-01

It has previously been reported that cold water-extracts of Moringa oleifera leaf have anticancer activity against various human cancer cell lines, including non-small cell lung cancer. In the present study, the anticancer activity of M. oleifera leaf extracts was investigated in human hepatocellular carcinoma HepG2 cells. By the analysis of apoptotic signals, including the induction of caspase or poly(ADP-ribose) polymerase cleavage, and the Annexin V and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays, it was demonstrated that M. oleifera leaf extracts induce the apoptosis of HepG2 cells. In the hollow fiber assay, oral administration of the leaf extracts significantly reduced (44–52%) the proliferation of the HepG2 cells and A549 non-small cell lung cancer cells. These results support the potential of soluble extracts of M. oleifera leaf as orally administered therapeutics for the treatment of human liver and lung cancers. PMID:26622717

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

PubMed

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

2014-04-01

Mice repopulated with human hematopoietic cells are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, existing humanized mouse models cannot support development of human innate immune cells, including myeloid cells and natural killer (NK) cells. Here we describe two mouse strains called MITRG and MISTRG, in which human versions of four genes encoding cytokines important for innate immune cell development are knocked into their respective mouse loci. The human cytokines support the development and function of monocytes, macrophages and NK cells derived from human fetal liver or adult CD34(+) progenitor cells injected into the mice. Human macrophages infiltrated a human tumor xenograft in MITRG and MISTRG mice in a manner resembling that observed in tumors obtained from human patients. This humanized mouse model may be used to model the human immune system in scenarios of health and pathology, and may enable evaluation of therapeutic candidates in an in vivo setting relevant to human physiology.

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

PubMed Central

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

2014-01-01

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

Histological observation of goblet cells following topical rebamipide treatment of the human ocular surface: A case report.

PubMed

Kase, Satoru; Shinohara, Toshiya; Kase, Manabu

2015-02-01

The topical administration of rebamipide (Mucosta®), an antiulcer agent, clinically increases the mucin level of tear film. The aim of this study was to report the histological changes of goblet cells following the topical administration of rebamipide to a patient with nevus of the lacrimal caruncle. A 62-year-old male exhibited a pigmented nodule located in the lacrimal caruncle in the left eye. An excisional biopsy and subsequent surgical resection were conducted at the caruncle, prior to and three months after topical rebamipide administration. Histologically, a biopsy specimen revealed a pigmented nevus beneath the caruncle epithelium containing a few goblet cells [4 cells/high power field (HPF)]. A few nevus cells were present at the surgical margin. By contrast, the secondary resected specimen obtained three months after the initiation of topical rebamipide treatment revealed the epithelium and nevus, where numerous goblet cells were present (28 cells/HPF), and mucin-like substances were markedly secreted from the goblet cells. Topical rebamipide markedly increased the number of goblet cells and stimulated the secretion of mucin-like substances in the caruncular tissue of a human patient. These results suggest that topical rebamipide is useful in patients following surgery and/or biopsy to support tissue repair of the ocular surface.

Utilization of human amniotic mesenchymal cells as feeder layers to sustain propagation of human embryonic stem cells in the undifferentiated state.

PubMed

Zhang, Kehua; Cai, Zhe; Li, Yang; Shu, Jun; Pan, Lin; Wan, Fang; Li, Hong; Huang, Xiaojie; He, Chun; Liu, Yanqiu; Cui, Xiaohui; Xu, Yang; Gao, Yan; Wu, Liqun; Cao, Shanxia; Li, Lingsong

2011-08-01

Human embryonic stem (ES) cells are usually maintained in the undifferentiated state by culturing on feeder cells layers of mouse embryonic fibroblasts (MEFs). However, MEFs are not suitable to support human ES cells used for clinical purpose because of risk of zoonosis from animal cells. Therefore, human tissue-based feeder layers need to be developed for human ES cells for clinical purpose. Hereof we report that human amniotic mesenchymal cells (hAMCs) could act as feeder cells for human ES cells, because they are easily obtained and relatively exempt from ethical problem. Like MEFs, hAMCs could act as feeder cells for human ES cells to grow well on. The self-renewal rate of human ES cells cultured on hAMCs feeders was higher than that on MEFs and human amniotic epithelial cells determined by measurement of colonial diameters and growth curve as well as cell cycle analysis. Both immunofluorescence staining and immunoblotting showed that human ES cells cultured on hAMCs expressed stem cell markers such as Oct-3/4, Sox2, and NANOG. Verified by embryoid body formation in vitro and teratoma formation in vivo, we found out that after 20 passages of culture, human ES cells grown on hAMCs feeders could still retain the potency of differentiating into three germ layers. Taken together, our data suggested hAMCs may be safe feeder cells to sustain the propagation of human ES cells in undifferentiated state for future therapeutic use.

Age-related increase in Wnt inhibitor causes a senescence-like phenotype in human cardiac stem cells

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

Nakamura, Tamami; Hosoyama, Tohru; Regenerative Medicine Institute, Yamaguchi University Graduate School of Medicine

Aging of cardiac stem/progenitor cells (CSCs) impairs heart regeneration and leads to unsatisfactory outcomes of cell-based therapies. As the precise mechanisms underlying CSC aging remain unclear, the use of therapeutic strategies for elderly patients with heart failure is severely delayed. In this study, we used human cardiosphere-derived cells (CDCs), a subtype of CSC found in the postnatal heart, to identify secreted factor(s) associated with CSC aging. Human CDCs were isolated from heart failure patients of various ages (2–83 years old). Gene expression of key soluble factors was compared between CDCs derived from young and elderly patients. Among these factors, SFRP1,more » a gene encoding a Wnt antagonist, was significantly up-regulated in CDCs from elderly patients (≥65 years old). sFRP1 levels was increased significantly also in CDCs, whose senescent phenotype was induced by anti-cancer drug treatment. These results suggest the participation of sFRP1 in CSC aging. We show that the administration of recombinant sFRP1 induced cellular senescence in CDCs derived from young patients, as indicated by increased levels of markers such as p16, and a senescence-associated secretory phenotype. In addition, co-administration of recombinant sFRP1 could abrogate the accelerated CDC proliferation induced by Wnt3A. Taken together, our results suggest that canonical Wnt signaling and its antagonist, sFRP1, regulate proliferation of human CSCs. Furthermore, excess sFRP1 in elderly patients causes CSC aging. - Highlights: • Wnt signaling regulates proliferation of human cardiac stem cells. • Expression of sFRP1, which is a Wnt antagonist, is up-regulated in elderly patients with heart failure. • Expression of sFRP1 is increased in anti-cancer drug-induced senescent human cardiac stem cells. • sFRP1 causes cellular senescence of young patients-derived cardiac stem cells.« less

Human monoclonal antibodies reactive with human myelomonocytic leukemia cells.

PubMed

Posner, M R; Santos, D J; Elboim, H S; Tumber, M B; Frackelton, A R

1989-04-01

Peripheral blood mononuclear cells from a patient with chronic myelogenous leukemia (CML), in remission, were depleted of CD8-positive T-cells and cultured with Epstein-Barr virus. Four of 20 cultures (20%) secreted human IgG antibodies selectively reactive with the cell surfaces of certain human leukemia cell lines. Three polyclonal, Epstein-Barr virus-transformed, B-cell lines were expanded and fused with the human-mouse myeloma analogue HMMA2.11TG/O. Antibody from secreting clones HL 1.2 (IgG1), HL 2.1 (IgG3), and HL 3.1 (IgG1) have been characterized. All three react with HL-60 (promyelocytic), RWLeu4 (CML promyelocytic), and U937 (monocytic), but not with KG-1 (myeloblastic) or K562 (CML erythroid). There is no reactivity with T-cell lines, Burkitt's cell lines, pre-B-leukemia cell lines, or an undifferentiated CML cell line, BV173. Leukemic cells from two of seven patients with acute myelogenous leukemia and one of five with acute lymphocytic leukemia react with all three antibodies. Normal lymphocytes, monocytes, polymorphonuclear cells, red blood cells, bone marrow cells, and platelets do not react. Samples from patients with other diverse hematopoietic malignancies showed no reactivity. Immunoprecipitations suggest that the reactive antigen(s) is a lactoperoxidase iodinatable series of cell surface proteins with molecular weights of 42,000-54,000 and a noniodinatable protein with a molecular weight of 82,000. Based on these data these human monoclonal antibodies appear to react with myelomonocytic leukemic cells and may detect a leukemia-specific antigen or a highly restricted differentiation antigen.

Cell-to-Cell Contact and Nectin-4 Govern Spread of Measles Virus from Primary Human Myeloid Cells to Primary Human Airway Epithelial Cells.

PubMed

Singh, Brajesh K; Li, Ni; Mark, Anna C; Mateo, Mathieu; Cattaneo, Roberto; Sinn, Patrick L

2016-08-01

Measles is a highly contagious, acute viral illness. Immune cells within the airways are likely first targets of infection, and these cells traffic measles virus (MeV) to lymph nodes for amplification and subsequent systemic dissemination. Infected immune cells are thought to return MeV to the airways; however, the mechanisms responsible for virus transfer to pulmonary epithelial cells are poorly understood. To investigate this process, we collected blood from human donors and generated primary myeloid cells, specifically, monocyte-derived macrophages (MDMs) and dendritic cells (DCs). MDMs and DCs were infected with MeV and then applied to primary cultures of well-differentiated airway epithelial cells from human donors (HAE). Consistent with previous results obtained with free virus, infected MDMs or DCs were incapable of transferring MeV to HAE when applied to the apical surface. Likewise, infected MDMs or DCs applied to the basolateral surface of HAE grown on small-pore (0.4-μm) support membranes did not transfer virus. In contrast, infected MDMs and DCs applied to the basolateral surface of HAE grown on large-pore (3.0-μm) membranes successfully transferred MeV. Confocal microscopy demonstrated that MDMs and DCs are capable of penetrating large-pore membranes but not small-pore membranes. Further, by using a nectin-4 blocking antibody or recombinant MeV unable to enter cells through nectin-4, we demonstrated formally that transfer from immune cells to HAE occurs in a nectin-4-dependent manner. Thus, both infected MDMs and DCs rely on cell-to-cell contacts and nectin-4 to efficiently deliver MeV to the basolateral surface of HAE. Measles virus spreads rapidly and efficiently in human airway epithelial cells. This rapid spread is based on cell-to-cell contact rather than on particle release and reentry. Here we posit that MeV transfer from infected immune cells to epithelial cells also occurs by cell-to-cell contact rather than through cell-free particles. In

Variability of human pluripotent stem cell lines.

PubMed

Ortmann, Daniel; Vallier, Ludovic

2017-10-01

Human pluripotent stem cells derived from embryos (human Embryonic Stem Cells or hESCs) or generated by direct reprogramming of somatic cells (human Induced Pluripotent Stem Cells or hiPSCs) can proliferate almost indefinitely in vitro while maintaining the capacity to differentiate into a broad diversity of cell types. These two properties (self-renewal and pluripotency) confers human pluripotent stem cells a unique interest for clinical applications since they could allow the production of infinite quantities of cells for disease modelling, drug screening and cell based therapy. However, recent studies have clearly established that human pluripotent stem cell lines can display variable capacity to differentiate into specific lineages. Consequently, the development of universal protocols of differentiation which could work efficiently with any human pluripotent cell line is complicated substantially. As a consequence, each protocol needs to be adapted to every cell line thereby limiting large scale applications and precluding personalised therapies. Here, we summarise our knowledge concerning the origin of this variability and describe potential solutions currently available to bypass this major challenge. Copyright © 2017 Elsevier Ltd. All rights reserved.

Signaling hierarchy regulating human endothelial cell development.

PubMed

Kelly, Melissa A; Hirschi, Karen K

2009-05-01

Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these studies. Using human embryonic stem cells as a model system, we were able to reproducibly and robustly generate differentiated endothelial cells via coculture on OP9 marrow stromal cells. We found that, in contrast to studies in the mouse, bFGF and VEGF had no specific effects on the initiation of human vasculogenesis. However, exogenous Ihh promoted endothelial cell differentiation, as evidenced by increased production of cells with cobblestone morphology that coexpress multiple endothelial-specific genes and proteins, form lumens, and exhibit DiI-AcLDL uptake. Inhibition of BMP signaling using Noggin or BMP4, specifically, using neutralizing antibodies suppressed endothelial cell formation; whereas, addition of rhBMP4 to cells treated with the hedgehog inhibitor cyclopamine rescued endothelial cell development. Our studies revealed that Ihh promoted human endothelial cell differentiation from pluripotent hES cells via BMP signaling, providing novel insights applicable to modulating human endothelial cell formation and vascular regeneration for human clinical therapies.

HMGB1-stimulated human primary cardiac fibroblasts exert a paracrine action on human and murine cardiac stem cells.

PubMed

Rossini, Alessandra; Zacheo, Antonella; Mocini, David; Totta, Pierangela; Facchiano, Antonio; Castoldi, Raffaella; Sordini, Paolo; Pompilio, Giulio; Abeni, Damiano; Capogrossi, Maurizio C; Germani, Antonia

2008-04-01

High Mobility Box 1 Protein (HMGB1) is a cytokine released into the extracellular space by necrotic cells and activated macrophages in response to injury. We recently demonstrated that HMGB1 administration into the mouse heart during acute myocardial infarction induces cardiac tissue regeneration by activating resident cardiac c-kit+ cells (CSCs) and significantly enhances left ventricular function. In the present study it was analyzed the hypothesis that human cardiac fibroblasts (cFbs) exposed to HMGB1 may exert a paracrine effect on mouse and human CSCs. Human cFbs expressed the HMGB1 receptor RAGE. Luminex technology and ELISA assays revealed that HMGB1 significantly enhanced VEGF, PlGF, Mip-1alpha, IFN-gamma, GM-CSF, Il-10, Il-1beta, Il-4, Il-1ra, Il-9 and TNF-alpha in cFbs cell culture medium. HMGB1-stimulated cFbs conditioned media induced CSC migration and proliferation. These effects were significantly higher to those obtained when HMGB1 was added directly to the culture medium. In conclusion, we provide evidence that HMGB1 may act in a paracrine manner stimulating growth factor, cytokine and chemokine release by cFbs which, in turn, modulate CSC function. Via this mechanism HMGB1 may contribute to cardiac tissue regeneration.

Inhibition of human mast cell growth and differentiation by interferon gamma-1b.

PubMed

Kirshenbaum, A S; Worobec, A S; Davis, T A; Goff, J P; Semere, T; Metcalfe, D D

1998-03-01

In an effort to identify cytokines that inhibit human mast cell growth, we cultured HMC-1 cells and recombinant human stem cell factor (rhSCF)-dependent human bone marrow-derived mast cells (HBMCs) in the presence of interferon gamma (IFNgamma)-1b and interferon alpha (IFNalpha)-2b. HMC-1 cell numbers decreased in the presence of 1000 U/mL IFNgamma-1b but were unaffected by 1000 U/mL of IFNalpha-2b. HBMCs were then cultured for 0 to 7 days with 100 ng/mL rhSCF and 10 ng/mL recombinant human IL-3 (rhIL-3), followed by culture in rhSCF and administration of either 1000 U/mL IFNalpha-2b or 1000 U/mL IFNgamma-1b. HBMCs appearing in cultures with rhSCF alone or in combination with IFNalpha-2b were virtually identical in number through 8 weeks of culture. In cultures supplemented with IFNgamma-1b, HBMCs significantly decreased in number and incidence of granular metachromasia by 4 to 5 weeks (p<0.001). Similar results were obtained when human marrow was cultured from day 0 with rhSCF and IFNgamma-1b. Mature rhSCF-dependent HBMCs were also cultured at 5 weeks with rhSCF alone or in combination with IFNgamma-1b. Compared with cells cultured in rhSCF, mature 5-week HBMC cultures treated with rhSCF plus IFNgamma-1b revealed a decrease in mast cells, and those mast cells that remained had fewer toluidine blue- and tryptase-positive granules after 5 to 8 weeks. FACS analysis of rhSCF plus IFNgamma-1b-treated mature HBMCs revealed increased c-kit and Fc(epsilon)RI expression. Mast cell releasibility was not increased. IFNgamma-lb was thus able to suppress mast cell growth from CD34+ cells, suggesting that this agent should be considered as a candidate cytokine for the treatment of disorders of mast cell proliferation.

Congratulations or Condolences? The Role of Human Capital in the Cultivation of a University Administrator

ERIC Educational Resources Information Center

McDowell, John; Singell, Larry D., Jr.; Stater, Mark

2009-01-01

Administrative skill is essential to organizational effectiveness. Yet, few studies examine how human capital investments over a career affect selection into administration. We use panel data for economists to estimate the probability of choosing administration over a pure academic track. The results show that, while research-specific human…

Embryonic Stem Cell Patents and Human Dignity

PubMed Central

Resnik, David B.

2009-01-01

This article examines the assertion that human embryonic stem cells patents are immoral because they violate human dignity. After analyzing the concept of human dignity and its role in bioethics debates, this article argues that patents on human embryos or totipotent embryonic stem cells violate human dignity, but that patents on pluripotent or multipotent stem cells do not. Since patents on pluripotent or multipotent stem cells may still threaten human dignity by encouraging people to treat embryos as property, patent agencies should carefully monitor and control these patents to ensure that patents are not inadvertently awarded on embryos or totipotent stem cells. PMID:17922198

Human Cells Display Reduced Apoptotic Function Relative to Chimpanzee Cells

PubMed Central

McDonald, John F.

2012-01-01

Previously published gene expression analyses suggested that apoptotic function may be reduced in humans relative to chimpanzees and led to the hypothesis that this difference may contribute to the relatively larger size of the human brain and the increased propensity of humans to develop cancer. In this study, we sought to further test the hypothesis that humans maintain a reduced apoptotic function relative to chimpanzees by conducting a series of apoptotic function assays on human, chimpanzee and macaque primary fibroblastic cells. Human cells consistently displayed significantly reduced apoptotic function relative to the chimpanzee and macaque cells. These results are consistent with earlier findings indicating that apoptotic function is reduced in humans relative to chimpanzees. PMID:23029431

Aripiprazole maintenance increases smoked cocaine self-administration in humans

PubMed Central

Rubin, Eric; Foltin, Richard W.

2011-01-01

Rationale Partial dopamine receptor agonists have been proposed as candidate pharmacotherapies for cocaine dependence. Objective This 42-day, within-subject, human laboratory study assessed how maintenance on aripiprazole, a partial D2 receptor agonist, influenced smoked cocaine self-administration, cardiovascular measures, subjective effects, and cocaine craving in nontreatment-seeking, cocaine-dependent volunteers. Methods In order to achieve steady-state concentrations, participants (n=8 men) were administered placebo and aripiprazole (15 mg/day) capsules in counter-balanced order for 21 days. A smoked cocaine dose–response curve (0, 12, 25, 50 mg) was determined twice under placebo and aripiprazole maintenance. Sessions comprised a “sample” trial, when participants smoked the cocaine dose available that session, and five choice trials, when they responded on a progressive-ratio schedule of reinforcement to receive the cocaine dose or receive $5.00. Results Cocaine’s reinforcing, subjective, and cardiovascular effects were dose-dependent. Aripiprazole significantly increased cocaine (12, 25 mg) self-administration. Following a single administration of cocaine (25 mg), aripiprazole decreased ratings of how much participants would pay for that dose. Following repeated cocaine (50 mg) self-administration, aripiprazole decreased ratings of cocaine quality, craving, and good drug effect as compared to placebo. Conclusions These data suggest that aripiprazole may have increased self-administration to compensate for a blunted subjective cocaine effect. Overall, the findings do not suggest aripiprazole would be useful for treating cocaine dependence. PMID:21373790

Hybrid clone cells derived from human breast epithelial cells and human breast cancer cells exhibit properties of cancer stem/initiating cells.

PubMed

Gauck, Daria; Keil, Silvia; Niggemann, Bernd; Zänker, Kurt S; Dittmar, Thomas

2017-08-02

The biological phenomenon of cell fusion has been associated with cancer progression since it was determined that normal cell × tumor cell fusion-derived hybrid cells could exhibit novel properties, such as enhanced metastatogenic capacity or increased drug resistance, and even as a mechanism that could give rise to cancer stem/initiating cells (CS/ICs). CS/ICs have been proposed as cancer cells that exhibit stem cell properties, including the ability to (re)initiate tumor growth. Five M13HS hybrid clone cells, which originated from spontaneous cell fusion events between M13SV1-EGFP-Neo human breast epithelial cells and HS578T-Hyg human breast cancer cells, and their parental cells were analyzed for expression of stemness and EMT-related marker proteins by Western blot analysis and confocal laser scanning microscopy. The frequency of ALDH1-positive cells was determined by flow cytometry using AldeRed fluorescent dye. Concurrently, the cells' colony forming capabilities as well as the cells' abilities to form mammospheres were investigated. The migratory activity of the cells was analyzed using a 3D collagen matrix migration assay. M13HS hybrid clone cells co-expressed SOX9, SLUG, CK8 and CK14, which were differently expressed in parental cells. A variation in the ALDH1-positive putative stem cell population was observed among the five hybrids ranging from 1.44% (M13HS-7) to 13.68% (M13HS-2). In comparison to the parental cells, all five hybrid clone cells possessed increased but also unique colony formation and mammosphere formation capabilities. M13HS-4 hybrid clone cells exhibited the highest colony formation capacity and second highest mammosphere formation capacity of all hybrids, whereby the mean diameter of the mammospheres was comparable to the parental cells. In contrast, the largest mammospheres originated from the M13HS-2 hybrid clone cells, whereas these cells' mammosphere formation capacity was comparable to the parental breast cancer cells. All M13HS

Effects of Intravenous Administration of Human Umbilical Cord Blood Stem Cells in 3-Acetylpyridine-Lesioned Rats

PubMed Central

Calatrava-Ferreras, Lucía; Gonzalo-Gobernado, Rafael; Herranz, Antonio S.; Reimers, Diana; Montero Vega, Teresa; Jiménez-Escrig, Adriano; Richart López, Luis Alberto; Bazán, Eulalia

2012-01-01

Cerebellar ataxias include a heterogeneous group of infrequent diseases characterized by lack of motor coordination caused by disturbances in the cerebellum and its associated circuits. Current therapies are based on the use of drugs that correct some of the molecular processes involved in their pathogenesis. Although these treatments yielded promising results, there is not yet an effective therapy for these diseases. Cell replacement strategies using human umbilical cord blood mononuclear cells (HuUCBMCs) have emerged as a promising approach for restoration of function in neurodegenerative diseases. The aim of this work was to investigate the potential therapeutic activity of HuUCBMCs in the 3-acetylpyridine (3-AP) rat model of cerebellar ataxia. Intravenous administered HuUCBMCs reached the cerebellum and brain stem of 3-AP ataxic rats. Grafted cells reduced 3-AP-induced neuronal loss promoted the activation of microglia in the brain stem, and prevented the overexpression of GFAP elicited by 3-AP in the cerebellum. In addition, HuUCBMCs upregulated the expression of proteins that are critical for cell survival, such as phospho-Akt and Bcl-2, in the cerebellum and brain stem of 3-AP ataxic rats. As all these effects were accompanied by a temporal but significant improvement in motor coordination, HuUCBMCs grafts can be considered as an effective cell replacement therapy for cerebellar disorders. PMID:23150735

Human ES cells – haematopoiesis and transplantation strategies*

PubMed Central

Kaufman, DS; Thomson, JA

2002-01-01

Human embryonic stem (ES) cells provide a novel opportunity to study early developmental events in a human system. We have used human ES cell lines, including clonally derived lines, to evaluate haematopoiesis. Co-culture of the human ES cells with irradiated bone marrow stromal cell lines in the presence of fetal bovine serum (FBS), but without other exogenous cytokines, leads to differentiation of the human ES cells within a matter of days. A portion of these differentiated cells express CD34, the best-defined marker for early haematopoietic cells. Haematopoietic colony-forming cells (CFCs) are demonstrated by methylcellulose assay. Myeloid, erythroid, megakaryocyte and multipotential CFCs can all be derived under these conditions. Enrichment of CD34+ cells derived from the human ES cells markedly increases the yield of CFCs, as would be expected for cells derived from adult bone marrow or umbilical cord blood. Transcription factors are also expressed in a manner consistent with haematopoietic differentiation. This system now presents the potential to evaluate specific conditions needed to induce or support events in early human blood development. Human ES cells are also a novel source of cells for transplantation therapies. The immunogenicity of ES cell-derived cells is unknown. The unique properties of ES cells afford the opportunity to explore novel mechanisms to prevent immune-mediated rejection. Potential strategies to overcome rejection will be presented, including creation of haematopoietic chimerism as a means to successfully transplant cells and tissues derived from human ES cells. PMID:12033728

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

Human Hepatocyte Growth Factor (hHGF)-Modified Hepatic Oval Cells Improve Liver Transplant Survival

PubMed Central

Li, Li; Ran, Jiang-Hua; Li, Xue-Hua; Liu, Zhi-Heng; Liu, Gui-Jie; Gao, Yan-Chao; Zhang, Xue-Li; Sun, Hiu-Dong

2012-01-01

Despite progress in the field of immunosuppression, acute rejection is still a common postoperative complication following liver transplantation. This study aims to investigate the capacity of the human hepatocyte growth factor (hHGF) in modifying hepatic oval cells (HOCs) administered simultaneously with orthotopic liver transplantation as a means of improving graft survival. HOCs were activated and isolated using a modified 2-acetylaminofluorene/partial hepatectomy (2-AAF/PH) model in male Lewis rats. A HOC line stably expressing the HGF gene was established following stable transfection of the pBLAST2-hHGF plasmid. Our results demonstrated that hHGF-modified HOCs could efficiently differentiate into hepatocytes and bile duct epithelial cells in vitro. Administration of HOCs at the time of liver transplantation induced a wider distribution of SRY-positive donor cells in liver tissues. Administration of hHGF-HOC at the time of transplantation remarkably prolonged the median survival time and improved liver function for recipients compared to these parameters in the other treatment groups (P<0.05). Moreover, hHGF-HOC administration at the time of liver transplantation significantly suppressed elevation of interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) levels while increasing the production of IL-10 and TGF-β1 (P<0.05). HOC or hHGF-HOC administration promoted cell proliferation, reduced cell apoptosis, and decreased liver allograft rejection rates. Furthermore, hHGF-modified HOCs more efficiently reduced acute allograft rejection (P<0.05 versus HOC transplantation only). Our results indicate that the combination of hHGF-modified HOCs with liver transplantation decreased host anti-graft immune responses resulting in a reduction of allograft rejection rates and prolonging graft survival in recipient rats. This suggests that HOC-based cell transplantation therapies can be developed as a means of treating severe liver injuries. PMID

Functional characterization of human pluripotent stem cell-derived arterial endothelial cells.

PubMed

Zhang, Jue; Chu, Li-Fang; Hou, Zhonggang; Schwartz, Michael P; Hacker, Timothy; Vickerman, Vernella; Swanson, Scott; Leng, Ning; Nguyen, Bao Kim; Elwell, Angela; Bolin, Jennifer; Brown, Matthew E; Stewart, Ron; Burlingham, William J; Murphy, William L; Thomson, James A

2017-07-25

Here, we report the derivation of arterial endothelial cells from human pluripotent stem cells that exhibit arterial-specific functions in vitro and in vivo. We combine single-cell RNA sequencing of embryonic mouse endothelial cells with an EFNB2-tdTomato/EPHB4-EGFP dual reporter human embryonic stem cell line to identify factors that regulate arterial endothelial cell specification. The resulting xeno-free protocol produces cells with gene expression profiles, oxygen consumption rates, nitric oxide production levels, shear stress responses, and TNFα-induced leukocyte adhesion rates characteristic of arterial endothelial cells. Arterial endothelial cells were robustly generated from multiple human embryonic and induced pluripotent stem cell lines and have potential applications for both disease modeling and regenerative medicine.

Susceptibility of human liver cells to porcine endogenous retrovirus.

PubMed

Lin, Xinzi; Qi, Lin; Li, Zhiguo; Chi, Hao; Lin, Wanjun; Wang, Yan; Jiang, Zesheng; Pan, Mingxin; Gao, Yi

2013-12-01

The risk of porcine endogenous retrovirus infection is a major barrier for pig-to-human xenotransplant. Porcine endogenous retrovirus, present in porcine cells, can infect many human and nonhuman primate cells in vitro, but there is no evidence available about in vitro infection of human liver cells. We investigated the susceptibility of different human liver cells to porcine endogenous retrovirus. The supernatant from a porcine kidney cell line was added to human liver cells, including a normal hepatocyte cell line (HL-7702 cells), primary hepatocytes (Phh cells), and a liver stellate cell line (Lx-2 cells), and to human embryonic kidney cells as a reference control. Expression of the porcine endogenous retrovirus antigen p15E in the human cells was evaluated with polymerase chain reaction, reverse transcription-polymerase chain reaction, and Western blot. The porcine endogenous retrovirus antigen p15E was not expressed in any human liver cells (HL-7702, Phh, or Lx-2 cells) that had been exposed to supernatants from porcine kidney cell lines. Porcine endogenous retrovirus-specific fragments were amplified in human kidney cells. Human liver cells tested were not susceptible to infection by porcine endogenous retrovirus. Therefore, not all human cells are susceptible to porcine endogenous retrovirus.

Generation and characteristics of human Sertoli cell line immortalized by overexpression of human telomerase

PubMed Central

Wen, Liping; Yuan, Qingqing; Sun, Min; Niu, Minghui; Wang, Hong; Fu, Hongyong; Zhou, Fan; Yao, Chencheng; Wang, Xiaobo; Li, Zheng; He, Zuping

2017-01-01

Sertoli cells are required for normal spermatogenesis and they can be reprogrammed to other types of functional cells. However, the number of primary Sertoli cells is rare and human Sertoli cell line is unavailable. In this study, we have for the first time reported a stable human Sertoli cell line, namely hS1 cells, by overexpression of human telomerase. The hS1 cells expressed a number of hallmarks for human Sertoli cells, including SOX9, WT1, GDNF, SCF, BMP4, BMP6, GATA4, and VIM, and they were negative for 3β-HSD, SMA, and VASA. Higher levels of AR and FSHR were observed in hS1 cells compared to primary human Sertoli cells. Microarray analysis showed that 70.4% of global gene profiles of hS1 cells were similar to primary human Sertoli cells. Proliferation assay demonstrated that hS1 cells proliferated rapidly and they could be passaged for more than 30 times in 6 months. Neither Y chromosome microdeletion nor tumorgenesis was detected in this cell line and 90% normal karyotypes existed in hS1 cells. Collectively, we have established the first human Sertoli cell line with phenotype of primary human Sertoli cells, an unlimited proliferation potential and high safety, which could offer sufficient human Sertoli cells for basic research as well as reproductive and regenerative medicine. PMID:28152522

Targeting PI3K-AKT-mTOR by LY3023414 inhibits human skin squamous cell carcinoma cell growth in vitro and in vivo.

PubMed

Zou, Ying; Ge, Minggai; Wang, Xuemin

2017-08-19

Abnormal activation of PI3K-AKT-mTOR signaling is detected in human skin squamous cell carcinoma (SCC). LY3023414 is a novel, potent, and orally bio-available PI3K-AKT-mTOR inhibitor. Its activity against human skin SCC cells was tested. We demonstrated that LY3023414 was cytotoxic when added to established (A431 line) and primary (patient-derived) human skin SCC cells. LY3023414 induced G0/1-S arrest and inhibited proliferation of skin SCC cells. Moreover, LY3023414 induced activation of caspase-3/-9 and apoptosis in skin SCC cells. Intriguingly, LY3023414 was yet non-cytotoxic nor pro-apoptotic to normal human skin cells (melanocytes, keratinocytes and fibroblasts). At the molecular level, LY3023414 blocked PI3K-AKT-mTOR activation in skin SCC cells, as it dephosphorylated PI3K-AKT-mTOR substrates: P85, AKT and S6K1. In vivo studies showed that oral administration of LY3023414 at well-tolerated doses inhibited A431 xenograft tumor growth in severe combined immunodeficiency (SCID) mice. AKT-mTOR activation in LY3023414-treated tumors was also largely inhibited. Together, these results suggest that targeting PI3K-AKT-mTOR by LY3023414 inhibits human skin SCC cell growth in vitro and in vivo, establishing the rationale for further clinical testing. Copyright © 2017 Elsevier Inc. All rights reserved.

Th17 cells in human disease

PubMed Central

Tesmer, Laura A.; Lundy, Steven K.; Sarkar, Sujata; Fox, David A.

2012-01-01

Summary Our understanding of the role of T cells in human disease is undergoing revision as a result of the discovery of T-helper 17 (Th17) cells, a unique CD4+ T-cell subset characterized by production of interleukin-17 (IL-17). IL-17 is a highly inflammatory cytokine with robust effects on stromal cells in many tissues. Recent data in humans and mice suggest that Th17 cells play an important role in the pathogenesis of a diverse group of immune-mediated diseases, including psoriasis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, and asthma. Initial reports also propose a role for Th17 cells in tumorigenesis and transplant rejection. Important differences, as well as many similarities, are emerging when the biology of Th17 cells in the mouse is compared with corresponding phenomena in humans. As our understanding of human Th17 biology grows, the mechanisms underlying many diseases are becoming more apparent, resulting in a new appreciation for both previously known and more recently discovered cytokines, chemokines, and feedback mechanisms. Given the strong association between excessive Th17 activity and human disease, new therapeutic approaches targeting Th17 cells are highly promising, but the potential safety of such treatments may be limited by the role of these cells in normal host defenses against infection. PMID:18613831

Clinical concerns of immunogenicity produced at cellular levels by biopharmaceuticals following their parenteral administration into human body.

PubMed

Tamilvanan, Shunmugaperumal; Raja, Natarajan Livingston; Sa, Biswanath; Basu, Sanat Kumar

2010-08-01

Similar to the low molecular weight traditional drugs, biopharmaceuticals are capable of producing not only therapeutic effects but also side effects provided if the dose of these compounds exceeds certain concentration and/or if the exposure duration of these compounds at subtoxic doses is being lengthened. In addition, a major drawback of biopharmaceuticals is the risk of antibody formation. Following the administration of biopharmaceuticals into human body, the formation of antidrug-antibody (ADA) or neutralizing antibody and other general immune system effects (including allergy, anaphylaxis, or serum sickness) are of clinical concern regarding therapeutic efficacy and patient safety. For example, drug-induced neutralizing antibodies to erythropoietin (EPO) result in pure red cell aplasia, whereas drug-induced acquired anti-factor VIII antibodies worsen the pathology associated with hemophilia. Since most of the already developed or under development biopharmaceuticals are to some extent immunogenic, the regulatory agencies insist to conduct potential ADA formation during the drug development process itself. This review encompasses a short overview on the clinical concerns of immunogenicity produced at cellular levels by growth hormone, interferon-alpha, EPO, factor VIII, and factor IX following their parenteral administration into human body. Clinical concerns related to immunogenicity produced by the biosimilar versions of these drugs are also presented wherever possible.

3 CFR - Guidelines for Human Stem Cell Research

Code of Federal Regulations, 2010 CFR

2010-01-01

... 3 The President 1 2010-01-01 2010-01-01 false Guidelines for Human Stem Cell Research Presidential Documents Other Presidential Documents Memorandum of July 30, 2009 Guidelines for Human Stem Cell Research..., scientifically worthy human stem cell research, including human embryonic stem cell research, to the extent...

Pluripotent stem cells in translation: a Food and Drug Administration-National Institutes of Health collaboration.

PubMed

Kleitman, Naomi; Rao, Mahendra S; Owens, David F

2013-07-01

Recently, the U.S. Food and Drug Administration (FDA), the U.S. National Institutes of Health, and the stem cell research community have collaborated on a series of workshops that address moving pluripotent stem cell therapies into the clinic. The first two workshops in the series focused on preclinical science, and a third, future workshop will focus on clinical trials. This summary addresses major points from both of the recent preclinically focused meetings. When entering into a therapeutics developmental program based on pluripotent cells, investigators must make decisions at the very early stages that will have major ramifications during later phases of development. Presentations and discussions from both invited participants and FDA staff described the need to characterize and document the quality, variability, and suitability of the cells and commercial reagents used at every translational stage. This requires consideration of future regulatory requirements, ranging from donor eligibility of the original source material to the late-stage manufacturing protocols. Federal, industrial, and academic participants agreed that planning backward is the best way to anticipate what evidence will be needed to justify human testing of novel therapeutics and to eliminate wasted efforts.

Pluripotent Stem Cells in Translation: A Food and Drug Administration-National Institutes of Health Collaboration

PubMed Central

Kleitman, Naomi; Owens, David F.

2013-01-01

Recently, the U.S. Food and Drug Administration (FDA), the U.S. National Institutes of Health, and the stem cell research community have collaborated on a series of workshops that address moving pluripotent stem cell therapies into the clinic. The first two workshops in the series focused on preclinical science, and a third, future workshop will focus on clinical trials. This summary addresses major points from both of the recent preclinically focused meetings. When entering into a therapeutics developmental program based on pluripotent cells, investigators must make decisions at the very early stages that will have major ramifications during later phases of development. Presentations and discussions from both invited participants and FDA staff described the need to characterize and document the quality, variability, and suitability of the cells and commercial reagents used at every translational stage. This requires consideration of future regulatory requirements, ranging from donor eligibility of the original source material to the late-stage manufacturing protocols. Federal, industrial, and academic participants agreed that planning backward is the best way to anticipate what evidence will be needed to justify human testing of novel therapeutics and to eliminate wasted efforts. PMID:23757505

Large-scale production of embryonic red blood cells from human embryonic stem cells.

PubMed

Olivier, Emmanuel N; Qiu, Caihong; Velho, Michelle; Hirsch, Rhoda Elison; Bouhassira, Eric E

2006-12-01

To develop a method to produce in culture large number of erythroid cells from human embryonic stem cells. Human H1 embryonic stem cells were differentiated into hematopoietic cells by coculture with a human fetal liver cell line, and the resulting CD34-positive cells were expanded in vitro in liquid culture using a three-step method. The erythroid cells produced were then analyzed by light microscopy and flow cytometry. Globin expression was characterized by quantitative reverse-transcriptase polymerase chain reaction and by high-performance liquid chromatography. CD34-positive cells produced from human embryonic stem cells could be efficiently differentiated into erythroid cells in liquid culture leading to a more than 5000-fold increase in cell number. The erythroid cells produced are similar to primitive erythroid cells present in the yolk sac of early human embryos and did not enucleate. They are fully hemoglobinized and express a mixture of embryonic and fetal globins but no beta-globin. We have developed an experimental protocol to produce large numbers of primitive erythroid cells starting from undifferentiated human embryonic stem cells. As the earliest human erythroid cells, the nucleated primitive erythroblasts, are not very well characterized because experimental material at this stage of development is very difficult to obtain, this system should prove useful to answer a number of experimental questions regarding the biology of these cells. In addition, production of mature red blood cells from human embryonic stem cells is of great potential practical importance because it could eventually become an alternate source of cell for transfusion.

Modeling human infertility with pluripotent stem cells.

PubMed

Chen, Di; Gell, Joanna J; Tao, Yu; Sosa, Enrique; Clark, Amander T

2017-05-01

Human fertility is dependent upon the correct establishment and differentiation of the germline. This is because no other cell type in the body is capable of passing a genome and epigenome from parent to child. Terminally differentiated germline cells in the adult testis and ovary are called gametes. However, the initial specification of germline cells occurs in the embryo around the time of gastrulation. Most of our knowledge regarding the cell and molecular events that govern human germline specification involves extrapolating scientific principles from model organisms, most notably the mouse. However, recent work using next generation sequencing, gene editing and differentiation of germline cells from pluripotent stem cells has revealed that the core molecular mechanisms that regulate human germline development are different from rodents. Here, we will discuss the major molecular pathways required for human germline differentiation and how pluripotent stem cells have revolutionized our ability to study the earliest steps in human embryonic lineage specification in order to understand human fertility. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Human innate lymphoid cells.

PubMed

Mjösberg, Jenny; Spits, Hergen

2016-11-01

Innate lymphoid cells (ILCs) are increasingly acknowledged as important mediators of immune homeostasis and pathology. ILCs act as early orchestrators of immunity, responding to epithelium-derived signals by expressing an array of cytokines and cell-surface receptors, which shape subsequent immune responses. As such, ILCs make up interesting therapeutic targets for several diseases. In patients with allergy and asthma, group 2 innate lymphoid cells produce high amounts of IL-5 and IL-13, thereby contributing to type 2-mediated inflammation. Group 3 innate lymphoid cells are implicated in intestinal homeostasis and psoriasis pathology through abundant IL-22 production, whereas group 1 innate lymphoid cells are accumulated in chronic inflammation of the gut (inflammatory bowel disease) and lung (chronic obstructive pulmonary disease), where they contribute to IFN-γ-mediated inflammation. Although the ontogeny of mouse ILCs is slowly unraveling, the development of human ILCs is far from understood. In addition, the growing complexity of the human ILC family in terms of previously unrecognized functional heterogeneity and plasticity has generated confusion within the field. Here we provide an updated view on the function and plasticity of human ILCs in tissue homeostasis and disease. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

GENETICS OF HUMAN CELL LINES

PubMed Central

Djordjevic, B.; Szybalski, Waclaw

1960-01-01

The human cell line D98S can be cultivated indefinitely in the presence of up to 3 x 10–5 M 5-bromodeoxyuridine (BUDR), without loss of cell viability. During this time, BUDR is incorporated into both strands of the DNA molecules, replacing up to 45 per cent of the thymidine and thereby rendering the cells highly sensitive to UV light and to x-rays. Cells grown for a limited period of time in the presence of 5-iododeoxyuridine (IUDR) become UV-sensitized, while prolonged cultivation with IUDR results in the loss of cell viability. The properties of the BUDR label permitted the demonstration that: (a) human DNA replicates in a "semiconservative" manner; (b) the degree of radiosensitization of BUDR-treated cells depends on whether the DNA has been substituted in one strand only ("unifilarly") or in both strands ("bifilarly"); (c) functional human DNA is produced during partial inhibition of protein synthesis. The potential applicability of this new rational principle of radiosensitization to the radiotherapy of neoplastic diseases is discussed. PMID:13723177

Some Ethical Concerns About Human Induced Pluripotent Stem Cells.

PubMed

Zheng, Yue Liang

2016-10-01

Human induced pluripotent stem cells can be obtained from somatic cells, and their derivation does not require destruction of embryos, thus avoiding ethical problems arising from the destruction of human embryos. This type of stem cell may provide an important tool for stem cell therapy, but it also results in some ethical concerns. It is likely that abnormal reprogramming occurs in the induction of human induced pluripotent stem cells, and that the stem cells generate tumors in the process of stem cell therapy. Human induced pluripotent stem cells should not be used to clone human beings, to produce human germ cells, nor to make human embryos. Informed consent should be obtained from patients in stem cell therapy.

Expression cloning of human B cell immunoglobulins.

PubMed

Wardemann, Hedda; Kofer, Juliane

2013-01-01

The majority of lymphomas originate from B cells at the germinal center stage or beyond. Preferential selection of B cell clones by a limited set of antigens has been suggested to drive lymphoma development. However, little is known about the specificity of the antibodies expressed by lymphoma cells, and the role of antibody-specificity in lymphomagenesis remains elusive. Here, we describe a strategy to characterize the antibody reactivity of human B cells. The approach allows the unbiased characterization of the human antibody repertoire on a single cell level through the generation of recombinant monoclonal antibodies from single primary human B cells of defined origin. This protocol offers a detailed description of the method starting from the flow cytometric isolation of single human B cells, to the RT-PCR-based amplification of the expressed Igh, Igκ, and Igλ chain genes, and Ig gene expression vector cloning for the in vitro production of monoclonal antibodies. The strategy may be used to obtain information on the clonal evolution of B cell lymphomas by single cell Ig gene sequencing and on the antibody reactivity of human lymphoma B cells.

Comprehensive evaluation of leukocyte lineage derived from human hematopoietic cells in humanized mice.

PubMed

Takahashi, Masayuki; Tsujimura, Noriyuki; Otsuka, Kensuke; Yoshino, Tomoko; Mori, Tetsushi; Matsunaga, Tadashi; Nakasono, Satoshi

2012-04-01

Recently, humanized animals whereby a part of the animal is biologically engineered using human genes or cells have been utilized to overcome interspecific differences. Herein, we analyzed the detail of the differentiation states of various human leukocyte subpopulations in humanized mouse and evaluated comprehensively the similarity of the leukocyte lineage between humanized mice and humans. Humanized mice were established by transplanting human CD34(+) cord blood cells into irradiated severely immunodeficient NOD/Shi-scid/IL2Rγ(null) (NOG) mice, and the phenotypes of human cells contained in bone marrow, thymus, spleen and peripheral blood from the mice were analyzed at monthly intervals until 4 months after cell transplantation. The analysis revealed that transplanted human hematopoietic stem cells via the caudal vein homed and engrafted themselves successfully at the mouse bone marrow. Subsequently, the differentiated leukocytes migrated to the various tissues. Almost all of the leukocytes within the thymus were human cells. Furthermore, analysis of the differentiation states of human leukocytes in various tissues and organs indicated that it is highly likely that the human-like leukocyte lineage can be developed in mice. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Only Follow-Up of Memory B Cells Helps Monitor Rituximab Administration to Patients with Neuromyelitis Optica Spectrum Disorders.

PubMed

Lebrun, Christine; Cohen, Mikael; Rosenthal-Allieri, Maria Alessandra; Bresch, Saskia; Benzaken, Sylvia; Marignier, Romain; Seitz-Polski, Barbara; Ticchioni, Michel

2018-06-07

Neuromyelitis optica spectrum disorders (NMOSD) are identified as a spectrum of inflammatory demyelinating disorders involving the brain, spinal cord and optic nerves. These disorders require early diagnosis and highly active immunosuppressive treatment. Rituximab (RTX) has demonstrated efficacy in limiting relapse in NMOSD when using several administration schedules. We questioned if the CD19+ CD27+ memory B cell count was a more reliable marker to monitor RTX administration than the RTX plasma level and CD19+ B cell count. We analyzed 125 blood samples from 17 NMOSD patients treated with RTX and also measured the level of anti-aquaporine-4 antibodies (anti-AQP-4 Abs), human anti-chimeric antibodies to the murine fragment of RTX (HACA-RTX Abs), and the RTX concentration. The mean follow-up time of the cohort was 7.4 (2-16) years. All patients improved with a mean EDSS going from 4 (1-8.5) to 2.7 (1-5.5). The mean interval between RTX infusions was 9.6 months with identification of prolonged responders. Total CD19+ B cell detection with the routine technique did not correlate to re-emergence of CD19+ CD27+ memory B cells. The RTX residual concentration did not correlate with the CD19+ CD27+ memory B cell count or with anti-RTX antibody production. In contrast to total CD19+ cell, detected with the routine technique, CD19+ CD27+ memory B cells are a reliable marker for biological relapse and allow a decrease in the frequency of infusions.

Assessment of oral transmission using cell-free human immunodeficiency virus-1 in mice reconstituted with human peripheral blood leucocyte

PubMed Central

Nakao, Ryoma; Hanada, Nobuhiro; Asano, Toshihiko; Hara, Takashi; Abdus Salam, MD; Matin, Khairul; Shimazu, Yoshihito; Nakasone, Tadashi; Horibata, Shigeo; Aoba, Takaaki; Honda, Mitsuo; Amagasa, Teruo; Senpuku, Hidenobu

2003-01-01

Oral–genital contact is one of the risk factors for the transmission of human immunodeficiency virus (HIV) in adults. In recent reports, oral exposure to simian immunodeficiency virus (SIV) was found to have important implications for the achievement of mucosal transmission of HIV in a rhesus monkey animal model. In the present study, we aimed first to establish a small animal model which did not develop tonsils suitable for HIV oral mucosa transmission, using non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice and NOD/SCID B2mnull mice grafted with human peripheral blood leucocytes (hu-PBL) and stimulated with interleukin (IL)-4, and second to investigate whether oral exposure to cell-free R5 and X4 HIV-1 could lead to oral transmission of HIV through intact or traumatized mucosal tissues in humanized mice. Both low and high concentrations of cell-free R5 and X4 viruses failed to cause oral transmission with or without trauma in hu-PBL-NOD/SCID and NOD/SCID Β2mnull mice, which presented a number of CD4+ cells in gingival tissues and oral cavities with or without tissue injury. The present results show that IL-4-administrated NOD/SCID B2mnull mice are useful as a small-humanized model for the study of HIV oral infection, which may help to define the window of opportunity for oral transmission by the HIV virus in animal model experiments. PMID:12757623

Haematopoietic stem and progenitor cells from human pluripotent stem cells

PubMed Central

Sugimura, Ryohichi; Jha, Deepak Kumar; Han, Areum; Soria-Valles, Clara; da Rocha, Edroaldo Lummertz; Lu, Yi-Fen; Goettel, Jeremy A.; Serrao, Erik; Rowe, R. Grant; Malleshaiah, Mohan; Wong, Irene; Sousa, Patricia; Zhu, Ted N.; Ditadi, Andrea; Keller, Gordon; Engelman, Alan N.; Snapper, Scott B.; Doulatov, Sergei; Daley, George Q.

2018-01-01

A variety of tissue lineages can be differentiated from pluripotent stem cells by mimicking embryonic development through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors. Here, to yield functional human haematopoietic stem cells, we perform morphogen-directed differentiation of human pluripotent stem cells into haemogenic endothelium followed by screening of 26 candidate haematopoietic stem-cell-specifying transcription factors for their capacity to promote multi-lineage haematopoietic engraftment in mouse hosts. We recover seven transcription factors (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1 and SPI1) that are sufficient to convert haemogenic endothelium into haematopoietic stem and progenitor cells that engraft myeloid, B and T cells in primary and secondary mouse recipients. Our combined approach of morphogen-driven differentiation and transcription-factor-mediated cell fate conversion produces haematopoietic stem and progenitor cells from pluripotent stem cells and holds promise for modelling haematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders. PMID:28514439

New perspectives in human stem cell therapeutic research.

PubMed

Trounson, Alan

2009-06-11

Human stem cells are in evaluation in clinical stem cell trials, primarily as autologous bone marrow studies, autologous and allogenic mesenchymal stem cell trials, and some allogenic neural stem cell transplantation projects. Safety and efficacy are being addressed for a number of disease state applications. There is considerable data supporting safety of bone marrow and mesenchymal stem cell transplants but the efficacy data are variable and of mixed benefit. Mechanisms of action of many of these cells are unknown and this raises the concern of unpredictable results in the future. Nevertheless there is considerable optimism that immune suppression and anti-inflammatory properties of mesenchymal stem cells will be of benefit for many conditions such as graft versus host disease, solid organ transplants and pulmonary fibrosis. Where bone marrow and mesenchymal stem cells are being studied for heart disease, stroke and other neurodegenerative disorders, again progress is mixed and mostly without significant benefit. However, correction of multiple sclerosis, at least in the short term is encouraging. Clinical trials on the use of embryonic stem cell derivatives for spinal injury and macular degeneration are beginning and a raft of other clinical trials can be expected soon, for example, the use of neural stem cells for killing inoperable glioma and embryonic stem cells for regenerating beta islet cells for diabetes. The change in attitude to embryonic stem cell research with the incoming Obama administration heralds a new co-operative environment for study and evaluation of stem cell therapies. The Californian stem cell initiative (California Institute for Regenerative Medicine) has engendered global collaboration for this new medicine that will now also be supported by the US Federal Government. The active participation of governments, academia, biotechnology, pharmaceutical companies, and private investment is a powerful consortium for advances in health.

Reprogramming human gallbladder cells into insulin-producing β-like cells

PubMed Central

Benedetti, Eric; Wang, Yuhan; Pelz, Carl; Schug, Jonathan; Kaestner, Klaus H.; Grompe, Markus

2017-01-01

The gallbladder and cystic duct (GBCs) are parts of the extrahepatic biliary tree and share a common developmental origin with the ventral pancreas. Here, we report on the very first genetic reprogramming of patient-derived human GBCs to β-like cells for potential autologous cell replacement therapy for type 1 diabetes. We developed a robust method for large-scale expansion of human GBCs ex vivo. GBCs were reprogrammed into insulin-producing pancreatic β-like cells by a combined adenoviral-mediated expression of hallmark pancreatic endocrine transcription factors PDX1, MAFA, NEUROG3, and PAX6 and differentiation culture in vitro. The reprogrammed GBCs (rGBCs) strongly induced the production of insulin and pancreatic endocrine genes and these responded to glucose stimulation in vitro. rGBCs also expressed an islet-specific surface marker, which was used to enrich for the most highly reprogrammed cells. More importantly, global mRNA and microRNA expression profiles and protein immunostaining indicated that rGBCs adopted an overall β-like state and these rGBCs engrafted in immunodeficient mice. Furthermore, comparative global expression analyses identified putative regulators of human biliary to β cell fate conversion. In summary, we have developed, for the first time, a reliable and robust genetic reprogramming and culture expansion of primary human GBCs—derived from multiple unrelated donors—into pancreatic β-like cells ex vivo, thus showing that human gallbladder is a potentially rich source of reprogrammable cells for autologous cell therapy in diabetes. PMID:28813430

Recombinant human SDF-1α administration accelerates aneurysm neck reendothelialization in rabbit saccular aneurysm after flow diverter treatment.

PubMed

Li, Zifu; Zhao, Rui; Fang, Xinggen; Huang, Qinghai; Liu, Jianmin

2017-03-01

Reendothelialization in the aneurysm neck is pivotal to vascular repair for intracranial aneurysm after flow diverter (FD) implantation. Recombinant human stromal cell-derived factor 1α (rhSDF-1α) is a vital chemoattractant to stem cells and potentially facilitates reendothelialization. Here, we sought to investigate the therapeutic effects of intravenous administration of rhSDF-1α and uncover its potential mechanism for promoting aneurysm neck reendothelialization. Recombinant pET32a-186 plasmid was transformed into Escherichia coli to produce the rhSDF-1α protein with biological activity. FD was implanted into the elastase-induced saccular aneurysm in New Zealand white rabbits. rhSDF-1α (50 μg/kg/day) was intravenously administrated for consecutive 7 days after FD implantation. After these procedures, aneurysms were harvested after 2 or 4 weeks. Scanning electron microscopy was used to measure the neointima thickness and count the endothelial-like cells at aneurysm neck. Four weeks later, the mRNA levels of endothelial markers in the neointima at aneurysm neck were examined. Migration assay showed that rhSDF-1α could induce migration of endothelial progenitor cells in a dose-dependent manner. Two weeks after stent implantation, follow-up angiography showed partial aneurysm occlusion in one of each group and total aneurysm occlusion in 17 saccular aneurysm rabbits (9 of the rhSDF-1α group and 8 of the control group). No significant change of neointima thickness at aneurysm neck was observed. Intriguingly, more endothelial-like cells were observed at aneurysm neck in the rhSDF-1α group at 2 weeks (55 vs 13 cells per high-power field) and 4 weeks (104 vs 60 cells per high-power field). The mRNA levels of Tie-2, VE-cadherin, KDR and E-selectin were significantly enhanced compared with those of the control group. These results showed that intravenous administration of rhSDF-1α can accelerate reendothelialization in the aneurysm neck after FD implantation

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

Induction of Skin-Derived Precursor Cells from Human Induced Pluripotent Stem Cells.

PubMed

Sugiyama-Nakagiri, Yoriko; Fujimura, Tsutomu; Moriwaki, Shigeru

2016-01-01

The generation of full thickness human skin from dissociated cells is an attractive approach not only for treating skin diseases, but also for treating many systemic disorders. However, it is currently not possible to obtain an unlimited number of skin dermal cells. The goal of this study was to develop a procedure to produce skin dermal stem cells from induced pluripotent stem cells (iPSCs). Skin-derived precursor cells (SKPs) were isolated as adult dermal precursors that could differentiate into both neural and mesodermal progenies and could reconstitute the dermis. Thus, we attempted to generate SKPs from iPSCs that could reconstitute the skin dermis. Human iPSCs were initially cultured with recombinant noggin and SB431542, an inhibitor of activin/nodal and TGFβ signaling, to induce neural crest progenitor cells. Those cells were then treated with SKP medium that included CHIR99021, a WNT signal activator. The induction efficacy from neural crest progenitor cells to SKPs was more than 97%. No other modifiers tested were able to induce those cells. Those human iPSC-derived SKPs (hiPSC-SKPs) showed a similar gene expression signature to SKPs isolated from human skin dermis. Human iPSC-SKPs differentiated into neural and mesodermal progenies, including adipocytes, skeletogenic cell types and Schwann cells. Moreover, they could be induced to follicular type keratinization when co-cultured with human epidermal keratinocytes. We here provide a new efficient protocol to create human skin dermal stem cells from hiPSCs that could contribute to the treatment of various skin disorders.

No influence of exogenous hyaluronan on the behavior of human cancer cells or endothelial cell capillary formation.

PubMed

Seino, Satoshi; Takeshita, Fumitaka; Asari, Akira; Masuda, Yasunobu; Kunou, Masaaki; Ochiya, Takahiro

2014-07-01

Hyaluronan (HA), a type of glycosaminoglycan used to construct the extracellular matrix, is involved in the proliferation and motility of cells, including cancer cells. The aim of this study was to determine whether exogenous HA has an influence on cancer in vitro and in vivo. High-molecular-weight HA (900 kDa) and low-molecular-weight HA (10 kDa) were added to several types of cancer cell lines in vitro, and proliferation and invasion were assessed. The effect of HA on capillary formation by human umbilical vein endothelial cells was also analyzed. The results showed that both types of HA had no apparent effect on cellular proliferation, invasion, or capillary formation. In an animal study, the 2 types of HA were orally administered to tumor-bearing mice at a dosage of 200 mg/kg/d for 4 wk. Analysis using an in vivo imaging system revealed that tumor proliferation and metastasis were not greatly altered by HA administration. Furthermore, CD31 immunohistochemical staining revealed no obvious change in tumor microvessels. Taken together, these results demonstrate that exogenously administered HA has little effect on cancer. This study may support the safety of various forms of HA administration, including oral intake. Orally administered hyaluronan was recently found to have beneficial effects. However, the effect of exogenous hyaluronan on cancer remains unclear. Our findings support the safety of orally administered hyaluronan and its use as a functional food ingredient. © 2014 Institute of Food Technologists®

DMSO Represses Inflammatory Cytokine Production from Human Blood Cells and Reduces Autoimmune Arthritis

PubMed Central

Elisia, Ingrid; Nakamura, Hisae; Lam, Vivian; Hofs, Elyse; Cederberg, Rachel; Cait, Jessica; Hughes, Michael R.; Lee, Leora; Jia, William; Adomat, Hans H.; Guns, Emma S.; McNagny, Kelly M.; Samudio, Ismael; Krystal, Gerald

2016-01-01

Dimethyl sulfoxide (DMSO) is currently used as an alternative treatment for various inflammatory conditions as well as for cancer. Despite its widespread use, there is a paucity of data regarding its safety and efficacy as well as its mechanism of action in human cells. Herein, we demonstrate that DMSO has ex-vivo anti-inflammatory activity using Escherichia coli- (E. coli) and herpes simplex virus-1 (HSV-1)-stimulated whole human blood. Specifically, we found that between 0.5%– 2%, DMSO significantly suppressed the expression of many pro-inflammatory cytokines/chemokines and prostaglandin E2 (PGE2). However, a significant reduction in monocyte viability was also observed at 2% DMSO, suggesting a narrow window of efficacy. Anti-inflammatory concentrations of DMSO suppressed E. coli-induced ERK1/2, p38, JNK and Akt phosphorylation, suggesting DMSO acts on these signaling pathways to suppress inflammatory cytokine/chemokine production. Although DMSO induces the differentiation of B16/F10 melanoma cells in vitro, topical administration of DMSO to mice subcutaneously implanted with B16 melanoma cells was ineffective at reducing tumor growth, DMSO was also found to block mouse macrophages from polarizing to either an M1- or an M2-phenotype, which may contribute to its inability to slow tumor growth. Topical administration of DMSO, however, significantly mitigated K/BxN serum-induced arthritis in mice, and this was associated with reduced levels of pro-inflammatory cytokines in the joints and white blood cell levels in the blood. Thus, while we cannot confirm the efficacy of DMSO as an anti-cancer agent, the use of DMSO in arthritis warrants further investigation to ascertain its therapeutic potential. PMID:27031833

Edaravone Protects against Methylglyoxal-Induced Barrier Damage in Human Brain Endothelial Cells

PubMed Central

Tóth, Andrea E.; Walter, Fruzsina R.; Bocsik, Alexandra; Sántha, Petra; Veszelka, Szilvia; Nagy, Lajos; Puskás, László G.; Couraud, Pierre-Olivier; Takata, Fuyuko; Dohgu, Shinya; Kataoka, Yasufumi; Deli, Mária A.

2014-01-01

Background Elevated level of reactive carbonyl species, such as methylglyoxal, triggers carbonyl stress and activates a series of inflammatory responses leading to accelerated vascular damage. Edaravone is the active substance of a Japanese medicine, which aids neurological recovery following acute brain ischemia and subsequent cerebral infarction. Our aim was to test whether edaravone can exert a protective effect on the barrier properties of human brain endothelial cells (hCMEC/D3 cell line) treated with methylglyoxal. Methodology Cell viability was monitored in real-time by impedance-based cell electronic sensing. The barrier function of the monolayer was characterized by measurement of resistance and flux of permeability markers, and visualized by immunohistochemistry for claudin-5 and β-catenin. Cell morphology was also examined by holographic phase imaging. Principal Findings Methylglyoxal exerted a time- and dose-dependent toxicity on cultured human brain endothelial cells: a concentration of 600 µM resulted in about 50% toxicity, significantly reduced the integrity and increased the permeability of the barrier. The cell morphology also changed dramatically: the area of cells decreased, their optical height significantly increased. Edaravone (3 mM) provided a complete protection against the toxic effect of methylglyoxal. Co-administration of edaravone restored cell viability, barrier integrity and functions of brain endothelial cells. Similar protection was obtained by the well-known antiglycating molecule, aminoguanidine, our reference compound. Conclusion These results indicate for the first time that edaravone is protective in carbonyl stress induced barrier damage. Our data may contribute to the development of compounds to treat brain endothelial dysfunction in carbonyl stress related diseases. PMID:25033388

AMPK/p53 Axis Is Essential for α-Lipoic Acid-Regulated Metastasis in Human and Mouse Colon Cancer Cells.

PubMed

Park, Sunmi; Choi, Seung Kug; Choi, Yura; Moon, Hyun-Seuk

2015-10-01

α-Lipoic acid (ALA) has an anticancer property of lung, cervix, and prostate cancer cells. However, direct evidence that ALA contributes to the development of colon cancer has not been fully elucidated. In addition, no previous studies have evaluated whether ALA may regulate malignant potential, such as adhesion, invasion, and colony formation of colon cancer cells. To address the aforementioned questions, we conducted in vitro ALA signaling studies using human (HT29) and mouse (MCA38) colon cancer cell lines. We observed that cell proliferation is reduced by ALA administration in a dose-dependent manner in human and mouse colon cancer cell lines. Specifically, 0.5 to 1 mM concentration of ALA significantly decreased cell proliferation when compared with control. Similarly, we found that ALA downregulates adhesion, invasion, and colony formation. Finally, we observed that ALA activates p53 and AMPK signaling pathways in human and mouse colon cancer cells. We found for the first time that ALA suppresses cell proliferation and malignant potential via p53 and AMPK signaling pathways in human and mouse colon cancer cells. These new and early mechanistic studies provide a causal role of ALA in colon cancer, suggesting that ALA might be a useful agent in the management or chemoprevention of colon cancer.

Activated mesenchymal stem cell administration inhibits chronic alcohol drinking and suppresses relapse-like drinking in high-alcohol drinker rats.

PubMed

Ezquer, Fernando; Quintanilla, María Elena; Morales, Paola; Ezquer, Marcelo; Lespay-Rebolledo, Carolyne; Herrera-Marschitz, Mario; Israel, Yedy

2017-10-18

Neuroinflammation has been reported to follow chronic ethanol intake and may perpetuate alcohol consumption. Present studies determined the effect of human mesenchymal stem cells (hMSCs), known for their anti-inflammatory action, on chronic ethanol intake and relapse-like ethanol intake in a post-deprivation condition. Rats were allowed 12-17 weeks of chronic voluntary ethanol (10% and 20% v/v) intake, after which a single dose of activated hMSCs (5 × 10 5 ) was injected into a brain lateral ventricle. Control animals were administered vehicle. After assessing the effect of hMSCs on chronic ethanol intake for 1 week, animals were deprived of ethanol for 2 weeks and thereafter an ethanol re-access of 60 min was allowed to determine relapse-like intake. A single administration of activated hMSCs inhibited chronic alcohol consumption by 70% (P < 0.001), an effect seen within the first 24 hours of hMSCs administration, and reduced relapse-like drinking by 80% (P < 0.001). In the relapse-like condition, control animals attain blood ethanol ('binge-like') levels >80 mg/dl. The single hMSC administration reduced relapse-like blood ethanol levels to 20 mg/dl. Chronic ethanol intake increased by 250% (P < 0.001) the levels of reactive oxygen species in hippocampus, which were markedly reduced by hMSC administration. Astrocyte glial acidic fibrillary protein immunoreactivity, a hallmark of neuroinflammation, was increased by 60-80% (P < 0.001) by chronic ethanol intake, an effect that was fully abolished by the administration of hMSCs. This study supports the neuroinflammation-chronic ethanol intake hypothesis and suggest that mesenchymal stem cell administration may be considered in the treatment of alcohol use disorders. © 2017 Society for the Study of Addiction.

Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells

PubMed Central

Xiong, Jimin; Menicanin, Danijela; Marino, Victor

2016-01-01

The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein “spots” were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population. PMID:27579043

Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells.

PubMed

Xiong, Jimin; Menicanin, Danijela; Zilm, Peter S; Marino, Victor; Bartold, P Mark; Gronthos, Stan

2016-01-01

The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein "spots" were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population.

Cell sources for in vitro human liver cell culture models

PubMed Central

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

2016-01-01

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

Aggregation of Human Eyelid Adipose-derived Stem Cells by Human Body Fluids

PubMed Central

Song, Yeonhwa; Yun, Sujin; Yang, Hye Jin; Yoon, A Young; Kim, Haekwon

2012-01-01

Fetal bovine serum (FBS) is the most frequently used serum for the cultivation of mammalian cells. However, since animal-derived materials might not be appropriate due to safety issues, allogeneic human serum (HS) has been used to replace FBS, particularly for the culture of human cells. While there has been a debate about the advantages of HS, its precise effect on human adult stem cells have not been clarified. The present study aimed to investigate the effect of HS on the human eyelid adipose stem cells (HEACs) in vitro. When HEACs were cultivated in a medium containing 10% HS, many cells moved into several spots and aggregated there. The phenomenon was observed as early as 9 days following 10% HS treatment, and 12 days following 5% HS plus 5% FBS treatment. However, the aggregation was never observed when the same cells were cultivated with 10% FBS or bovine serum albumin. To examine whether cell density might affect the aggregation, cells were seeded with different densities on 12-well dish. Until the beginning of aggregation, cells seeded at low densities exhibited the longest culture period of 16 days whereas cells seeded at high densities showed the shortest period of 9 days to form aggregation. The number of cells was 15.1±0.2×104 as the least for the low density group, and 29.3±2.8×104 as the greatest for the high density group. When human cord blood serum or normal bovine serum was examined for the same effect on HEACs, interestingly, cord blood serum induced the aggregation of cells whereas bovine serum treatment has never induced. When cells were cultivated with 10% HS for 9 days, they were obtained and analyzed by RT-PCR. Compared to FBS-cultivated HEACs, HS-cultivated HEACs did not express VIM, and less expressed GATA4, PALLD. On the other hand, HS-cultivated HEACs expressed MAP2 more than FBS-cultivated HEACs. In conclusion, human adult stem cells could move and form aggregates by the treatment with human body fluids. PMID:25949109

Regulatory, biosafety and safety challenges for novel cells as substrates for human vaccines.

PubMed

Hess, Ralf D; Weber, Friedemann; Watson, Keith; Schmitt, Siegfried

2012-04-05

In the development of novel substrates used for production of human vaccines there has been significant progress made in recent years. Emerging and re-emerging infectious diseases like the recent porcine Influenza A virus (H1N1) pandemic necessitated the availability of unprecedented amounts of vaccines. In addition, the high demand for vaccines in the industrialised countries has also been paralleled by a steep increase in demand in developing countries. The manufacturing capability for viral vaccines produced in embryonated hen eggs and conventional/classical cell substrates, such as chicken embryo fibroblasts, has now reached its capacity limit. This constraint may be overcome by utilising other recognised cell substrates such as Madin Darby Canine Kidney (MDCK) (dog origin), Chinese Hamster Ovary (CHO) (hamster cells) or Vero cells (monkey origin) or as an alternative, introduce new cell substrates of human or avian origin. Using new cell substrates may prove to be a highly replication-proficient way of producing live viral vaccines such as Influenza A viruses. Despite some advantages, cell substrates may pose a small residual risk to humans since some of them are known to be tumourigenic in immunosuppressed animals. However, this residual risk should be considered acceptable by regulators. Safety testing requirements for cell substrates used in the manufacture of vaccines is mandated by published guidance from organisations such as World Health Organization (WHO), United States Food and Drug Administration (FDA), European Medicines Agency (EMA) and International Conferences on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human use (ICH) as well as requirements laid down in compendial monographs (Ph. Eur. and USP). This paper considers the guidance contained in these regulatory documents. In addition, the safety challenges and almost arbitrary risk-based classification of cell substrates used in the production of human

The effect of cell phones on human health

NASA Astrophysics Data System (ADS)

Abu-Isbeih, Ibrahim N.; Saad, Dina

2011-10-01

The effect of cell phone radiation on human health is the subject of recent interest and study, as a result of the enormous increase in cell phone usage throughout the world. Cell phones use electromagnetic radiation in the microwave range, which some believe may be harmful to human health. Other digital wireless systems, such as data communication networks, produce similar radiation. The objective of this survey is to review the effects of cell phones on human health: A large body of research exists, both epidemiological and experimental, in non-human animals and in humans, of which the majority shows no definite causative relationship between exposure to cell phones and harmful biological effects in humans. This is often paraphrased simply as the balance of evidence showing no harm to humans from cell phones, although a significant number of individual studies do suggest such a relationship, or are inconclusive.

Development of hematopoietic stem and progenitor cells from human pluripotent stem cells.

PubMed

Chen, Tong; Wang, Fen; Wu, Mengyao; Wang, Zack Z

2015-07-01

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), provide a new cell source for regenerative medicine, disease modeling, drug discovery, and preclinical toxicity screening. Understanding of the onset and the sequential process of hematopoietic cells from differentiated hPSCs will enable the achievement of personalized medicine and provide an in vitro platform for studying of human hematopoietic development and disease. During embryogenesis, hemogenic endothelial cells, a specified subset of endothelial cells in embryonic endothelium, are the primary source of multipotent hematopoietic stem cells. In this review, we discuss current status in the generation of multipotent hematopoietic stem and progenitor cells from hPSCs via hemogenic endothelial cells. We also review the achievements in direct reprogramming from non-hematopoietic cells to hematopoietic stem and progenitor cells. Further characterization of hematopoietic differentiation in hPSCs will improve our understanding of blood development and expedite the development of hPSC-derived blood products for therapeutic purpose. © 2015 Wiley Periodicals, Inc.

Human stem cells for craniomaxillofacial reconstruction.

PubMed

Jalali, Morteza; Kirkpatrick, William Niall Alexander; Cameron, Malcolm Gregor; Pauklin, Siim; Vallier, Ludovic

2014-07-01

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.

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

Profiling human breast epithelial cells using single cell RNA sequencing identifies cell diversity.

PubMed

Nguyen, Quy H; Pervolarakis, Nicholas; Blake, Kerrigan; Ma, Dennis; Davis, Ryan Tevia; James, Nathan; Phung, Anh T; Willey, Elizabeth; Kumar, Raj; Jabart, Eric; Driver, Ian; Rock, Jason; Goga, Andrei; Khan, Seema A; Lawson, Devon A; Werb, Zena; Kessenbrock, Kai

2018-05-23

Breast cancer arises from breast epithelial cells that acquire genetic alterations leading to subsequent loss of tissue homeostasis. Several distinct epithelial subpopulations have been proposed, but complete understanding of the spectrum of heterogeneity and differentiation hierarchy in the human breast remains elusive. Here, we use single-cell mRNA sequencing (scRNAseq) to profile the transcriptomes of 25,790 primary human breast epithelial cells isolated from reduction mammoplasties of seven individuals. Unbiased clustering analysis reveals the existence of three distinct epithelial cell populations, one basal and two luminal cell types, which we identify as secretory L1- and hormone-responsive L2-type cells. Pseudotemporal reconstruction of differentiation trajectories produces one continuous lineage hierarchy that closely connects the basal lineage to the two differentiated luminal branches. Our comprehensive cell atlas provides insights into the cellular blueprint of the human breast epithelium and will form the foundation to understand how the system goes awry during breast cancer.

Multilineage response in aplastic anemia patients following long-term administration of filgrastim (recombinant human granulocyte colony stimulating factor).

PubMed

Sonoda, Y; Ohno, Y; Fujii, H; Takahashi, T; Nakayama, S; Haruyama, H; Nasu, K; Shimazaki, C; Hara, H; Kanamaru, A

1993-11-01

The present multicenter study was undertaken to confirm whether filgrastim/recombinant human granulocyte colony stimulating factor (rhG-CSF) could mobilize residual multipotential stem cells by its G0-shortening effect in patients with aplastic anemia (AA) and induce a multilineage response. Twenty-seven patients with acquired severe or moderate AA received long-term administration (2 to 12+ months) of rhG-CSF in doses from 100 to 400 micrograms/body/day by s.c. injection or 250 to 1,500 micrograms/body/day by i.v. infusion. Twenty-six out of the 27 evaluable patients showed a substantial increase in neutrophils associated with a recovery of myeloid precursors in bone marrow within one month of therapy. Interestingly, 10 out of the 27 patients showed a dramatic improvement in severe anemia after two to ten months of therapy. Moreover, severe thrombocytopenia improved after two to four months of therapy in three out of these ten patients accompanied by a significant increase in megakaryocytes in bone marrow. Clonal cultures of bone marrow cells revealed a recovery in myeloid as well as erythroid precursors in most of these ten patients. In two patients who showed a trilineage response, mixed and megakaryocyte colony formations also recovered. These results suggest that long-term administration of rhG-CSF mobilizes myeloid, erythroid, megakaryocyte and multipotential progenitor cells and induces a multilineage response in some patients with AA.

The contribution of human/non-human animal chimeras to stem cell research.

PubMed

Levine, Sonya; Grabel, Laura

2017-10-01

Chimeric animals are made up of cells from two separate zygotes. Human/non-human animal chimeras have been used for a number of research purposes, including human disease modeling. Pluripotent stem cell (PSC) research has relied upon the chimera approach to examine the developmental potential of stem cells, to determine the efficacy of cell replacement therapies, and to establish a means of producing human organs. Based on ethical issues, this work has faced pushback from various sources including funding agencies. We discuss here the essential role these studies have played, from gaining a better understanding of human biology to providing a stepping stone to human disease treatments. We also consider the major ethical issues, as well as the current status of support for this work in the United States. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Salidroside induces cell-cycle arrest and apoptosis in human breast cancer cells

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

Hu, Xiaolan, E-mail: huxiaolan1998@yahoo.com.cn; Zhang, Xianqi; Qiu, Shuifeng

2010-07-16

Research highlights: {yields} Salidroside inhibits the growth of human breast cancer cells. {yields} Salidroside induces cell-cycle arrest of human breast cancer cells. {yields} Salidroside induces apoptosis of human breast cancer cell lines. -- Abstract: Recently, salidroside (p-hydroxyphenethyl-{beta}-D-glucoside) has been identified as one of the most potent compounds isolated from plants of the Rhodiola genus used widely in traditional Chinese medicine, but pharmacokinetic data on the compound are unavailable. We were the first to report the cytotoxic effects of salidroside on cancer cell lines derived from different tissues, and we found that human breast cancer MDA-MB-231 cells (estrogen receptor negative) weremore » sensitive to the inhibitory action of low-concentration salidroside. To further investigate the cytotoxic effects of salidroside on breast cancer cells and reveal possible ER-related differences in response to salidroside, we used MDA-MB-231 cells and MCF-7 cells (estrogen receptor-positive) as models to study possible molecular mechanisms; we evaluated the effects of salidroside on cell growth characteristics, such as proliferation, cell cycle duration, and apoptosis, and on the expression of apoptosis-related molecules. Our results demonstrated for the first time that salidroside induces cell-cycle arrest and apoptosis in human breast cancer cells and may be a promising candidate for breast cancer treatment.« less

Characterization of human pancreatic progenitor cells.

PubMed

Noguchi, Hirofumi; Naziruddin, Bashoo; Jackson, Andrew; Shimoda, Masayuki; Ikemoto, Tetsuya; Fujita, Yasutaka; Chujo, Daisuke; Takita, Morihito; Kobayashi, Naoya; Onaca, Nicholas; Hayashi, Shuji; Levy, Marlon F; Matsumoto, Shinichi

2010-01-01

β-Cell replacement therapy via islet transplantation is an effective treatment for diabetes mellitus, but its widespread use is severely limited by the shortage of donor organs. Because pancreatic stem/progenitor cells are abundantly available in the pancreas of these patients and in donor organs, the cells could become a useful target for β-cell replacement therapy. We previously established a mouse pancreatic stem cell line without genetic manipulation. In this study, we used the techniques to identify and isolate human pancreatic stem/progenitor cells. The cells from a duct-rich population were cultured in 23 kinds of culture media, based on media for mouse pancreatic stem cells or for human embryonic stem cells. The cells in serum-free media formed "cobblestone" morphologies, similar to a mouse pancreatic stem cell line. On the other hand, the cells in serum-containing medium and the medium for human embryonic stem cells formed "fibroblast-like" morphologies. The cells divided actively until day 30, and the population doubling level (PDL) was 6-10. However, the cells stopped dividing after 30 days in any culture conditions. During the cultures, the nucleus/cytoplasm (N/C) ratio decreased, suggesting that the cells entered senescence. Exendin-4 treatment and transduction of PDX-1 and NeuroD proteins by protein transduction technology into the cells induced insulin and pancreas-related gene expression. Although the duplications of these cells were limited, this approach could provide a potential new source of insulin-producing cells for transplantation.

New Monoclonal Antibodies to Defined Cell Surface Proteins on Human Pluripotent Stem Cells.

PubMed

O'Brien, Carmel M; Chy, Hun S; Zhou, Qi; Blumenfeld, Shiri; Lambshead, Jack W; Liu, Xiaodong; Kie, Joshua; Capaldo, Bianca D; Chung, Tung-Liang; Adams, Timothy E; Phan, Tram; Bentley, John D; McKinstry, William J; Oliva, Karen; McMurrick, Paul J; Wang, Yu-Chieh; Rossello, Fernando J; Lindeman, Geoffrey J; Chen, Di; Jarde, Thierry; Clark, Amander T; Abud, Helen E; Visvader, Jane E; Nefzger, Christian M; Polo, Jose M; Loring, Jeanne F; Laslett, Andrew L

2017-03-01

The study and application of human pluripotent stem cells (hPSCs) will be enhanced by the availability of well-characterized monoclonal antibodies (mAbs) detecting cell-surface epitopes. Here, we report generation of seven new mAbs that detect cell surface proteins present on live and fixed human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. These mAbs are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs, providing useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. In addition, we report that subsets of the seven new mAbs are also immunoreactive to human bone marrow-derived mesenchymal stem cells (MSCs), normal human breast subsets and both normal and tumorigenic colorectal cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types. Stem Cells 2017;35:626-640. © 2016 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

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.

DNA Repair in Human Pluripotent Stem Cells Is Distinct from That in Non-Pluripotent Human Cells

PubMed Central

Luo, Li Z.; Park, Sang-Won; Bates, Steven E.; Zeng, Xianmin; Iverson, Linda E.; O'Connor, Timothy R.

2012-01-01

The potential for human disease treatment using human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells (iPSCs), also carries the risk of added genomic instability. Genomic instability is most often linked to DNA repair deficiencies, which indicates that screening/characterization of possible repair deficiencies in pluripotent human stem cells should be a necessary step prior to their clinical and research use. In this study, a comparison of DNA repair pathways in pluripotent cells, as compared to those in non-pluripotent cells, demonstrated that DNA repair capacities of pluripotent cell lines were more heterogeneous than those of differentiated lines examined and were generally greater. Although pluripotent cells had high DNA repair capacities for nucleotide excision repair, we show that ultraviolet radiation at low fluxes induced an apoptotic response in these cells, while differentiated cells lacked response to this stimulus, and note that pluripotent cells had a similar apoptotic response to alkylating agent damage. This sensitivity of pluripotent cells to damage is notable since viable pluripotent cells exhibit less ultraviolet light-induced DNA damage than do differentiated cells that receive the same flux. In addition, the importance of screening pluripotent cells for DNA repair defects was highlighted by an iPSC line that demonstrated a normal spectral karyotype, but showed both microsatellite instability and reduced DNA repair capacities in three out of four DNA repair pathways examined. Together, these results demonstrate a need to evaluate DNA repair capacities in pluripotent cell lines, in order to characterize their genomic stability, prior to their pre-clinical and clinical use. PMID:22412831

CELLPEDIA: a repository for human cell information for cell studies and differentiation analyses.

PubMed

Hatano, Akiko; Chiba, Hirokazu; Moesa, Harry Amri; Taniguchi, Takeaki; Nagaie, Satoshi; Yamanegi, Koji; Takai-Igarashi, Takako; Tanaka, Hiroshi; Fujibuchi, Wataru

2011-01-01

CELLPEDIA is a repository database for current knowledge about human cells. It contains various types of information, such as cell morphologies, gene expression and literature references. The major role of CELLPEDIA is to provide a digital dictionary of human cells for the biomedical field, including support for the characterization of artificially generated cells in regenerative medicine. CELLPEDIA features (i) its own cell classification scheme, in which whole human cells are classified by their physical locations in addition to conventional taxonomy; and (ii) cell differentiation pathways compiled from biomedical textbooks and journal papers. Currently, human differentiated cells and stem cells are classified into 2260 and 66 cell taxonomy keys, respectively, from which 934 parent-child relationships reported in cell differentiation or transdifferentiation pathways are retrievable. As far as we know, this is the first attempt to develop a digital cell bank to function as a public resource for the accumulation of current knowledge about human cells. The CELLPEDIA homepage is freely accessible except for the data submission pages that require authentication (please send a password request to cell-info@cbrc.jp). Database URL: http://cellpedia.cbrc.jp/

Cell sources for in vitro human liver cell culture models.

PubMed

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

2016-09-01

In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described. © 2016 by the Society for Experimental Biology and Medicine.

Establishment of human hair follicle mesenchymal stem cells with overexpressed human hepatocyte growth factor.

PubMed

Zhou, Dan; Cheng, Hongjing; Liu, Jinyu; Zhang, Lei

2017-06-01

Chronic liver disease has become a major health problem that causes serious damage to human health. Since the existing treatment effect was not ideal, we need to seek new treatment methods. We utilized the gene recombination technology to obtain the human hair mesenchymal stem cells which overexpression of human hepatocyte growth factor (hHGF). Furthermore, we verified the property of transfected cells through detecting surface marker by flow cytometry. We show here establishment of the hHGF-overexpressing lentivirus vector, and successfully transfection to human hair follicle mesenchymal stem cells. The verified experiments could demonstrate the human hair follicle mesenchymal stem cells which have been transfected still have the properties of stem cells. We successfully constructed human hair follicle mesenchymal stem cells which overexpression hHGF, and maintain the same properties compared with pro-transfected cells.

Interluekin-12 enhances the function and anti-tumor activity in murine and human CD8+ T cells

PubMed Central

Rubinstein, Mark P.; Su, Ee Wern; Suriano, Samantha; Cloud, Colleen A.; Andrijauskaite, Kristina; Kesarwani, Pravin; Schwartz, Kristina M.; Williams, Katelyn; Johnson, C. Bryce; Li, Mingli; Scurti, Gina M.; Salem, Mohamed L.; Paulos, Chrystal M.; Garrett-Mayer, Elizabeth; Mehrotra, Shikhar; Cole, David J.

2016-01-01

Mouse CD8+ T cells conditioned with Interleukin (IL)-12 ex vivo mediate the potent regression of established melanoma when transferred into lymphodepleted mice. However, the quantitative and qualitative changes induced by IL-12 in the responding mouse CD8+ T cells have not been well defined. Moreover, the mechanisms by which IL-12-conditioning impacts human CD8+ T cells, and how such cells might be expanded prior to infusion into patients is not known. We found that ex vivo IL-12-conditioning of mouse CD8+ T cells led to a 10- to 100-fold increase in persistence and anti-tumor efficacy upon adoptive transfer into lymphodepleted mice. The enhancing effect of IL-12 was associated with maintenance of functional avidity. Importantly, in the context of ongoing ACT clinical trials, human CD8+ T cells genetically modified with a tyrosinase-specific T-cell receptor exhibited significantly enhanced functional activity when conditioned with IL-12 as indicated by heightened granzyme B expression and elevated peptide-specific CD107a degranulation. This effect was sustainable despite the 20 days of in vitro cellular expansion required to expand cells over 1,000-fold allowing adequate cell numbers for administration to cancer patients. Overall, these findings support the efficacy and feasibility of ex vivo IL-12-conditioning of TCR-modified human CD8+ T cells for adoptive transfer and cancer therapy. PMID:25676709

Constitutive expression of HCA(2) in human retina and primary human retinal pigment epithelial cells.

PubMed

Yu, Alice L; Birke, Kerstin; Lorenz, Reinhard L; Welge-Lussen, Ulrich

2014-05-01

HCA2, a receptor of β-hydroxybutyrate and niacin, has recently been described in mouse retina and immortalized human retinal pigment epithelial (RPE) cell lines. As HCA2 might be a pharmacologic target, e.g. in diabetic retinopathy, we studied its expression in human retina and primary human RPE cells. Paraffin sections of human retina and primary human RPE cells were obtained from human donor eyes. Expression of HCA2 in human retina was investigated by immunohistochemistry of paraffin sections and by RT-PCR. HCA2 expression in primary human RPE cells was examined by immunocytochemistry and by Western-blot analysis. Positive immunohistochemical staining for HCA2 was found in paraffin sections of human retina, and positive immunocytochemical staining for HCA2 in primary human RPE cells. RT-PCR analysis detected mRNA expression of HCA2 in human retina. The expression of HCA2 protein was found in primary human RPE cells. Based on these results, HCA2 appears to be constitutively expressed in human retina and in primary human RPE cells. Although its functional role is still unknown, HCA2 may be potentially involved in the pathogenesis of various retinopathies and may offer a new therapeutic target.

Human cells and cell cultures: availability, authentication and future prospects.

PubMed

Hay, R J

1996-09-01

The availability of well characterized, viable human cells, tissues and cell lines along with pertinent data on the specific patient donors is a prerequisite for much current transplantation and biomedical research. In the USA, institutional and multi-center networks have been established for provision of primary human cells and tissues to qualified clinicians and research scientists. Monetary support derives from government, university, institutional and fee sources. Problems involved include concern for the rights and privacy of tissue donors, cultural reservations relating to tissue provision, the need for safe and expeditious transport, short term survival and limited supply, adequate correlation of patient data with samples provided, presence of infectious viruses and microorganisms, as well as state or government regulations regarding national or international shipping. The use of human cell lines with continuous or even somewhat limited doubling potentials overcomes many of the above difficulties. National cell banks have been established to provide reference lines for use by multiple investigators. Use of such cell lines assures improved research comparability both geographically and with time. Authentication procedures are critically important for all of these programs. Verification of tissue types and conditions is required through histological, biochemical and immunological assays. Tests for microbial and viral contaminants must be applied. In addition to such procedures utilized for tissues, with cell lines the banking agency must also verify species and where possible identity, properties and functions. The literature is replete with descriptions documenting incorrect identifications and infections of proliferating cell strains used for research. The availability of viable tissue through local sources and distribution agencies in the USA is becoming more commonplace even including full family participation and collection of related, detailed histories

Possibilities of vaccine manufacture in human diploid cell strains with a serum replacement factor.

PubMed

Candal, F J; George, V G; Ades, E W

1991-07-01

Cell lines MDCK (canine kidney), BGM (Buffalo green monkey kidney) and human embryonic lung fibroblast will support viral growth efficiently in medium without serum. Both MRC-5 and WI-38 cell strains have been approved by the Food and Drug Administration for manufacturing viral vaccines against cytomegalovirus and varicella-zoster virus. In this study we examine these two cell lines and viruses for their ability to grow in medium containing a serum replacement. The serum substitute used is LPSR-1 (low protein serum replacement). Using LPSR-1 in defined medium, we demonstrate multipassage cell growth and viral cultivation and replication equivalent to those obtained in medium containing fetal bovine serum (FBS). Viral growth after complete elimination of FBS varies and depends on cell line and virus. Serum substitutes can eliminate FBS in the viral growth phase of vaccine production and reduce costs.

Human genome project and sickle cell disease.

PubMed

Norman, Brenda J; Miller, Sheila D

2011-01-01

Sickle cell disease is one of the most common genetic blood disorders in the United States that affects 1 in every 375 African Americans. Sickle cell disease is an inherited condition caused by abnormal hemoglobin in the red blood cells. The Human Genome Project has provided valuable insight and extensive research advances in the understanding of the human genome and sickle cell disease. Significant progress in genetic knowledge has led to an increase in the ability for researchers to map and sequence genes for diagnosis, treatment, and prevention of sickle cell disease and other chronic illnesses. This article explores some of the recent knowledge and advances about sickle cell disease and the Human Genome Project.

T-cell receptor transfer into human T cells with ecotropic retroviral vectors.

PubMed

Koste, L; Beissert, T; Hoff, H; Pretsch, L; Türeci, Ö; Sahin, U

2014-05-01

Adoptive T-cell transfer for cancer immunotherapy requires genetic modification of T cells with recombinant T-cell receptors (TCRs). Amphotropic retroviral vectors (RVs) used for TCR transduction for this purpose are considered safe in principle. Despite this, TCR-coding and packaging vectors could theoretically recombine to produce replication competent vectors (RCVs), and transduced T-cell preparations must be proven free of RCV. To eliminate the need for RCV testing, we transduced human T cells with ecotropic RVs so potential RCV would be non-infectious for human cells. We show that transfection of synthetic messenger RNA encoding murine cationic amino-acid transporter 1 (mCAT-1), the receptor for murine retroviruses, enables efficient transient ecotropic transduction of human T cells. mCAT-1-dependent transduction was more efficient than amphotropic transduction performed in parallel, and preferentially targeted naive T cells. Moreover, we demonstrate that ecotropic TCR transduction results in antigen-specific restimulation of primary human T cells. Thus, ecotropic RVs represent a versatile, safe and potent tool to prepare T cells for the adoptive transfer.

Gammaherpesvirus Infection of Human Neuronal Cells

PubMed Central

Jha, Hem Chandra; Mehta, Devan; Lu, Jie; El-Naccache, Darine; Shukla, Sanket K.; Kovacsics, Colleen; Kolson, Dennis

2015-01-01

ABSTRACT Gammaherpesviruses human herpesvirus 4 (HHV4) and HHV8 are two prominent members of the herpesvirus family associated with a number of human cancers. HHV4, also known as Epstein-Barr virus (EBV), a ubiquitous gammaherpesvirus prevalent in 90 to 95% of the human population, is clinically associated with various neurological diseases such as primary central nervous system lymphoma, multiple sclerosis, Alzheimer’s disease, cerebellar ataxia, and encephalitis. However, the possibility that EBV and Kaposi’s sarcoma-associated herpesvirus (KSHV) can directly infect neurons has been largely overlooked. This study has, for the first time, characterized EBV infection in neural cell backgrounds by using the Sh-Sy5y neuroblastoma cell line, teratocarcinoma Ntera2 neurons, and primary human fetal neurons. Furthermore, we also demonstrated KSHV infection of neural Sh-Sy5y cells. These neuronal cells were infected with green fluorescent protein-expressing recombinant EBV or KSHV. Microscopy, genetic analysis, immunofluorescence, and Western blot analyses for specific viral antigens supported and validated the infection of these cells by EBV and KSHV and showed that the infection was efficient and productive. Progeny virus produced from infected neuronal cells efficiently infected fresh neuronal cells, as well as peripheral blood mononuclear cells. Furthermore, acyclovir was effective at inhibiting the production of virus from neuronal cells similar to lymphoblastoid cell lines; this suggests active lytic replication in infected neurons in vitro. These studies represent a potentially new in vitro model of EBV- and KSHV-associated neuronal disease development and pathogenesis. PMID:26628726

Brain transplantation of immortalized human neural stem cells promotes functional recovery in mouse intracerebral hemorrhage stroke model.

PubMed

Lee, Hong J; Kim, Kwang S; Kim, Eun J; Choi, Hyun B; Lee, Kwang H; Park, In H; Ko, Yong; Jeong, Sang W; Kim, Seung U

2007-05-01

We have generated stable, immortalized cell lines of human NSCs from primary human fetal telencephalon cultures via a retroviral vector encoding v-myc. HB1.F3, one of the human NSC lines, expresses a normal human karyotype of 46, XX, and nestin, a cell type-specific marker for NSCs. F3 has the ability to proliferate continuously and differentiate into cells of neuronal and glial lineage. The HB1.F3 human NSC line was used for cell therapy in a mouse model of intracerebral hemorrhage (ICH) stroke. Experimental ICH was induced in adult mice by intrastriatal administration of bacterial collagenase; 1 week after surgery, the rats were randomly divided into two groups so as to receive intracerebrally either human NSCs labeled with beta-galactosidase (n = 31) or phosphate-buffered saline (PBS) (n = 30). Transplanted NSCs were detected by 5-bromo-4-chloro-3-indolyl-beta-d-galactoside histochemistry or double labeling with beta-galactosidase (beta-gal) and mitogen-activated protein (MAP)2, neurofilaments (both for neurons), or glial fibrillary acidic protein (GFAP) (for astrocytes). Behavior of the animals was evaluated for period up to 8 weeks using modified Rotarod tests and a limb placing test. Transplanted human NSCs were identified in the perihematomal areas and differentiated into neurons (beta-gal/MAP2(+) and beta-gal/NF(+)) or astrocytes (beta-gal/GFAP(+)). The NSC-transplanted group showed markedly improved functional performance on the Rotarod test and limb placing after 2-8 weeks compared with the control PBS group (p < .001). These results indicate that the stable immortalized human NSCs are a valuable source of cells for cell replacement and gene transfer for the treatment of ICH and other human neurological disorders. Disclosure of potential conflicts of interest is found at the end of this article.

Identification and characterization of enhancer agonist human cytotoxic T-cell epitopes of the human papillomavirus type 16 (HPV16) E6/E7

PubMed Central

Tsang, Kwong Y.; Fantini, Massimo; Fernando, Romaine I.; Palena, Claudia; David, Justin M.; Hodge, James W.; Gabitzsch, Elizabeth S.; Jones, Frank R.; Schlom, Jeffrey

2017-01-01

Human papillomavirus (HPV) is associated with the etiology of cervical carcinoma, head and neck squamous cell carcinoma, and several other cancer types. Vaccines directed against HPV virus-like particles and coat proteins have been extremely successful in the prevention of cervical cancer through the activation of host HPV-specific antibody responses; however, HPV-associated cancers remain a major public health problem. The development of a therapeutic vaccine will require the generation of T-cell responses directed against early HPV proteins (E6/E7) expressed in HPV-infected tumor cells. Clinical studies using various vaccine platforms have demonstrated that both HPV-specific human T cells can be generated and patient benefit can be achieved. However, no HPV therapeutic vaccine has been approved by the Food and Drug Administration to date. One method of enhancing the potential efficacy of a therapeutic vaccine is the generation of agonist epitopes. We report the first description of enhancer cytotoxic T lymphocyte agonist epitopes for HPV E6 and E7. While the in silico algorithm revealed six epitopes with potentially improved binding to human leukocyte antigen–A2 allele (HLA-A2)–Class I, 5/6 demonstrated enhanced binding to HLA-Class I in cell-based assays and only 3/6 had a greater ability to activate HPV-specific T cells which could lyse tumor cells expressing native HPV, compared to their native epitope counterparts. These agonist epitopes have potential for use in a range of HPV therapeutic vaccine platforms and for use in HPV-specific adoptive T- or natural killer–cell platforms. PMID:28389098

Effects of vasopressin administration on diuresis of water immersion in normal humans

NASA Technical Reports Server (NTRS)

Epstein, M.; Denunzio, A. G.; Loutzenhiser, R. D.

1981-01-01

The influence of vasopressin suppression on the diuresis encountered during water immersion is investigated in studies on normal humans immersed to the neck. Six hydrated male subjects were studied on two occasions while undergoing 6 h of immersion without or during the administration of aqueous vasopressin for the initial 4 h. Neck immersion is found to result in a significant increase in urinary flow rate beginning in the first hour and persisting throughout the immersion. The administration of vasopressin markedly attenuated the diuretic response throughout the period of infusion, while cessation of vasopressin administration during the final 2 h of immersion resulted in a marked offset of the antidiuresis. Results thus support the view that the suppression of antidiuretic hormone contributes to the immersion diuresis of hydrated subjects.

Self-administration of cocaine, cannabis and heroin in the human laboratory: benefits and pitfalls.

PubMed

Haney, Margaret

2009-01-01

The objective of this review is to describe self-administration procedures for modeling addiction to cocaine, cannabis and heroin in the human laboratory, the benefits and pitfalls of the approach, and the methodological issues unique to each drug. In addition, the predictive validity of the model for testing treatment medications will be addressed. The results show that all three drugs of abuse are reliably and robustly self-administered by non-treatment-seeking research volunteers. In terms of pharmacotherapies, cocaine use is extraordinarily difficult to disrupt either in the laboratory or in the clinic. A range of medications has been shown to significantly decrease cocaine's subjective effects and craving without decreasing either cocaine self-administration or cocaine abuse by patients. These negative data combined with recent positive findings with modafinil suggest that self-administration procedures are an important intermediary step between pre-clinical and clinical studies. In terms of cannabis, a recent study suggests that medications that improve sleep and mood during cannabis withdrawal decrease the resumption of marijuana self-administration in abstinent volunteers. Clinical data on patients seeking treatment for their marijuana use are needed to validate these laboratory findings. Finally, in contrast to cannabis or cocaine dependence, there are three efficacious Food and Drug Administration-approved medications to treat opioid dependence, all of which decrease both heroin self-administration and subjective effects in the human laboratory. In summary, self-administration procedures provide meaningful behavioral data in a small number of individuals. These studies contribute to our understanding of the variables maintaining cocaine, marijuana and heroin intake, and are important in guiding the development of more effective drug treatment programs.

Human papillomavirus-mediated carcinogenesis and HPV-associated oral and oropharyngeal squamous cell carcinoma. Part 2: Human papillomavirus associated oral and oropharyngeal squamous cell carcinoma

PubMed Central

2010-01-01

Human papillomavirus (HPV) infection of the mouth and oropharynx can be acquired by a variety of sexual and social forms of transmission. HPV-16 genotype is present in many oral and oropharyngeal squamous cell carcinomata. It has an essential aetiologic role in the development of oropharyngeal squamous cell carcinoma in a subset of subjects who are typically younger, are more engaged with high-risk sexual behaviour, have higher HPV-16 serum antibody titer, use less tobacco and have better survival rates than in subjects with HPV-cytonegative oropharyngeal squamous cell carcinoma. In this subset of subjects the HPV-cytopositive carcinomatous cells have a distinct molecular profile. In contrast to HPV-cytopositive oropharyngeal squamous cell carcinoma, the causal association between HPV-16 and other high-risk HPV genotypes and squamous cell carcinoma of the oral mucosa is weak, and the nature of the association is unclear. It is likely that routine administration of HPV vaccination against high-risk HPV genotypes before the start of sexual activity will bring about a reduction in the incidence of HPV-mediated oral and oropharyngeal squamous cell carcinoma. This article focuses on aspects of HPV infection of the mouth and the oropharynx with emphasis on the link between HPV and squamous cell carcinoma, and on the limitations of the available diagnostic tests in identifying a cause-and-effect relationship of HPV with squamous cell carcinoma of the mouth and oropharynx. PMID:20633288

Single-Cell RNA Sequencing of Human T Cells.

PubMed

Villani, Alexandra-Chloé; Shekhar, Karthik

2017-01-01

Understanding how populations of human T cells leverage cellular heterogeneity, plasticity, and diversity to achieve a wide range of functional flexibility, particularly during dynamic processes such as development, differentiation, and antigenic response, is a core challenge that is well suited for single-cell analysis. Hypothesis-free evaluation of cellular states and subpopulations by transcriptional profiling of single T cells can identify relationships that may be obscured by targeted approaches such as FACS sorting on cell-surface antigens, or bulk expression analysis. While this approach is relevant to all cell types, it is of particular interest in the study of T cells for which classical phenotypic criteria are now viewed as insufficient for distinguishing different T cell subtypes and transitional states, and defining the changes associated with dysfunctional T cell states in autoimmunity and tumor-related exhaustion. This unit describes a protocol to generate single-cell transcriptomic libraries of human blood CD4 + and CD8 + T cells, and also introduces the basic bioinformatic steps to process the resulting sequence data for further computational analysis. We show how cellular subpopulations can be identified from transcriptional data, and derive characteristic gene expression signatures that distinguish these states. We believe single-cell RNA-seq is a powerful technique to study the cellular heterogeneity in complex tissues, a paradigm that will be of great value for the immune system.

Assay for mutagenesis in heterozygous diploid human lymphoblasts

DOEpatents

Skopek, Thomas R.; Liber, Howard L.; Penman, Bruce W.; Thilly, William G.; Hoppe, IV, Henry

1981-01-01

An assay is disclosed for determining mutagenic damage caused by the administration of a known or suspected mutagen to diploid human lymphoblastoid cell lines. The gene locus employed for this assay is the gene for thymidine kinase, uridine kinase, or cytidine deaminase. Since human lymphoblastoid cells contain two genes for these enzymes, heterozygotes of human lymphoblastoid cells are used in this assay.

Review article: stem cells in human reproduction.

PubMed

Gargett, Caroline E

2007-07-01

The derivation of human embryonic stem (hES) cells heralds a new era in stem cell research, generating excitement for their therapeutic potential in regenerative medicine. Pioneering work of embryologists, developmental biologists, and reproductive medicine practitioners in in vitro fertilization clinics has facilitated hES cell research. This review summarizes current research focused on optimizing hES cell culture conditions for good manufacturing practice, directing hES cell differentiation toward trophectoderm and germ cells, and approaches used to reprogram cells for pluripotent cell derivation. The identification of germ stem cells in the testis and the recent controversy over their existence in the ovary raise the possibility of harnessing them for treating young cancer survivors. There is also the potential to harvest fetal stem cells with pluripotent cell-like properties from discarded placental tissues. The recent identification of adult stem/progenitor cell activity in the human endometrium offers a new understanding of common gynecological diseases. Discoveries resulting from research into embryonic, germ, fetal, and adult stem cells are highly relevant to human reproduction.

Human cytokine response to Texas crotaline envenomation before and after antivenom administration.

PubMed

Crocker, Patrick; Zad, Omid; Milling, Truman; Maxson, Todd; King, Benjamin; Whorton, Elbert

2010-10-01

The aim of this study was to characterize the human cytokine response to Texas crotaline envenomation before and after antivenom administration. This study enrolled crotaline bite victims presenting to a regional trauma center and children's hospital from March to November 2007 and age-matched unbitten controls. Blood spot cards were obtained from bite victims at presentation and at 1 and 6 hours after antivenom administration. One control sample was drawn from each of the age-matched controls selected from urgent care patients presenting for minor complaints. Samples were delivered to a laboratory using a proprietary method for quantitative evaluation of a large number of biomarkers in parallel with bead-based multiplex immunoassays. After obtaining informed consent, 14 crotaline bite victims (age range, 5-85 years; median age, 45 years; 50% female) (Snakebite Severity Score, 2-7; median, 3) and 14 age-matched controls were enrolled. There were 7 copperhead (Agkistrodon contortrix) bites, 4 rattlesnake (probably Western Diamondback Crotalus atrox) bites, 2 cottonmouth (Agkistrodon piscivorus) bites, and 1 bite from a snake that was not identified by the victim. In t tests, the means in the presentation samples for apolipoprotein A-I (Apo A-I), Apo C3, interleukin 4 (IL-4), myeloperoxidase, plasminogen activator inhibitor 1 (PAI-1), epidermal growth factor, and regulated upon activation, normal t-cell expressed and secreted were significantly lower and Apo H was significantly higher in the bite patients than in the controls. In the 1-hour sample, α(1)-antitrypsin, Apo A-I, Apo C3, eotaxin, IL-4, myeloperoxidase, and PAI-1 levels were lower and prostatic acid phosphatase and cancer antigen 125 levels were higher in the bite patients than in the controls. And in the 6-hour sample, α(1)-antitrypsin, Apo A-I, Apo C3, endothelin-1, IL-4, macrophage inflammatory protein 1β, myeloperoxidase, and epidermal growth factor levels were lower and Apo H level was higher in

21 CFR 864.2280 - Cultured animal and human cells.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Cultured animal and human cells. 864.2280 Section... Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro cultivated cell lines from the tissue of humans or other animals which are used in various diagnostic...

21 CFR 864.2280 - Cultured animal and human cells.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Cultured animal and human cells. 864.2280 Section... Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro cultivated cell lines from the tissue of humans or other animals which are used in various diagnostic...

21 CFR 864.2280 - Cultured animal and human cells.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Cultured animal and human cells. 864.2280 Section... Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro cultivated cell lines from the tissue of humans or other animals which are used in various diagnostic...

21 CFR 864.2280 - Cultured animal and human cells.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Cultured animal and human cells. 864.2280 Section... Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro cultivated cell lines from the tissue of humans or other animals which are used in various diagnostic...

Interaction of Human Enterochromaffin Cells with Human Enteric Adenovirus 41 Leads to Serotonin Release and Subsequent Activation of Enteric Glia Cells.

PubMed

Westerberg, Sonja; Hagbom, Marie; Rajan, Anandi; Loitto, Vesa; Persson, B David; Allard, Annika; Nordgren, Johan; Sharma, Sumit; Magnusson, Karl-Eric; Arnberg, Niklas; Svensson, Lennart

2018-04-01

Human adenovirus 41 (HAdV-41) causes acute gastroenteritis in young children. The main characteristics of HAdV-41 infection are diarrhea and vomiting. Nevertheless, the precise mechanism of HAdV-41-induced diarrhea is unknown, as a suitable small-animal model has not been described. In this study, we used the human midgut carcinoid cell line GOT1 to investigate the effect of HAdV-41 infection and the individual HAdV-41 capsid proteins on serotonin release by enterochromaffin cells and on enteric glia cell (EGC) activation. We first determined that HAdV-41 could infect the enterochromaffin cells. Immunofluorescence staining revealed that the cells expressed HAdV-41-specific coxsackievirus and adenovirus receptor (CAR); flow cytometry analysis supported these findings. HAdV-41 infection of the enterochromaffin cells induced serotonin secretion dose dependently. In contrast, control infection with HAdV-5 did not induce serotonin secretion in the cells. Confocal microscopy studies of enterochromaffin cells infected with HAdV-41 revealed decreased serotonin immunofluorescence compared to that in uninfected cells. Incubation of the enterochromaffin cells with purified HAdV-41 short fiber knob and hexon proteins increased the serotonin levels in the harvested cell supernatant significantly. HAdV-41 infection could also activate EGCs, as shown in the significantly altered expression of glia fibrillary acidic protein (GFAP) in EGCs incubated with HAdV-41. The EGCs were also activated by serotonin alone, as shown in the significantly increased GFAP staining intensity. Likewise, EGCs were activated by the cell supernatant of HAdV-41-infected enterochromaffin cells. IMPORTANCE The nonenveloped human adenovirus 41 causes diarrhea, vomiting, dehydration, and low-grade fever mainly in children under 2 years of age. Even though acute gastroenteritis is well described, how human adenovirus 41 causes diarrhea is unknown. In our study, we analyzed the effect of human adenovirus 41

Culture of human cell lines by a pathogen-inactivated human platelet lysate.

PubMed

Fazzina, R; Iudicone, P; Mariotti, A; Fioravanti, D; Procoli, A; Cicchetti, E; Scambia, G; Bonanno, G; Pierelli, L

2016-08-01

Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety.

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

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

Varga, Nora; Vereb, Zoltan; Rajnavoelgyi, Eva

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 ofmore » 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.« less

Effects of cellular origin on differentiation of human induced pluripotent stem cell–derived endothelial cells

PubMed Central

Zhao, Ming-Tao; Jahanbani, Fereshteh; Lee, Won Hee; Snyder, Michael P.

2016-01-01

Human induced pluripotent stem cells (iPSCs) can be derived from various types of somatic cells by transient overexpression of 4 Yamanaka factors (OCT4, SOX2, C-MYC, and KLF4). Patient-specific iPSC derivatives (e.g., neuronal, cardiac, hepatic, muscular, and endothelial cells [ECs]) hold great promise in drug discovery and regenerative medicine. In this study, we aimed to evaluate whether the cellular origin can affect the differentiation, in vivo behavior, and single-cell gene expression signatures of human iPSC–derived ECs. We derived human iPSCs from 3 types of somatic cells of the same individuals: fibroblasts (FB-iPSCs), ECs (EC-iPSCs), and cardiac progenitor cells (CPC-iPSCs). We then differentiated them into ECs by sequential administration of Activin, BMP4, bFGF, and VEGF. EC-iPSCs at early passage (10 < P < 20) showed higher EC differentiation propensity and gene expression of EC-specific markers (PECAM1 and NOS3) than FB-iPSCs and CPC-iPSCs. In vivo transplanted EC-iPSC–ECs were recovered with a higher percentage of CD31+ population and expressed higher EC-specific gene expression markers (PECAM1, KDR, and ICAM) as revealed by microfluidic single-cell quantitative PCR (qPCR). In vitro EC-iPSC–ECs maintained a higher CD31+ population than FB-iPSC–ECs and CPC-iPSC–ECs with long-term culturing and passaging. These results indicate that cellular origin may influence lineage differentiation propensity of human iPSCs; hence, the somatic memory carried by early passage iPSCs should be carefully considered before clinical translation. PMID:27398408

Evodiamine Induces Cell Growth Arrest, Apoptosis and Suppresses Tumorigenesis in Human Urothelial Cell Carcinoma Cells.

PubMed

Shi, Chung-Sheng; Li, Jhy-Ming; Chin, Chih-Chien; Kuo, Yi-Hung; Lee, Ying-Ray; Huang, Yun-Ching

2017-03-01

Evodiamine, an indole alkaloid derived from Evodia rutaecarpa, exhibits pharmacological activities including vasodilatation, analgesia, anti-cardiovascular disease, anti-Alzheimer's disease, anti-inflammation, and anti-tumor activity. This study analyzes the anti-tumor effects of evodiamine on cellular growth, tumorigenesis, cell cycle and apoptosis induction of human urothelial cell carcinoma (UCC) cells. The present study showed that evodiamine significantly inhibited the proliferation of UCC cells in a dose- and time-dependent manner. Also, evodiamine suppressed the tumorigenesis of UCC cells in vitro. Moreover, evodiamine caused G 2 /M cell-cycle arrest and induced caspase-dependent apoptosis in UCC cells. Finally, we demonstrated that evodiamine exhibits better cytotoxic than 5-fluorouracil, a clinical chemotherapeutic drug, for UCC cells. Evodiamine induces growth inhibition, tumorigenesis suppression, cell-cycle arrest, and apoptosis induction in human UCC cells. Therefore, this agent displays a therapeutic potential for treating human UCC cells and is worthy for further investigation. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

[COMPARISON OF CYTOPROTECTIVE EFFECTS OF HEMANTANE AND AMANTADINE UNDER CONDITIONS OF 6-HYDROXYDOPAMINE NEUROTOXIN ACTION ON CULTURED HUMAN NEUROBLASTOMA CELLS].

PubMed

Logvinov, I O; Antipova, T A; Nepoklonov, A V; Valdman, E A

2016-01-01

Potential neuroprotective activity of the novel antiparkinsonian drug hemantane (hydrochloride N-2-(adamantyl)-hexamethylenimine) in comparison to amantadine has been studied in various regimes of administration on human neuroblastoma SH-SY5Y cell line injury induced by 6-hydroxydopamine (6-OHDA), which is used as in vitro model of dopaminergic neurons for Parkinson's disease. Two regimes of hemantane and amantadine administration in a range of final concentrations 10⁻⁶-10⁻⁸ M were used either prior to or immediately after 6-OHDA introduction. MTT colorimetric assay was used to assess the viability of test cells. Significant decrease in viability of SH-SY5Y cells treated with 6-OHDA was observed. The addition of hemantane to cell medium produced cytoprotective effects in both regimes of administration--before and after 6-OHDA--at concentrations 10⁻⁷ M and 10⁻⁶-10⁻⁸ M, respectively. Amantadine in con- centrations 10⁻⁷-10⁻⁸ M was effective to increase cell survival only when administered after 6-OHDA. These results show that hemantane has a greater neu-roprotective potential in comparison to amantadine.

Oral Administration of Probiotics Increases Paneth Cells and Intestinal Antimicrobial Activity.

PubMed

Cazorla, Silvia I; Maldonado-Galdeano, Carolina; Weill, Ricardo; De Paula, Juan; Perdigón, Gabriela D V

2018-01-01

The huge amount of intestinal bacteria represents a continuing threat to the intestinal barrier. To meet this challenge, gut epithelial cells produce antimicrobial peptides (AMP) that act at the forefront of innate immunity. We explore whether this antimicrobial activity and Paneth cells, the main intestinal cell responsible of AMP production, are influenced by probiotics administration, to avoid the imbalance of intestinal microbiota and preserve intestinal barrier. Administration of Lactobacillus casei CRL 431 (Lc 431) and L. paracasei CNCM I-1518 (Lp 1518) to 42 days old mice, increases the number of Paneth cells on small intestine, and the antimicrobial activity against the pathogens Staphylococcus aureus and Salmonella Typhimurium in the intestinal fluids. Specifically, strong damage of the bacterial cell with leakage of cytoplasmic content, and cellular fragmentation were observed in S. Typhimurium and S. aureus . Even more important, probiotics increase the antimicrobial activity of the intestinal fluids at the different ages, from weaning (21 days old) to old age (180 days old). Intestinal antimicrobial activity stimulated by oral probiotics, do not influence significantly the composition of total anaerobic bacteria, lactobacilli and enterobacteria in the large intestine, at any age analyzed. This result, together with the antimicrobial activity observed against the same probiotic bacteria; endorse the regular consumption of probiotics without adverse effect on the intestinal homeostasis in healthy individuals. We demonstrate that oral probiotics increase intestinal antimicrobial activity and Paneth cells in order to strengthen epithelial barrier against pathogens. This effect would be another important mechanism by which probiotics protect the host mainly against infectious diseases.

Human interleukin for DA cells or leukemia inhibitory factor is released by Vero cells in human embryo coculture.

PubMed

Papaxanthos-Roche, A; Taupin, J L; Mayer, G; Daniel, J Y; Moreau, J F

1994-09-01

In the light of the newly discovered implications of human interleukin for DA cells and leukemia inhibitory factor in embryology, we searched for the presence of this soluble cytokine in the supernatant of Vero cell coculture systems. Using a bioassay as well as a specific ELISA, we demonstrated that Vero cells are able to release large quantities of human interleukin for DA cells and leukemia inhibitory factor in the embryo-growing medium of such cocultures.

Autophagy in Human Embryonic Stem Cells

PubMed Central

Tra, Thien; Gong, Lan; Kao, Lin-Pin; Li, Xue-Lei; Grandela, Catarina; Devenish, Rodney J.; Wolvetang, Ernst; Prescott, Mark

2011-01-01

Autophagy (macroautophagy) is a degradative process that involves the sequestration of cytosolic material including organelles into double membrane vesicles termed autophagosomes for delivery to the lysosome. Autophagy is essential for preimplantation development of mouse embryos and cavitation of embryoid bodies. The precise roles of autophagy during early human embryonic development, remain however largely uncharacterized. Since human embryonic stem cells constitute a unique model system to study early human embryogenesis we investigated the occurrence of autophagy in human embryonic stem cells. We have, using lentiviral transduction, established multiple human embryonic stem cell lines that stably express GFP-LC3, a fluorescent marker for the autophagosome. Each cell line displays both a normal karyotype and pluripotency as indicated by the presence of cell types representative of the three germlayers in derived teratomas. GFP expression and labelling of autophagosomes is retained after differentiation. Baseline levels of autophagy detected in cultured undifferentiated hESC were increased or decreased in the presence of rapamycin and wortmannin, respectively. Interestingly, autophagy was upregulated in hESCs induced to undergo differentiation by treatment with type I TGF-beta receptor inhibitor SB431542 or removal of MEF secreted maintenance factors. In conclusion we have established hESCs capable of reporting macroautophagy and identify a novel link between autophagy and early differentiation events in hESC. PMID:22110659

Research on DNA methylation of human osteosarcoma cell MGMT and its relationship with cell resistance to alkylating agents.

PubMed

Guo, Jun; Cui, Qiu; Jiang, WeiHao; Liu, Cheng; Li, DingFeng; Zeng, Yanjun

2013-08-01

The objective of this study was to explore the O(6)-methylguanine-DNA methyltransferase (MGMT) gene methylation status and its protein expression, as well as the effects of demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-CdR) on MGMT gene expression and its resistance to alkylating agents, and to elucidate MGMT expression mechanism and significance in osteosarcoma. The human osteosarcoma cell lines Saos-2 and MG-63 were collected and treated with 5-Aza-CdR for 6 days. The cells not treated with 5-Aza-CdR were set as a negative control. The genomic DNA was extracted from the Saos-2 and MG-63 cells using methylation-specific PCR to detect the promoter CpG island methylation status of the MGMT gene. Cell sensitivity to alkylating agents before and after drug administration was detected by the MTT method. The variation in MGMT gene mRNA and protein was detected by reverse transcription PCR (RT-PCR) and Western blotting. The MGMT promoter gene of normal Saos-2 cells was methylated, with reduced MGMT mRNA and protein expression; the MGMT mRNA and protein expression of Saos-2 cells treated with 5-Aza-CdR was obviously enhanced, and its sensitivity to alkylating agents was reversed. Meanwhile, with promoter CpG island unmethylation of the MGMT gene, MGMT protein was expressed in the normal MG-63 cells and the MG-63 cells treated with 5-Aza-CdR, and both showed resistance to alkylating agents. The methylation status of the MGMT gene promoter in human osteosarcoma cells reflected the cells' ability to induce MGMT protein expression and can be used as a molecular marker to project the sensitivity of cancer tissues to alkylating agent drugs.

Composition and function of macroencapsulated human embryonic stem cell-derived implants: comparison with clinical human islet cell grafts.

PubMed

Motté, Evi; Szepessy, Edit; Suenens, Krista; Stangé, Geert; Bomans, Myriam; Jacobs-Tulleneers-Thevissen, Daniel; Ling, Zhidong; Kroon, Evert; Pipeleers, Daniel

2014-11-01

β-Cells generated from large-scale sources can overcome current shortages in clinical islet cell grafts provided that they adequately respond to metabolic variations. Pancreatic (non)endocrine cells can develop from human embryonic stem (huES) cells following in vitro derivation to pancreatic endoderm (PE) that is subsequently implanted in immune-incompetent mice for further differentiation. Encapsulation of PE increases the proportion of endocrine cells in subcutaneous implants, with enrichment in β-cells when they are placed in TheraCyte-macrodevices and predominantly α-cells when they are alginate-microencapsulated. At posttransplant (PT) weeks 20-30, macroencapsulated huES implants presented higher glucose-responsive plasma C-peptide levels and a lower proinsulin-over-C-peptide ratio than human islet cell implants under the kidney capsule. Their ex vivo analysis showed the presence of single-hormone-positive α- and β-cells that exhibited rapid secretory responses to increasing and decreasing glucose concentrations, similar to isolated human islet cells. However, their insulin secretory amplitude was lower, which was attributed in part to a lower cellular hormone content; it was associated with a lower glucose-induced insulin biosynthesis, but not with lower glucagon-induced stimulation, which together is compatible with an immature functional state of the huES-derived β-cells at PT weeks 20-30. These data support the therapeutic potential of macroencapsulated huES implants but indicate the need for further functional analysis. Their comparison with clinical-grade human islet cell grafts sets references for future development and clinical translation. Copyright © 2014 the American Physiological Society.

Rapamycin-treated human endothelial cells preferentially activate allogeneic regulatory T cells

PubMed Central

Wang, Chen; Yi, Tai; Qin, Lingfeng; Maldonado, Roberto A.; von Andrian, Ulrich H.; Kulkarni, Sanjay; Tellides, George; Pober, Jordan S.

2013-01-01

Human graft endothelial cells (ECs) can act as antigen-presenting cells to initiate allograft rejection by host memory T cells. Rapamycin, an mTOR inhibitor used clinically to suppress T cell responses, also acts on DCs, rendering them tolerogenic. Here, we report the effects of rapamycin on EC alloimmunogenicity. Compared with mock-treated cells, rapamycin-pretreated human ECs (rapa-ECs) stimulated less proliferation and cytokine secretion from allogeneic CD4+ memory cells, an effect mimicked by shRNA knockdown of mTOR or raptor in ECs. The effects of rapamycin persisted for several days and were linked to upregulation of the inhibitory molecules PD-L1 and PD-L2 on rapa-ECs. Additionally, rapa-ECs produced lower levels of the inflammatory cytokine IL-6. CD4+ memory cells activated by allogeneic rapa-ECs became hyporesponsive to restimulation in an alloantigen-specific manner and contained higher percentages of suppressive CD4+CD25hiCD127loFoxP3+ cells that did not produce effector cytokines. In a human-mouse chimeric model of allograft rejection, rapamycin pretreatment of human arterial allografts increased graft EC expression of PD-L1 and PD-L2 and reduced subsequent infiltration of allogeneic effector T cells into the artery intima and intimal expansion. Preoperative conditioning of allograft ECs with rapamycin could potentially reduce immune-mediated rejection. PMID:23478407

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

PubMed Central

2012-01-01

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

Bidirectional enhancing activities between human T cell leukemia-lymphoma virus type I and human cytomegalovirus in human term syncytiotrophoblast cells cultured in vitro.

PubMed

Tóth, F D; Aboagye-Mathiesen, G; Szabó, J; Liu, X; Mosborg-Petersen, P; Kiss, J; Hager, H; Zdravkovic, M; Andirkó, I; Aranyosi, J

1995-12-01

The syncytiotrophoblast layer of the human placenta has an important role in limiting transplacental viral spread from mother to fetus. Human cytomegalovirus (HCMV) is capable of establishing a latent infection in syncytiotrophoblast cells, with restriction of gene expression to immediate-early and early proteins. We analyzed the extent of replication of human T cell leukemia-lymphoma virus type I (HTLV-I) in human term syncytiotrophoblasts infected with HTLV-I alone or coinfected with HTLV-I and HCMV. Although syncytiotrophoblasts could be infected with cell-free HTLV-I, no viral protein expression was found in the singly infected cells. On the contrary, coinfection of the cells with HTLV-I and HCMV resulted in simultaneous replication of both viruses. Bidirectional enhancing activities between HTLV-I and HCMV were mediated primarily by the Tax and immediate-early proteins, respectively. The stimulatory effect of HTLV-I Tax on HCMV replication appeared to be mediated partly by tumor necrosis factor beta and transforming growth factor beta-1. We observed formation of pseudotypes with HTLV-I nucleocapsids within HCMV envelopes, whereas HCMV was not pseudotyped by HTLV-I envelopes in dually infected syncytiotrophoblast cells. Our data suggest that in vivo dual infection of syncytiotrophoblast cells with HTLV-I and HCMV may facilitate the transplacental transmission of both viruses.

Phenotypic and Functional Properties of Human Steady State CD14+ and CD1a+ Antigen Presenting Cells and Epidermal Langerhans Cells.

PubMed

Fehres, Cynthia M; Bruijns, Sven C M; Sotthewes, Brigit N; Kalay, Hakan; Schaffer, Lana; Head, Steven R; de Gruijl, Tanja D; Garcia-Vallejo, Juan J; van Kooyk, Yvette

2015-01-01

Cutaneous antigen presenting cells (APCs) are critical for the induction and regulation of skin immune responses. The human skin contains phenotypically and functionally distinct APCs subsets that are present at two separated locations. While CD1ahigh LCs form a dense network in the epidermis, the CD14+ and CD1a+ APCs reside in the dermal compartment. A better understanding of the biology of human skin APC subsets is necessary for the improvement of vaccine strategies that use the skin as administration route. In particular, progress in the characterization of uptake and activatory receptors will certainly improve APC-targeting strategies in vaccination. Here we performed a detailed analysis of the expression and function of glycan-binding and pattern-recognition receptors in skin APC subsets. The results demonstrate that under steady state conditions human CD1a+ dermal dendritic cells (DCs) were phenotypically most mature as measured by the expression of CD83 and CD86, whereas the CD14+ cells showed a higher expression of the CLRs DC-SIGN, mannose receptor and DCIR and had potent antigen uptake capacity. Furthermore, steady state LCs showed superior antigen cross-presentation as compared to the dermal APC subsets. Our results also demonstrate that the TLR3 ligand polyribosinic-polyribocytidylic acid (pI:C) was the most potent stimulator of cytokine production by both LCs and dDCs. These studies warrant further exploration of human CD1a+ dDCs and LCs as target cells for cancer vaccination to induce anti-tumor immune responses.

Influence of hydrogen-occluding-silica on migration and apoptosis in human esophageal cells in vitro.

PubMed

Li, Qiang; Tanaka, Yoshiharu; Miwa, Nobuhiko

2017-01-01

In the last decade, many studies have shown that hydrogen gas or hydrogen water can reduce the levels of reactive oxygen species in the living body. Molecular hydrogen has antioxidant and antiapoptotic effects and a preventive effect on oxidative stress-induced cell death. In the present study, we investigated solidified hydrogen-occluding-silica (H 2 -silica) that can release molecular hydrogen into cell culture medium because the use of hydrogen gas has strict handling limitations in hospital and medical facilities and laboratories, owing to its physicochemical characteristics. Human esophageal squamous cell carcinoma (KYSE-70) cells and normal human esophageal epithelial cells (HEEpiCs) were used to investigate the effects of H 2 -silica on cell viability and proliferation. Cell migration was examined with wound healing and culture-insert migration assays. The intracellular levels of reactive oxygen species were evaluated with a nitroblue tetrazolium assay. To assess the apoptotic status of the cells, the Bax/Bcl-2 ratio and cleaved caspase-3 were analyzed by western blot. The results showed that KYSE-70 cells and HEEpiCs were generally inhibited by H 2 -silica administration, and there was a significant proliferation-inhibitory effect in an H 2 -silica concentration-dependent manner compared with the control group ( P < 0.05) in KYSE-70. Apoptosis-inducing effect on KYSE-70 cells was observed in 10, 300, 600, and 1,200 ppm H 2 -silica, and only 1,200 ppm H 2 -silica caused a 2.4-fold increase in apoptosis in HEEpiCs compared with the control group as the index of Bax/Bcl-2. H 2 silica inhibited cell migration in KYSE-70 cells, and high concentrations had a cytotoxic effect on normal cells. These findings should provide insights into the mechanism of inhibition of H 2 -silica on human cancer cells in vitro .

Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor

NASA Technical Reports Server (NTRS)

Parks, Kelsey

2009-01-01

Space flight has been shown to alter the astronauts immune systems. Because immune performance is complex and reflects the influence of multiple organ systems within the host, scientists sought to understand the potential impact of microgravity alone on the cellular mechanisms critical to immunity. Lymphocytes and their differentiated immature form, lymphoblasts, play an important and integral role in the body's defense system. T cells, one of the three major types of lymphocytes, play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors. Reported studies have shown that spaceflight can affect the expression of cell surface markers. Cell surface markers play an important role in the ability of cells to interact and to pass signals between different cells of the same phenotype and cells of different phenotypes. Recent evidence suggests that cell-cycle regulators are essential for T-cell function. To trigger an effective immune response, lymphocytes must proliferate. The objective of this project is to investigate the changes in growth of human cells cultured in rotating bioreactors and to measure the growth rate and the cell cycle distribution for different human cell types. Human lymphocytes and lymphoblasts will be cultured in a bioreactor to simulate aspects of microgravity. The bioreactor is a cylindrical culture vessel that incorporates the aspects of clinostatic rotation of a solid fluid body around a horizontal axis at a constant speed, and compensates gravity by rotation and places cells within the fluid body into a sustained free-fall. Cell cycle progression and cell proliferation of the lymphocytes will be measured for a number of days. In addition, RNA from the cells will be isolated for expression of genes related in cell cycle regulations.

Human Skin Cells Are More Sensitive than Human Lung Cells to the Cytotoxic and Cell Cycle Arresting Impacts of Particulate and Soluble Hexavalent Chromium

PubMed Central

Xie, Hong; Holmes, Amie L.; Wise, Sandra S.; Young, Jamie L.; Wise, James T. F.; Wise, John Pierce

2015-01-01

Hexavalent chromium Cr(VI) is a known human lung carcinogen, with solubility playing an important role in its carcinogenic potency. Dermal exposure to Cr(VI) is common and has been associated with skin damage; however, no link between chromate exposure and skin cancer has been found. In this study, we compared the cytotoxic and clastogenic effects of Cr(VI) and its impacts on cell cycle progression in human lung and skin fibroblasts. We found human skin cells arrested earlier in their cell cycle and exhibit more cytotoxicity than human lung cells, despite taking up similar amounts of Cr. These outcomes are consistent with a hypothesis that different cellular and molecular responses underlie the differences in carcinogenic outcome in these two tissues. PMID:25805272

Human growth hormone treatment: synthesis of literature on product delivery systems and administration practices.

PubMed

Bhosle, Monali; Klingman, David; Aagren, Mark; Wisniewski, Tami; Lee, Won Chan

2011-01-01

To synthesize current literature on recombinant human growth hormone (rhGH) use and to identify areas of research that have received little to no attention in light of administration practice and patient perception/behavior. Relevant articles for a systematic review were identified through PubMed. A total of 43 articles were identified: 9 (15.9%) studies on product administration practices and 34 (84.1%) on patient behavior patterns. Patients primarily preferred simple, convenient, and easy-to-use delivery devices. However, literature addressing the effect of convenient product administration practices on treatment outcomes using real-world patient/caregiver data is lacking. Better understanding of real-world product administration practices will help nurses identify areas of improvement in patient education and training. © 2010, Wiley Periodicals, Inc.

New frontiers in human cell biology and medicine: can pluripotent stem cells deliver?

PubMed

Goldstein, Lawrence S B

2012-11-12

Human pluripotent stem cells provide enormous opportunities to treat disease using cell therapy. But human stem cells can also drive biomedical and cell biological discoveries in a human model system, which can be directly linked to understanding disease or developing new therapies. Finally, rigorous scientific studies of these cells can and should inform the many science and medical policy issues that confront the translation of these technologies to medicine. In this paper, I discuss these issues using amyotrophic lateral sclerosis as an example.

45 CFR 1177.8 - Administrative offset.

Code of Federal Regulations, 2010 CFR

2010-10-01

... HUMANITIES NATIONAL ENDOWMENT FOR THE HUMANITIES CLAIMS COLLECTION § 1177.8 Administrative offset. (a) The... administrative offset, the National Endowment for the Humanities will send the debtor written notice of the... interest. (d) The National Endowment for the Humanities may effect an administrative offset against a...

45 CFR 1177.8 - Administrative offset.

Code of Federal Regulations, 2011 CFR

2011-10-01

... HUMANITIES NATIONAL ENDOWMENT FOR THE HUMANITIES CLAIMS COLLECTION § 1177.8 Administrative offset. (a) The... administrative offset, the National Endowment for the Humanities will send the debtor written notice of the... interest. (d) The National Endowment for the Humanities may effect an administrative offset against a...

Pharmacokinetic and pharmacodynamic comparisons between human granulocyte colony-stimulating factor purified from human bladder carcinoma cell line 5637 culture medium and recombinant human granulocyte colony-stimulating factor produced in Escherichia coli.

PubMed

Tanaka, H; Kaneko, T

1992-07-01

The pharmacokinetics and biological activities of recombinant human granulocyte colony-stimulating factor (hG-CSF) produced in Escherichia coli were compared with those of hG-CSF purified from human bladder carcinoma cell line 5637 culture medium (5637-hG-CSF). Recombinant hG-CSF was biologically active in a bone marrow cell proliferation assay in vitro, with a dose-response curve similar to that of 5637-hG-CSF. The effects of 5637- and recombinant hG-CSF administered via i.v. injection to rats showed similar response patterns of neutrophil counts in peripheral blood. From these results, it is concluded that the O-linked sugar chain of hG-CSF does not contribute to the in vitro and in vivo biological activities. The pharmacokinetics of both forms of hG-CSF in rats were investigated using a sandwich enzyme-linked immunosorbent assay. After i.v. administration, the serum concentration-time curves of 5637- and recombinant hG-CSF declined biexponentially. Total body clearance and steady-state volume of distribution of 5637-hG-CSF were smaller than those for the recombinant form. After s.c. administration, a lower peak serum level, smaller AUC, and lower bioavailability of 5637-hG-CSF were observed compared to recombinant hG-CSF.

Engraftment of Human Mesenchymal Stem Cells in a Rat Photothrombotic Cerebral Infarction Model : Comparison of Intra-Arterial and Intravenous Infusion Using MRI and Histological Analysis

PubMed Central

Byun, Jun Soo; Kim, Jae Kyun; Jung, Jisung; Ha, Bon Chul; Park, Serah

2013-01-01

Objective This study aimed to evaluate the hypotheses that administration routes [intra-arterial (IA) vs. intravenous (IV)] affect the early stage migration of transplanted human bone marrow-derived mesenchymal stem cells (hBM-MSCs) in acute brain infarction. Methods Male Sprague-Dawley rats (n=40) were subjected to photothrombotic infarction. Three days after photothrombotic infarction, rats were randomly allocated to one of four experimental groups [IA group : n=12, IV group : n=12, superparamagnetic iron oxide (SPIO) group : n=8, control group : n=8]. All groups were subdivided into 1, 6, 24, and 48 hours groups according to time point of sacrifice. Magnetic resonance imaging (MRI) consisting of T2 weighted image (T2WI), T2* weighted image (T2*WI), susceptibility weighted image (SWI), and diffusion weighted image of rat brain were obtained prior to and at 1, 6, 24, and 48 hours post-implantation. After final MRI, rats were sacrificed and grafted cells were analyzed in brain and lung specimen using Prussian blue and immunohistochemical staining. Results Grafted cells appeared as dark signal intensity regions at the peri-lesional zone. In IA group, dark signals in peri-lesional zone were more prominent compared with IV group. SWI showed largest dark signal followed by T2*WI and T2WI in both IA and IV groups. On Prussian blue staining, IA administration showed substantially increased migration and a large number of transplanted hBM-MSCs in the target brain than IV administration. The Prussian blue-positive cells were not detected in SPIO and control groups. Conclusion In a rat photothrombotic model of ischemic stroke, selective IA administration of human mesenchymal stem cells is more effective than IV administration. MRI and histological analyses revealed the time course of cell migration, and the numbers and distribution of hBM-MSCs delivered into the brain. PMID:24527188

Esculetin Inhibits the Survival of Human Prostate Cancer Cells by Inducing Apoptosis and Arresting the Cell Cycle.

PubMed

Turkekul, Kader; Colpan, R Dilsu; Baykul, Talha; Ozdemir, Mehmet D; Erdogan, Suat

2018-03-01

Prostate cancer (PCa) is one of the most important causes of death in men and thus new therapeutic approaches are needed. In this study, antiproliferative and anti-migration properties of a coumarin derivative esculetin were evaluated. Human PCa cell lines PC3, DU145, and LNCaP were treated with various concentrations of esculetin for 24 to 72 hours, and cell viability was determined by the MTT test. Cell cycle and apoptosis were analyzed by using cell-based cytometer. Gene expression levels were assessed by reverse transcription and quantitative real-time PCR, cell migration was determined by the wound healing assay. The protein expression was measured by Western blotting. Esculetin inhibited cell proliferation in a dose- and time-dependent manner. Cell migration was inhibited by esculetin treatment. Administration of esculetin significantly reduced the cells survival, induced apoptosis and caused the G1 phase cell cycle arrest shown by image-based cytometer. The induced expression of cytochrome c , p53, p21 and p27, and down-regulated CDK2 and CDK4 may be the underlying molecular mechanisms of esculetin effect. Esculetin suppressed phosphorylation of Akt and enhanced protein expression of tumor-suppressor phosphatase and tensin homologue. Our findings showed that the coumarin derivative esculetin could be used in the management of PCa. However, further in vivo research is needed.

Esculetin Inhibits the Survival of Human Prostate Cancer Cells by Inducing Apoptosis and Arresting the Cell Cycle

PubMed Central

Turkekul, Kader; Colpan, R. Dilsu; Baykul, Talha; Ozdemir, Mehmet D.

2018-01-01

Background Prostate cancer (PCa) is one of the most important causes of death in men and thus new therapeutic approaches are needed. In this study, antiproliferative and anti-migration properties of a coumarin derivative esculetin were evaluated. Methods Human PCa cell lines PC3, DU145, and LNCaP were treated with various concentrations of esculetin for 24 to 72 hours, and cell viability was determined by the MTT test. Cell cycle and apoptosis were analyzed by using cell-based cytometer. Gene expression levels were assessed by reverse transcription and quantitative real-time PCR, cell migration was determined by the wound healing assay. The protein expression was measured by Western blotting. Results Esculetin inhibited cell proliferation in a dose- and time-dependent manner. Cell migration was inhibited by esculetin treatment. Administration of esculetin significantly reduced the cells survival, induced apoptosis and caused the G1 phase cell cycle arrest shown by image-based cytometer. The induced expression of cytochrome c, p53, p21 and p27, and down-regulated CDK2 and CDK4 may be the underlying molecular mechanisms of esculetin effect. Esculetin suppressed phosphorylation of Akt and enhanced protein expression of tumor-suppressor phosphatase and tensin homologue. Conclusions Our findings showed that the coumarin derivative esculetin could be used in the management of PCa. However, further in vivo research is needed. PMID:29629344

Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells.

PubMed

Lee, Jonghyeob; Sugiyama, Takuya; Liu, Yinghua; Wang, Jing; Gu, Xueying; Lei, Ji; Markmann, James F; Miyazaki, Satsuki; Miyazaki, Jun-Ichi; Szot, Gregory L; Bottino, Rita; Kim, Seung K

2013-11-19

Pancreatic islet β-cell insufficiency underlies pathogenesis of diabetes mellitus; thus, functional β-cell replacement from renewable sources is the focus of intensive worldwide effort. However, in vitro production of progeny that secrete insulin in response to physiological cues from primary human cells has proven elusive. Here we describe fractionation, expansion and conversion of primary adult human pancreatic ductal cells into progeny resembling native β-cells. FACS-sorted adult human ductal cells clonally expanded as spheres in culture, while retaining ductal characteristics. Expression of the cardinal islet developmental regulators Neurog3, MafA, Pdx1 and Pax6 converted exocrine duct cells into endocrine progeny with hallmark β-cell properties, including the ability to synthesize, process and store insulin, and secrete it in response to glucose or other depolarizing stimuli. These studies provide evidence that genetic reprogramming of expandable human pancreatic cells with defined factors may serve as a general strategy for islet replacement in diabetes. DOI: http://dx.doi.org/10.7554/eLife.00940.001.

Human organomics: a fresh approach to understanding human development using single-cell transcriptomics.

PubMed

Camp, J Gray; Treutlein, Barbara

2017-05-01

Innovative methods designed to recapitulate human organogenesis from pluripotent stem cells provide a means to explore human developmental biology. New technologies to sequence and analyze single-cell transcriptomes can deconstruct these 'organoids' into constituent parts, and reconstruct lineage trajectories during cell differentiation. In this Spotlight article we summarize the different approaches to performing single-cell transcriptomics on organoids, and discuss the opportunities and challenges of applying these techniques to generate organ-level, mechanistic models of human development and disease. Together, these technologies will move past characterization to the prediction of human developmental and disease-related phenomena. © 2017. Published by The Company of Biologists Ltd.

[Primary culture of human normal epithelial cells].

PubMed

Tang, Yu; Xu, Wenji; Guo, Wanbei; Xie, Ming; Fang, Huilong; Chen, Chen; Zhou, Jun

2017-11-28

The traditional primary culture methods of human normal epithelial cells have disadvantages of low activity of cultured cells, the low cultivated rate and complicated operation. To solve these problems, researchers made many studies on culture process of human normal primary epithelial cell. In this paper, we mainly introduce some methods used in separation and purification of human normal epithelial cells, such as tissue separation method, enzyme digestion separation method, mechanical brushing method, red blood cell lysis method, percoll layered medium density gradient separation method. We also review some methods used in the culture and subculture, including serum-free medium combined with low mass fraction serum culture method, mouse tail collagen coating method, and glass culture bottle combined with plastic culture dish culture method. The biological characteristics of human normal epithelial cells, the methods of immunocytochemical staining, trypan blue exclusion are described. Moreover, the factors affecting the aseptic operation, the conditions of the extracellular environment, the conditions of the extracellular environment during culture, the number of differential adhesion, and the selection and dosage of additives are summarized.

Quantification of folic acid in human feces after administration of Bifidobacterium probiotic strains.

PubMed

Strozzi, G Paolo; Mogna, Luca

2008-09-01

Folic acid, or vitamin B9, is involved in appropriate regulation of DNA replication, synthesis of purines and deoxythymidine (dTMP), conversion of homocysteine to methionine, histidine catabolism, and correct differentiation of the neural tube during fetal organogenesis. Folic acid from food sources is almost completely absorbed in the small intestine, mostly in the jejunum, and does not reach the large intestine. The administration of probiotic strains able to synthesize folates de novo and release them in the extracellular space may provide an additional, constant endogenous source of this important vitamin in the intestinal lumen of humans. A pilot study involving 23 healthy volunteers was conducted to evaluate the ability of 3 probiotic strains, Bifidobacterium adolescentis DSM 18350, B. adolescentis DSM 18352, and Bifidobacterium pseudocatenulatum DSM 18353, to produce folates in the human intestine. Volunteers were randomly assigned to 1 of 3 groups for treatment with a specific probiotic strain (5 x 10(9) colony forming units/d). Strain effectiveness was evaluated by determination of the folate concentration in feces evacuated within 48 hours before and after administration of the probiotics. Quantification of microorganisms belonging to the genus Bifidobacterium was performed in parallel to folate analysis. Ingestion of these probiotic strains resulted in a significant increase of folic acid concentration in human feces in all treated groups. Analysis of the fecal Bifidobacteria confirmed the potential of all strains, especially B. adolescentis DSM 18352, to colonize the intestinal environment. The demonstrated ability of the probiotic microorganisms B. adolescentis DSM 18350, B. adolescentis DSM 18352, and B. pseudocatenulatum DSM 18353 to synthesize and secrete folates in the human intestinal environment may provide a complementary endogenous source of such molecules, which is especially useful for the homeostasis of mucosal enterocytes of the colon and

Human umbilical cord blood mesenchymal stem cells reduce colitis in mice by activating NOD2 signaling to COX2.

PubMed

Kim, Hyung-Sik; Shin, Tae-Hoon; Lee, Byung-Chul; Yu, Kyung-Rok; Seo, Yoojin; Lee, Seunghee; Seo, Min-Soo; Hong, In-Sun; Choi, Soon Won; Seo, Kwang-Won; Núñez, Gabriel; Park, Jong-Hwan; Kang, Kyung-Sun

2013-12-01

Decreased levels or function of nucleotide-binding oligomerization domain 2 (NOD2) are associated with Crohn's disease. NOD2 regulates intestinal inflammation, and also is expressed by human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs), to regulate their differentiation. We investigated whether NOD2 is required for the anti-inflammatory activities of MSCs in mice with colitis. Colitis was induced in mice by administration of dextran sulfate sodium or trinitrobenzene sulfonic acid. Mice then were given intraperitoneal injections of NOD2-activated hUCB-MSCs; colon tissues and mesenteric lymph nodes were collected for histologic analyses. A bromodeoxyuridine assay was used to determine the ability of hUCB-MSCs to inhibit proliferation of human mononuclear cells in culture. Administration of hUCB-MSCs reduced the severity of colitis in mice. The anti-inflammatory effects of hUCB-MSCs were greatly increased by activation of NOD2 by its ligand, muramyl dipeptide (MDP). Administration of NOD2-activated hUCB-MSCs increased anti-inflammatory responses in colons of mice, such as production of interleukin (IL)-10 and infiltration by T regulatory cells, and reduced production of inflammatory cytokines. Proliferation of mononuclear cells was inhibited significantly by co-culture with hUCB-MSCs that had been stimulated with MDP. MDP induced prolonged production of prostaglandin (PG)E2 in hUCB-MSCs via the NOD2-RIP2 pathway, which suppressed proliferation of mononuclear cells derived from hUCB. PGE2 produced by hUCB-MSCs in response to MDP increased production of IL-10 and T regulatory cells. In mice, production of PGE2 by MSCs and subsequent production of IL-10 were required to reduce the severity of colitis. Activation of NOD2 is required for the ability of hUCB-MSCs to reduce the severity of colitis in mice. NOD2 signaling increases the ability of these cells to suppress mononuclear cell proliferation by inducing production of PGE2. Copyright © 2013 AGA

Development of a new live attenuated mumps virus vaccine in human diploid cells.

PubMed

Sassani, A; Mirchamsy, H; Shafyi, A; Ahourai, P; Razavi, J; Gholami, M R; Mohammadi, A; Ezzi, A; Rahmani, M; Fateh, G

1991-07-01

A new live attenuated mumps vaccine was developed in human diploid cells. The S-12 virus was isolated from a 10-year-old girl showing typical symptoms of mumps infection, the diagnosis was confirmed by a pediatrician. The virus was isolated in green monkey kidney cells, without passage in chick embryo cavity or chick embryo fibroblasts. Attenuation of the wild virus was performed by serial passages in human diploid cells (MRC-5). The attenuated virus was characterized by identity tests, as well as by a reduction in plaque size, as marker tests. The virus was free from adventitious agents and safe for laboratory animals as well as for monkeys. The reactogenicity and immunogenicity of the S-12 virus for man was investigated by administration of a monovalent vaccine to 20 seronegative adult male volunteers and 30 children aged 1 to 5 years without history of mumps infection or vaccination. Seroconversion was obtained in 95% of the vaccinees. The new vaccine has the advantage of not requiring specific pathogen-free eggs, and being free from avian proteins and therefore can be used in sensitized patients.

Promotion or suppression of experimental metastasis of B16 melanoma cells after oral administration of lapachol

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

Maeda, Masayo; Murakami, Manabu; Takegami, Tsutomu

Lapachol [2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone] is a vitamin K antagonist with antitumor activity. The effect of lapachol on the experimental metastasis of murine B16BL6 melanoma cells was examined. A single oral administration of a high toxic dose of lapachol (80-100 mg/kg) 6 h before iv injection of tumor cells drastically promoted metastasis. This promotion of metastasis was also observed in T-cell-deficient mice and NK-suppressed mice. In vitro treatment of B16BL6 cells with lapachol promoted metastasis only slightly, indicating that lapachol promotes metastasis primarily by affecting host factors other than T cells and NK cells. A single oral administration of warfarin, the most commonlymore » used vitamin K antagonist, 6 h before iv injection of tumor cells also drastically promoted the metastasis of B16BL6 cells. The promotion of metastasis by lapachol and warfarin was almost completely suppressed by preadministration of vitamin K3, indicating that the promotion of metastasis by lapachol was derived from vitamin K antagonism. Six hours after oral administration of lapachol or warfarin, the protein C level was reduced maximally, without elongation of prothrombin time. These observations suggest that a high toxic dose of lapachol promotes metastasis by inducing a hypercoagulable state as a result of vitamin K-dependent pathway inhibition. On the other hand, serial oral administration of low non-toxic doses of lapachol (5-20 mg/kg) weakly but significantly suppressed metastasis by an unknown mechanism, suggesting the possible use of lapachol as an anti-metastatic agent.« less

Repeated hematopoietic stem and progenitor cell mobilization without depletion of the bone marrow stem and progenitor cell pool in mice after repeated administration of recombinant murine G-CSF.

PubMed

de Kruijf, Evert-Jan F M; van Pel, Melissa; Hagoort, Henny; Kruysdijk, Donnée; Molineux, Graham; Willemze, Roel; Fibbe, Willem E

2007-05-01

Administration of recombinant-human G-CSF (rhG-CSF) is highly efficient in mobilizing hematopoietic stem and progenitor cells (HSC/HPC) from the bone marrow (BM) toward the peripheral blood. This study was designed to investigate whether repeated G-CSF-induced HSC/HPC mobilization in mice could lead to a depletion of the bone marrow HSC/HPC pool with subsequent loss of mobilizing capacity. To test this hypothesis Balb/c mice were treated with a maximum of 12 repeated 5-day cycles of either 10 microg rhG-CSF/day or 0.25 microg rmG-CSF/day. Repeated administration of rhG-CSF lead to strong inhibition of HSC/HPC mobilization toward the peripheral blood and spleen after >4 cycles because of the induction of anti-rhG-CSF antibodies. In contrast, after repeated administration of rmG-CSF, HSC/HPC mobilizing capacity remained intact for up to 12 cycles. The number of CFU-GM per femur did not significantly change for up to 12 cycles. We conclude that repeated administration of G-CSF does not lead to depletion of the bone marrow HSC/HPC pool.

Involvement of PI3K and ROCK signaling pathways in migration of bone marrow-derived mesenchymal stem cells through human brain microvascular endothelial cell monolayers.

PubMed

Lin, Mei-Na; Shang, De-Shu; Sun, Wei; Li, Bo; Xu, Xin; Fang, Wen-Gang; Zhao, Wei-Dong; Cao, Liu; Chen, Yu-Hua

2013-06-04

Bone marrow-derived mesenchymal stem cells (MSC) represent an important and easily available source of stem cells for potential therapeutic use in neurological diseases. The entry of circulating cells into the central nervous system by intravenous administration requires, firstly, the passage of the cells across the blood-brain barrier (BBB). However, little is known of the details of MSC transmigration across the BBB. In the present study, we employed an in vitro BBB model constructed using a human brain microvascular endothelial cell monolayer to study the mechanism underlying MSC transendothelial migration. Transmigration assays, transendothelial electrical resistance (TEER) and horseradish peroxidase (HRP) flux assays showed that MSC could transmigrate through human brain microvascular endothelial cell monolayers by a paracellular pathway. Cell fractionation and immunofluorescence assays confirmed the disruption of tight junctions. Inhibition assays showed that a Rho-kinase (ROCK) inhibitor (Y27632) effectively promoted MSC transendothelial migration; conversely, a PI3K inhibitor (LY294002) blocked MSC transendothelial migration. Interestingly, adenovirus-mediated interference with ROCK in MSC significantly increased MSC transendothelial migration, and overexpression of a PI3K dominant negative mutant in MSC cells could block transendothelial migration. Our findings provide clear evidence that the PI3K and ROCK pathways are involved in MSC migration through human brain microvascular endothelial cell monolayers. The information yielded by this study may be helpful in constructing gene-modified mesenchymal stem cells that are able to penetrate the BBB effectively for cell therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

Analyses of the peripheral immunome following multiple administrations of avelumab, a human IgG1 anti-PD-L1 monoclonal antibody.

PubMed

Donahue, Renee N; Lepone, Lauren M; Grenga, Italia; Jochems, Caroline; Fantini, Massimo; Madan, Ravi A; Heery, Christopher R; Gulley, James L; Schlom, Jeffrey

2017-01-01

Multiple anti-PD-L1/PD-1 checkpoint monoclonal antibodies (MAb) have shown clear evidence of clinical benefit. All except one have been designed or engineered to omit the possibility to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) as a second potential mode of anti-tumor activity; the reason for this is the concern of lysis of PD-L1 positive immune cells. Avelumab is a fully human IgG1 MAb which has been shown in prior in vitro studies to mediate ADCC versus a range of human tumor cells, and clinical studies have demonstrated anti-tumor activity versus a range of human cancers. This study was designed to investigate the effect on immune cell subsets in the peripheral blood of cancer patients prior to and following multiple administrations of avelumab. One hundred twenty-three distinct immune cell subsets in the peripheral blood of cancer patients ( n  = 28) in a phase I trial were analyzed by flow cytometry prior to and following one, three, and nine cycles of avelumab. Changes in soluble (s) CD27 and sCD40L in plasma were also evaluated. In vitro studies were also performed to determine if avelumab would mediate ADCC of PBMC. No statistically significant changes in any of the 123 immune cell subsets analyzed were observed at any dose level, or number of doses, of avelumab. Increases in the ratio of sCD27:sCD40L were observed, suggesting potential immune activation. Controlled in vitro studies also showed lysis of tumor cells by avelumab versus no lysis of PBMC from five donors. These studies demonstrate the lack of any significant effect on multiple immune cell subsets, even those expressing PD-L1, following multiple cycles of avelumab. These results complement prior studies showing anti-tumor effects of avelumab and comparable levels of adverse events with avelumab versus other anti-PD-1/PD-L1 MAbs. These studies provide the rationale to further exploit the potential ADCC mechanism of action of avelumab as well as other human IgG1 checkpoint

Xanthosine administration does not affect the proportion of epithelial stem cells in bovine mammary tissue, but has a latent negative effect on cell proliferation

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

Rauner, Gat, E-mail: gat.rauner@mail.huji.ac.il; The Robert H. Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem; Barash, Itamar, E-mail: itamar.barash@mail.huji.ac.il

The challenge in manipulating the proportion of somatic stem cells lies in having to override tissue homeostasis. Xanthosine infusion via the teat canal has been reported to augment the number of label-retaining cells in the mammary gland of 3-month-old bovine calves. To further delineate xanthosine's effect on defined stem cells in the mammary gland of heifers—which are candidates for increased prospective milk production following such manipulation—bovine mammary parenchymal tissue was transplanted and integrated into the cleared mammary fat pad of immunodeficient mice. Xanthosine administration for 14 days did not affect the number of label-retaining cells after 10- and 11-week chases.more » No change in stem cell proportion, analyzed according to CD49f and CD24 expression, was noted. Clone formation and propagation rate of cultured cells, as well as expression of stem cell markers, were also unaffected. In contrast, a latent 50% decrease in bovine mammary cell proliferation rate was observed 11 weeks after xanthosine administration. Tumor development in mice was also limited by xanthosine administration. These effects may have resulted from an initial decrease in expression of the rate-limiting enzyme in guanine synthesis, IMPDH. The data indicate that caution should be exerted when considering xanthosine for stem cell manipulation. - Highlights: • Novel “bovinized“ mouse model for exogenous effects on bovine mammary gland. • Xanthosine did not affect stem cell number/function in bovine mammary gland. • Xanthosine caused an immediate decrease in IMPDH expression in bovine mammary gland. • Xanthosine had latent negative effect on cell proliferation in bovine mammary gland. • Xanthosine administration limited mammary tumor growth.« less

Side population cells in the human vocal fold.

PubMed

Yamashita, Masaru; Hirano, Shigeru; Kanemaru, Shin-ichi; Tsuji, Shunichiro; Suehiro, Atsushi; Ito, Juichi

2007-11-01

The regenerative processes of the vocal fold, or the existence of stem cells in the folds, are unknown. Side population (SP) cells are defined as cells that have the ability to exclude the DNA binding dye, Hoechst 33342. They are regarded as a cell population enriched with stem cells and can be isolated from non-SP cells by a fluorescence-activated cell sorter. This study was designed to determine whether SP cells exist in the human vocal fold, as a first step in elucidating the regenerative mechanisms of the vocal fold. Seven human excised larynges were used in this study. Two were used for fluorescence-activated cell sorter analysis, and 5 were subjected to immunohistochemical analysis with antibodies against an adenosine triphosphate binding cassette transporter family member, ABCG2, which is expressed in SP cells. The number of SP cells in the human vocal fold was about 0.2% of the total number of cells. ABCG2-positive cells were identified in both the epithelium and subepithelial tissue throughout the entire vocal fold. This preliminary study demonstrated the existence of SP cells in the human vocal fold. Further studies are warranted to clarify how these cells work in the vocal fold, particularly in the regenerative process.

Comparative reactivity of human IgE to cynomolgus monkey and human effector cells and effects on IgE effector cell potency

PubMed Central

Saul, Louise; Saul, Louise; Josephs, Debra H; Josephs, Debra H; Cutler, Keith; Cutler, Keith; Bradwell, Andrew; Bradwell, Andrew; Karagiannis, Panagiotis; Karagiannis, Panagiotis; Selkirk, Chris; Selkirk, Chris; Gould, Hannah J; Gould, Hannah J; Jones, Paul; Jones, Paul; Spicer, James F; Spicer, James F; Karagiannis, Sophia N; Karagiannis, Sophia N

2014-01-01

Background: Due to genetic similarities with humans, primates of the macaque genus such as the cynomolgus monkey are often chosen as models for toxicology studies of antibody therapies. IgE therapeutics in development depend upon engagement with the FcεRI and FcεRII receptors on immune effector cells for their function. Only limited knowledge of the primate IgE immune system is available to inform the choice of models for mechanistic and safety evaluations.   Methods: The recognition of human IgE by peripheral blood lymphocytes from cynomolgus monkey and man was compared. We used effector cells from each species in ex vivo affinity, dose-response, antibody-receptor dissociation and potency assays. Results: We report cross-reactivity of human IgE Fc with cynomolgus monkey cells, and comparable binding kinetics to peripheral blood lymphocytes from both species. In competition and dissociation assays, however, human IgE dissociated faster from cynomolgus monkey compared with human effector cells. Differences in association and dissociation kinetics were reflected in effector cell potency assays of IgE-mediated target cell killing, with higher concentrations of human IgE needed to elicit effector response in the cynomolgus monkey system. Additionally, human IgE binding on immune effector cells yielded significantly different cytokine release profiles in each species. Conclusion: These data suggest that human IgE binds with different characteristics to human and cynomolgus monkey IgE effector cells. This is likely to affect the potency of IgE effector functions in these two species, and so has relevance for the selection of biologically-relevant model systems when designing pre-clinical toxicology and functional studies. PMID:24492303

Alternative Sources of Adult Stem Cells: Human Amniotic Membrane

NASA Astrophysics Data System (ADS)

Wolbank, Susanne; van Griensven, Martijn; Grillari-Voglauer, Regina; Peterbauer-Scherb, Anja

Human amniotic membrane is a highly promising cell source for tissue engineering. The cells thereof, human amniotic epithelial cells (hAEC) and human amniotic mesenchymal stromal cells (hAMSC), may be immunoprivileged, they represent an early developmental status, and their application is ethically uncontroversial. Cell banking strategies may use freshly isolated cells or involve in vitro expansion to increase cell numbers. Therefore, we have thoroughly characterized the effect of in vitro cultivation on both phenotype and differentiation potential of hAEC. Moreover, we present different strategies to improve expansion including replacement of animal-derived supplements by human platelet products or the introduction of the catalytic subunit of human telomerase to extend the in vitro lifespan of amniotic cells. Characterization of the resulting cultures includes phenotype, growth characteristics, and differentiation potential, as well as immunogenic and immunomodulatory properties.

The rat closely mimics oxidative stress and inflammation in humans after exercise but not after exercise combined with vitamin C administration.

PubMed

Veskoukis, Aristidis S; Goutianos, Georgios; Paschalis, Vassilis; Margaritelis, Nikos V; Tzioura, Aikaterini; Dipla, Konstantina; Zafeiridis, Andreas; Vrabas, Ioannis S; Kyparos, Antonios; Nikolaidis, Michalis G

2016-04-01

The purpose of the present study was to directly compare oxidative stress and inflammation responses between rats and humans. We contrasted rat and human oxidative stress and inflammatory responses to exercise (pro-oxidant stimulus) and/or vitamin C (anti-oxidant stimulus) administration. Vitamin C was administered orally in both species (16 mg kg(-1) of body weight). Twelve redox biomarkers and seven inflammatory biomarkers were determined in plasma and erythrocytes pre- and post-exercise or pre- and post-exercise combined with vitamin C administration. Exercise increased oxidative stress and induced an inflammatory state in rats and humans. There were only 1/19 significant species × exercise interactions (catalase), indicating similar responses to exercise between rats and humans in redox and inflammatory biomarkers. Vitamin C decreased oxidative stress and increased antioxidant capacity only in humans and did not affect the redox state of rats. In contrast, vitamin C induced an anti-inflammatory state only in rats and did not affect the inflammatory state of humans. There were 10/19 significant species × vitamin C interactions, indicating that rats poorly mimic human oxidative stress and inflammatory responses to vitamin C administration. Exercise after acute vitamin C administration altered redox state only in humans and did not affect the redox state of rats. On the contrary, inflammation biomarkers changed similarly after exercise combined with vitamin C in both rats and humans. The rat adequately mimics human responses to exercise in basic blood redox/inflammatory profile, yet this is not the case after exercise combined with vitamin C administration.

Repeated administration of an acetylcholinesterase inhibitor attenuates nicotine taking in rats and smoking behavior in human smokers

PubMed Central

Ashare, R L; Kimmey, B A; Rupprecht, L E; Bowers, M E; Hayes, M R; Schmidt, H D

2016-01-01

Tobacco smoking remains the leading cause of preventable death worldwide and current smoking cessation medications have limited efficacy. Thus, there is a clear need for translational research focused on identifying novel pharmacotherapies for nicotine addiction. Our previous studies demonstrated that acute administration of an acetylcholinesterase inhibitor (AChEI) attenuates nicotine taking and seeking in rats and suggest that AChEIs could be repurposed for smoking cessation. Here, we expand upon these findings with experiments designed to determine the effects of repeated AChEI administration on voluntary nicotine taking in rats as well as smoking behavior in human smokers. Rats were trained to self-administer intravenous infusions of nicotine (0.03 mg kg−1 per 0.59 ml) on a fixed-ratio-5 schedule of reinforcement. Once rats maintained stable nicotine taking, galantamine or donepezil was administered before 10 consecutive daily nicotine self-administration sessions. Repeated administration of 5.0 mg kg−1 galantamine and 3.0 mg kg−1 donepezil attenuated nicotine self-administration in rats. These effects were reinforcer-specific and not due to adverse malaise-like effects of drug treatment as repeated galantamine and donepezil administration had no effects on sucrose self-administration, ad libitum food intake and pica. The effects of repeated galantamine (versus placebo) on cigarette smoking were also tested in human treatment-seeking smokers. Two weeks of daily galantamine treatment (8.0 mg (week 1) and 16.0 mg (week 2)) significantly reduced smoking rate as well as smoking satisfaction and reward compared with placebo. This translational study indicates that repeated AChEI administration reduces nicotine reinforcement in rats and smoking behavior in humans at doses not associated with tolerance and/or adverse effects. PMID:26784967

21 CFR 864.2280 - Cultured animal and human cells.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cultured animal and human cells. 864.2280 Section... (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Cell And Tissue Culture Products § 864.2280 Cultured animal and human cells. (a) Identification. Cultured animal and human cells are in vitro...

Interaction of dacarbazine and imexon, in vitro and in vivo, in human A375 melanoma cells.

PubMed

Samulitis, Betty K; Dorr, Robert T; Chow, H-H Sherry

2011-09-01

We evaluated mechanisms of interaction between the alkyating agent dacarbazine (DTIC) and the pro-oxidant, imexon, in the human A375 melanoma cell line. The effect of DTIC and imexon, alone and in combination, was evaluated for growth inhibition (MTT), radiolabeled drug uptake, cellular thiol content (HPLC), and DNA strand breaks (Comet assay). Pharmacokinetic and antitumor effects were evaluated in mice. Growth inhibition in vitro was additive with the two drugs. There was no effect on drug uptake or on the number of DNA strand breaks. There was a >75% reduction in cellular glutathione and cysteine with imexon but not DTIC. Co-administration of the two drugs in mice caused an increase in the area under the curve of both drugs, but the combination was not effective in reducing human A375 melanoma tumors in vivo. Imexon and dacarbazine show additive effects in vitro but not in vivo in human A375 melanoma cells.

Human amniotic epithelial cells as feeder layer to derive and maintain human embryonic stem cells from poor-quality embryos.

PubMed

Ávila-González, Daniela; Vega-Hernández, Eva; Regalado-Hernández, Juan Carlos; De la Jara-Díaz, Julio Francisco; García-Castro, Irma Lydia; Molina-Hernández, Anayansi; Moreno-Verduzco, Elsa Romelia; Razo-Aguilera, Guadalupe; Flores-Herrera, Héctor; Portillo, Wendy; Díaz-Martínez, Néstor Emmanuel; García-López, Guadalupe; Díaz, Néstor Fabián

2015-09-01

Data from the literature suggest that human embryonic stem cell (hESC) lines used in research do not genetically represent all human populations. The derivation of hESC through conventional methods involve the destruction of viable human embryos, as well the use of mouse embryonic fibroblasts as a feeder layer, which has several drawbacks. We obtained the hESC line (Amicqui-1) from poor-quality (PQ) embryos derived and maintained on human amniotic epithelial cells (hAEC). This line displays a battery of markers of pluripotency and we demonstrated the capacity of these cells to produce derivates of the three germ layers. Copyright © 2015. Published by Elsevier B.V.

Differentiation and Transplantation of Human Embryonic Stem Cell-Derived Hepatocytes

PubMed Central

Basma, Hesham; Soto-Gutiérrez, Alejandro; Yannam, Govardhana Rao; Liu, Liping; Ito, Ryotaro; Yamamoto, Toshiyuki; Ellis, Ewa; Carson, Steven D.; Sato, Shintaro; Chen, Yong; Muirhead, David; Navarro-Álvarez, Nalu; Wong, Ron; Roy-Chowdhury, Jayanta; Platt, Jeffrey L.; Mercer, David F.; Miller, John D.; Strom, Stephen C.; Kobayashi, Noaya; Fox, Ira J.

2009-01-01

Background & Aims The ability to obtain unlimited numbers of human hepatocytes would improve development of cell-based therapies for liver diseases, facilitate the study of liver biology and improve the early stages of drug discovery. Embryonic stem cells are pluripotent, can potentially differentiate into any cell type and could therefore be developed as a source of human hepatocytes. Methods To generate human hepatocytes, human embryonic stem cells were differentiated by sequential culture in fibroblast growth factor 2 and human Activin-A, hepatocyte growth factor, and dexamethasone. Functional hepatocytes were isolated by sorting for surface asialoglycoprotein receptor expression. Characterization was performed by real-time PCR, imunohistochemistry, immunoblot, functional assays and transplantation. Results Embryonic stem cell-derived hepatocytes expressed liver-specific genes but not genes representing other lineages, secreted functional human liver-specific proteins similar to those of primary human hepatocytes and demonstrated human hepatocyte cytochrome P450 metabolic activity. Serum from rodents given injections of embryonic stem cell-derived hepatocytes contained significant amounts of human albumin and alpha-1-antitrypsin. Colonies of cytokeratin-18 and human albumin-expressing cells were present in the livers of recipient animals. Conclusion Human embryonic stem cells can be differentiated into cells with many characteristics of primary human hepatocytes. Hepatocyte-like cells can be enriched and recovered based on asialoglycoprotein receptor expression and could potentially be used in drug discovery research and developed as therapeutics. PMID:19026649

Human Satellite Cell Transplantation and Regeneration from Diverse Skeletal Muscles

PubMed Central

Xu, Xiaoti; Wilschut, Karlijn J.; Kouklis, Gayle; Tian, Hua; Hesse, Robert; Garland, Catharine; Sbitany, Hani; Hansen, Scott; Seth, Rahul; Knott, P. Daniel; Hoffman, William Y.; Pomerantz, Jason H.

2015-01-01

Summary Identification of human satellite cells that fulfill muscle stem cell criteria is an unmet need in regenerative medicine. This hurdle limits understanding how closely muscle stem cell properties are conserved among mice and humans and hampers translational efforts in muscle regeneration. Here, we report that PAX7 satellite cells exist at a consistent frequency of 2–4 cells/mm of fiber in muscles of the human trunk, limbs, and head. Xenotransplantation into mice of 50–70 fiber-associated, or 1,000–5,000 FACS-enriched CD56+/CD29+ human satellite cells led to stable engraftment and formation of human-derived myofibers. Human cells with characteristic PAX7, CD56, and CD29 expression patterns populated the satellite cell niche beneath the basal lamina on the periphery of regenerated fibers. After additional injury, transplanted satellite cells robustly regenerated to form hundreds of human-derived fibers. Together, these findings conclusively delineate a source of bona-fide endogenous human muscle stem cells that will aid development of clinical applications. PMID:26352798

Derivation, propagation and differentiation of human embryonic stem cells.

PubMed

Conley, Brock J; Young, Julia C; Trounson, Alan O; Mollard, Richard

2004-04-01

Embryonic stem (ES) cells are in vitro cultivated pluripotent cells derived from the inner cell mass (ICM) of the embryonic blastocyst. Attesting to their pluripotency, ES cells can be differentiated into representative derivatives of all three embryonic germ layers (endoderm, ectoderm and mesoderm) both in vitro and in vivo. Although mouse ES cells have been studied for many years, human ES cells have only more recently been derived and successfully propagated. Many biochemical differences and culture requirements between mouse and human ES cells have been described, yet despite these differences the study of murine ES cells has provided important insights into methodologies aimed at generating a greater and more in depth understanding of human ES cell biology. One common feature of both mouse and human ES cells is their capacity to undergo controlled differentiation into spheroid structures termed embryoid bodies (EBs). EBs recapitulate several aspects of early development, displaying regional-specific differentiation programs into derivatives of all three embryonic germ layers. For this reason, EB formation has been utilised as an initial step in a wide range of studies aimed at differentiating both mouse and human ES cells into a specific and desired cell type. Recent reports utilising specific growth factor combinations and cell-cell induction systems have provided alternative strategies for the directed differentiation of cells into a desired lineage. According to each one of these strategies, however, a relatively high cell lineage heterogeneity remains, necessitating subsequent purification steps including mechanical dissection, selective media or fluorescent or magnetic activated cell sorting (FACS and MACS, respectively). In the future, the ability to specifically direct differentiation of human ES cells at 100% efficiency into a desired lineage will allow us to fully explore the potential of these cells in the analysis of early human development, drug

Schedule-dependent cytotoxic synergism of pemetrexed and erlotinib in BXPC-3 and PANC-1 human pancreatic cancer cells.

PubMed

Wang, Lin; Zhu, Zhi-Xia; Zhang, Wen-Ying; Zhang, Wei-Min

2011-09-01

Previous studies have shown that both pemetrexed, a cytotoxic drug, and erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), inhibit the cell growth of pancreatic cancer cells. However, whether they exert a synergistic antitumor effect on pancreatic cancer cells remains unknown. The present study aimed to assess the synergistic effect of erlotinib in combination with pemetrexed using different sequential administration schedules on the proliferation of human pancreatic cancer BXPC-3 and PANC-1 cells and to probe its cellular mechanism. The EGFR and K-ras gene mutation status was examined by quantitative PCR high-resolution melting (qPCR-HRM) analysis. BXPC-3 and PANC-1 cells were incubated with pemetrexed and erlotinib using different administration schedules. MTT assay was used to determine cytotoxicity, and cell cycle distribution was determined by flow cytometry. The expression and phosphorylation of EGFR, HER3, AKT and MET were determined using Western blotting. Both pemetrexed and erlotinib inhibited the proliferation of BXPC-3 and PANC-1 cells in a dose- and time-dependent manner in vitro. Synergistic effects on cell proliferation were observed when pemetrexed was used in combination with erlotinib. The degree of the synergistic effects depended on the administration sequence, which was most obvious when erlotinib was sequentially administered at 24-h interval following pemetrexed. Cell cycle studies revealed that pemetrexed induced S arrest and erlotinib induced G(0)/G(1) arrest. The sequential administration of erlotinib following pemetrexed induced S arrest. Western blot analyses showed that pemetrexed increased and erlotinib decreased the phosphorylation of EGFR, HER3 and AKT, respectively. However, both pemetrexed and erlotinib exerted no significant effects on the phosphorylation of c-MET. The phosphorylation of EGFR, HER3 and AKT was significantly suppressed by scheduled incubation with pemetrexed followed by erlotinib

Enhanced Stem Cell Differentiation and Immunopurification of Genome Engineered Human Retinal Ganglion Cells.

PubMed

Sluch, Valentin M; Chamling, Xitiz; Liu, Melissa M; Berlinicke, Cynthia A; Cheng, Jie; Mitchell, Katherine L; Welsbie, Derek S; Zack, Donald J

2017-11-01

Human pluripotent stem cells have the potential to promote biological studies and accelerate drug discovery efforts by making possible direct experimentation on a variety of human cell types of interest. However, stem cell cultures are generally heterogeneous and efficient differentiation and purification protocols are often lacking. Here, we describe the generation of clustered regularly-interspaced short palindromic repeats(CRISPR)-Cas9 engineered reporter knock-in embryonic stem cell lines in which tdTomato and a unique cell-surface protein, THY1.2, are expressed under the control of the retinal ganglion cell (RGC)-enriched gene BRN3B. Using these reporter cell lines, we greatly improved adherent stem cell differentiation to the RGC lineage by optimizing a novel combination of small molecules and established an anti-THY1.2-based protocol that allows for large-scale RGC immunopurification. RNA-sequencing confirmed the similarity of the stem cell-derived RGCs to their endogenous human counterparts. Additionally, we developed an in vitro axonal injury model suitable for studying signaling pathways and mechanisms of human RGC cell death and for high-throughput screening for neuroprotective compounds. Using this system in combination with RNAi-based knockdown, we show that knockdown of dual leucine kinase (DLK) promotes survival of human RGCs, expanding to the human system prior reports that DLK inhibition is neuroprotective for murine RGCs. These improvements will facilitate the development and use of large-scale experimental paradigms that require numbers of pure RGCs that were not previously obtainable. Stem Cells Translational Medicine 2017;6:1972-1986. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Increasing cell culture population doublings for long-term growth of finite life span human cell cultures

DOEpatents

Stampfer, Martha R.; Garbe, James C.

2016-06-28

Cell culture media formulations for culturing human epithelial cells are herein described. Also described are methods of increasing population doublings in a cell culture of finite life span human epithelial cells and prolonging the life span of human cell cultures. Using the cell culture media disclosed alone and in combination with addition to the cell culture of a compound associated with anti-stress activity achieves extended growth of pre-stasis cells and increased population doublings and life span in human epithelial cell cultures.

Increasing cell culture population doublings for long-term growth of finite life span human cell cultures

DOEpatents

Stampfer, Martha R; Garbe, James C

2015-02-24

Cell culture media formulations for culturing human epithelial cells are herein described. Also described are methods of increasing population doublings in a cell culture of finite life span human epithelial cells and prolonging the life span of human cell cultures. Using the cell culture media disclosed alone and in combination with addition to the cell culture of a compound associated with anti-stress activity achieves extended growth of pre-stasis cells and increased population doublings and life span in human epithelial cell cultures.

Human embryonic stem cell-derived NK cells acquire functional receptors and cytolytic activity.

PubMed

Woll, Petter S; Martin, Colin H; Miller, Jeffrey S; Kaufman, Dan S

2005-10-15

Human embryonic stem cells (hESCs) provide a unique resource to analyze early stages of human hematopoiesis. However, little is known about the ability to use hESCs to evaluate lymphocyte development. In the present study, we use a two-step culture method to demonstrate efficient generation of functional NK cells from hESCs. The CD56(+)CD45(+) hESC-derived lymphocytes express inhibitory and activating receptors typical of mature NK cells, including killer cell Ig-like receptors, natural cytotoxicity receptors, and CD16. Limiting dilution analysis suggests that these cells can be produced from hESC-derived hemopoietic progenitors at a clonal frequency similar to CD34(+) cells isolated from cord blood. The hESC-derived NK cells acquire the ability to lyse human tumor cells by both direct cell-mediated cytotoxicity and Ab-dependent cellular cytotoxicity. Additionally, activated hESC-derived NK cells up-regulate cytokine production. hESC-derived lymphoid progenitors provide a novel means to characterize specific cellular and molecular mechanisms that lead to development of specific human lymphocyte populations. These cells may also provide a source for innovative cellular immune therapies.

Generation of Oligodendrogenic Spinal Neural Progenitor Cells From Human Induced Pluripotent Stem Cells.

PubMed

Khazaei, Mohamad; Ahuja, Christopher S; Fehlings, Michael G

2017-08-14

This unit describes protocols for the efficient generation of oligodendrogenic neural progenitor cells (o-NPCs) from human induced pluripotent stem cells (hiPSCs). Specifically, detailed methods are provided for the maintenance and differentiation of hiPSCs, human induced pluripotent stem cell-derived neural progenitor cells (hiPS-NPCs), and human induced pluripotent stem cell-oligodendrogenic neural progenitor cells (hiPSC-o-NPCs) with the final products being suitable for in vitro experimentation or in vivo transplantation. Throughout, cell exposure to growth factors and patterning morphogens has been optimized for both concentration and timing, based on the literature and empirical experience, resulting in a robust and highly efficient protocol. Using this derivation procedure, it is possible to obtain millions of oligodendrogenic-NPCs within 40 days of initial cell plating which is substantially shorter than other protocols for similar cell types. This protocol has also been optimized to use translationally relevant human iPSCs as the parent cell line. The resultant cells have been extensively characterized both in vitro and in vivo and express key markers of an oligodendrogenic lineage. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

In vitro and in vivo studies on antitumor effects of gossypol on human stomach adenocarcinoma (AGS) cell line and MNNG induced experimental gastric cancer

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

Gunassekaran, G.R., E-mail: gunassekaran@yahoo.co.in; Kalpana Deepa Priya, D.; Gayathri, R.

2011-08-12

Highlights: {yields} Gossypol is a well known polyphenolic compound used for anticancer studies but we are the first to report that gossypol has antitumor effect on MNNG induced gastric cancer in experimental animal models. {yields} Our study shows that gossypol inhibits the proliferation of AGS (human gastric adenocarcinoma) cell line. {yields} In animal models, gossypol extends the survival of cancer bearing animals and also protects the cells from carcinogenic effect. {yields} So we suggest that gossypol would be a potential chemotherapeutic and chemopreventive agent for gastric cancer. -- Abstract: The present study has evaluated the chemopreventive effects of gossypol onmore » N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis and on human gastric adenocarcinoma (AGS) cell line. Gossypol, C{sub 30}H{sub 30}O{sub 8}, is a polyphenolic compound that has anti proliferative effect and induces apoptosis in various cancer cells. The aim of this work was to delineate in vivo and in vitro anti-initiating mechanisms of orally administered gossypol in target (stomach) tissues and in human gastric adenocarcinoma (AGS) cell line. In vitro results prove that gossypol has potent cytotoxic effect and inhibit the proliferation of adenocarcinoma (AGS) cell line. In vivo results prove gossypol to be successful in prolonging the survival of MNNG induced cancer bearing animals and in delaying the onset of tumor in animals administrated with gossypol and MNNG simultaneously. Examination of the target (stomach) tissues in sacrificed experimental animals shows that administration of gossypol significantly reduces the level of tumor marker enzyme (carcino embryonic antigen) and pepsin. The level of Nucleic acid contents (DNA and RNA) significantly reduces, and the membrane damage of glycoprotein subsides, in the target tissues of cancer bearing animals, with the administration of gossypol. These data suggest that gossypol may create a beneficial effect in

The IGF-I/JAK2-STAT3/miR-21 signaling pathway may be associated with human renal cell carcinoma cell growth.

PubMed

Su, Ying; Zhao, An; Cheng, Guoping; Xu, Jingjing; Ji, Enming; Sun, Wenyong

2017-07-04

Renal cell carcinoma (RCC) is the highest mortality rate of the genitourinary cancers, and the treatment options are very limited. Thus, identification of molecular mechanisms underlying RCC tumorigenesis, is critical for identifying biomarkers for RCC diagnosis and prognosis. To validate whether the IGF-I/JAK2-STAT3/miR-21 signaling pathway is associated with human RCC cell growth. qRT-PCR and Western blotting were used to detect the mRNA and protein expression levels, respectively. The MTT assay was performed to determine cell survival rate. The Annexin V-FITC/PI apoptosis detection kit was used to detect cell apoptosis. We employed RCC tissues and cell lines (A498; ACHN; Caki-1; Caki-2 and 786-O) in the study. IGF-I, and its inhibitor (NT-157) were administrated to detect the effects of IGF-I on the expression of miR-21 and p-JAK2. JAK2 inhibitor (AG490), and si-STAT3 were used to detect the effects of JAK2/STAT3 signaling pathway on the expression of miR-21. In our study, we firstly showed that the expression levels of IGF-I and miR-21 were up-regulated in RCC tissues and cell lines. After exogenous IGF-I treatment, the expression levels of miR-21, p-IGF-IR and p-JAK2 were significantly increased, whereas NT-157 treatment showed the reversed results. Further study indicated that JAK2 inhibitor or si-STAT3 significantly reversed the IGF-I-induced miR-21 expression level. Finally, we found that IGF-I treatment significantly prompted human RCC cell survival and inhibited cell apoptosis, and NT-157 treatment showed the reversed results. The IGF-I/JAK2-STAT3/miR-21 signaling pathway may be associated with human RCC cell growth.

Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells

PubMed Central

Lee, Jonghyeob; Sugiyama, Takuya; Liu, Yinghua; Wang, Jing; Gu, Xueying; Lei, Ji; Markmann, James F; Miyazaki, Satsuki; Miyazaki, Jun-ichi; Szot, Gregory L; Bottino, Rita; Kim, Seung K

2013-01-01

Pancreatic islet β-cell insufficiency underlies pathogenesis of diabetes mellitus; thus, functional β-cell replacement from renewable sources is the focus of intensive worldwide effort. However, in vitro production of progeny that secrete insulin in response to physiological cues from primary human cells has proven elusive. Here we describe fractionation, expansion and conversion of primary adult human pancreatic ductal cells into progeny resembling native β-cells. FACS-sorted adult human ductal cells clonally expanded as spheres in culture, while retaining ductal characteristics. Expression of the cardinal islet developmental regulators Neurog3, MafA, Pdx1 and Pax6 converted exocrine duct cells into endocrine progeny with hallmark β-cell properties, including the ability to synthesize, process and store insulin, and secrete it in response to glucose or other depolarizing stimuli. These studies provide evidence that genetic reprogramming of expandable human pancreatic cells with defined factors may serve as a general strategy for islet replacement in diabetes. DOI: http://dx.doi.org/10.7554/eLife.00940.001 PMID:24252877

Genetic Causes of Human NK Cell Deficiency and Their Effect on NK Cell Subsets

PubMed Central

Mace, Emily M.; Orange, Jordan S.

2016-01-01

Human NK cells play critical roles in human host defense, particularly the control of viral infection and malignancy, and patients with congenital immunodeficiency affecting NK cell function or number can suffer from severe illness. The importance of NK cell function is particularly underscored in patients with primary immunodeficiency in which NK cells are the primary or sole affected population (NK cell deficiency, NKD). While NKD may lead to the absence of NK cells, we are also gaining an increasing appreciation of the effect that NKD may have on the generation of specific NK cell subsets. In turn, this leads to improved insights into the requirements for human NK cell subset generation, as well as their importance in immune homeostasis. The presence of inherently abnormally developed or functionally impaired NK cells, in particular, appears to be problematic in the way of interfering with normal human host defense and may be more impactful than low numbers of NK cells alone. Here, we review the known genetic causes of NKD and the insight that is derived by these into the requirements for human subset generation and, by extension, for NK cell-mediated immunity. PMID:27994588

Human stem cell decorated nanocellulose threads for biomedical applications.

PubMed

Mertaniemi, Henrikki; Escobedo-Lucea, Carmen; Sanz-Garcia, Andres; Gandía, Carolina; Mäkitie, Antti; Partanen, Jouni; Ikkala, Olli; Yliperttula, Marjo

2016-03-01

Upon surgery, local inflammatory reactions and postoperative infections cause complications, morbidity, and mortality. Delivery of human adipose mesenchymal stem cells (hASC) into the wounds is an efficient and safe means to reduce inflammation and promote wound healing. However, administration of stem cells by injection often results in low cell retention, and the cells deposit in other organs, reducing the efficiency of the therapy. Thus, it is essential to improve cell delivery to the target area using carriers to which the cells have a high affinity. Moreover, the application of hASC in surgery has typically relied on animal-origin components, which may induce immune reactions or even transmit infections due to pathogens. To solve these issues, we first show that native cellulose nanofibers (nanofibrillated cellulose, NFC) extracted from plants allow preparation of glutaraldehyde cross-linked threads (NFC-X) with high mechanical strength even under the wet cell culture or surgery conditions, characteristically challenging for cellulosic materials. Secondly, using a xenogeneic free protocol for isolation and maintenance of hASC, we demonstrate that cells adhere, migrate and proliferate on the NFC-X, even without surface modifiers. Cross-linked threads were not found to induce toxicity on the cells and, importantly, hASC attached on NFC-X maintained their undifferentiated state and preserved their bioactivity. After intradermal suturing with the hASC decorated NFC-X threads in an ex vivo experiment, cells remained attached to the multifilament sutures without displaying morphological changes or reducing their metabolic activity. Finally, as NFC-X optionally allows facile surface tailoring if needed, we anticipate that stem-cell-decorated NFC-X opens a versatile generic platform as a surgical bionanomaterial for fighting postoperative inflammation and chronic wound healing problems. Copyright © 2015 Elsevier Ltd. All rights reserved.

Chronic cadmium exposure in vitro induces cancer cell characteristics in human lung cells

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

Person, Rachel J.; Tokar, Erik J.; Xu, Yuanyuan

Cadmium is a known human lung carcinogen. Here, we attempt to develop an in vitro model of cadmium-induced human lung carcinogenesis by chronically exposing the peripheral lung epithelia cell line, HPL-1D, to a low level of cadmium. Cells were chronically exposed to 5 μM cadmium, a noncytotoxic level, and monitored for acquired cancer characteristics. By 20 weeks of continuous cadmium exposure, these chronic cadmium treated lung (CCT-LC) cells showed marked increases in secreted MMP-2 activity (3.5-fold), invasion (3.4-fold), and colony formation in soft agar (2-fold). CCT-LC cells were hyperproliferative, grew well in serum-free media, and overexpressed cyclin D1. The CCT-LCmore » cells also showed decreased expression of the tumor suppressor genes p16 and SLC38A3 at the protein levels. Also consistent with an acquired cancer cell phenotype, CCT-LC cells showed increased expression of the oncoproteins K-RAS and N-RAS as well as the epithelial-to-mesenchymal transition marker protein Vimentin. Metallothionein (MT) expression is increased by cadmium, and is typically overexpressed in human lung cancers. The major MT isoforms, MT-1A and MT-2A were elevated in CCT-LC cells. Oxidant adaptive response genes HO-1 and HIF-1A were also activated in CCT-LC cells. Expression of the metal transport genes ZNT-1, ZNT-5, and ZIP-8 increased in CCT-LC cells culminating in reduced cadmium accumulation, suggesting adaptation to the metal. Overall, these data suggest that exposure of human lung epithelial cells to cadmium causes acquisition of cancer cell characteristics. Furthermore, transformation occurs despite the cell's ability to adapt to chronic cadmium exposure. - Highlights: • Chronic cadmium exposure induces cancer cell characteristics in human lung cells. • This provides an in vitro model of cadmium-induced human lung cell transformation. • This occurred with general and lung specific changes typical for cancer cells. • These findings add insight to the

Generating trunk neural crest from human pluripotent stem cells

PubMed Central

Huang, Miller; Miller, Matthew L.; McHenry, Lauren K.; Zheng, Tina; Zhen, Qiqi; Ilkhanizadeh, Shirin; Conklin, Bruce R.; Bronner, Marianne E.; Weiss, William A.

2016-01-01

Neural crest cells (NCC) are stem cells that generate different lineages, including neuroendocrine, melanocytic, cartilage, and bone. The differentiation potential of NCC varies according to the level from which cells emerge along the neural tube. For example, only anterior “cranial” NCC form craniofacial bone, whereas solely posterior “trunk” NCC contribute to sympathoadrenal cells. Importantly, the isolation of human fetal NCC carries ethical and scientific challenges, as NCC induction typically occur before pregnancy is detectable. As a result, current knowledge of NCC biology derives primarily from non-human organisms. Important differences between human and non-human NCC, such as expression of HNK1 in human but not mouse NCC, suggest a need to study human NCC directly. Here, we demonstrate that current protocols to differentiate human pluripotent stem cells (PSC) to NCC are biased toward cranial NCC. Addition of retinoic acid drove trunk-related markers and HOX genes characteristic of a posterior identity. Subsequent treatment with bone morphogenetic proteins (BMPs) enhanced differentiation to sympathoadrenal cells. Our approach provides methodology for detailed studies of human NCC, and clarifies roles for retinoids and BMPs in the differentiation of human PSC to trunk NCC and to sympathoadrenal lineages. PMID:26812940

Activated glycoprotein A repetitions predominant (GARP)-expressing regulatory T cells inhibit allergen-induced intestinal inflammation in humanized mice.

PubMed

Eschborn, Melanie; Weigmann, Benno; Reissig, Sonja; Waisman, Ari; Saloga, Joachim; Bellinghausen, Iris

2015-07-01

Recently, we developed a humanized mouse model of allergen-induced IgE-dependent gut inflammation in PBMC-engrafted immunodeficient mice. In the present study, we wanted to investigate the role of regulatory T (Treg) cells and their activation status in this model. Nonobese diabetic-severe combined immunodeficiency-γc(-/-) mice were injected intraperitoneally with human PBMCs from allergic donors together with the respective allergen or NaCl as control in the presence or absence of different concentrations of CD4(+)CD25(+) Treg cells of the same donor. After an additional allergen boost 1 week later, mice were challenged with the allergen rectally on day 21 and gut inflammation was monitored by a high-resolution video mini-endoscopic system evaluating translucency, granularity, fibrin production, vascularity, and stool. Allergen-specific human IgE in mouse sera, which was detectable only in PBMC plus allergen-treated mice, was strongly inhibited by coinjection of Treg cells at a ratio of at least 1:10. Consequently, the presence of Treg cells significantly decreased IgE-dependent allergen-induced gut inflammation after rectal allergen challenge. In addition, Treg cells reduced allergen-specific proliferation and cytokine production of recovered human CD4(+) T cells in vitro. Activation of Treg cells before injection further increased all inhibitory effects. Prevention of gut inflammation also occurred by the administration of glycoprotein A repetitions predominant, a molecule expressed by activated Treg cells, whereas its blockade completely abrogated inhibition by Treg cells. These results demonstrate that allergen-specific gut inflammation in human PBMC-engrafted mice can be avoided by enhancing the numbers or activity of autologous Treg cells, which is of great interest for therapeutic intervention of allergic diseases of the intestine. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

New insights into human primordial germ cells and early embryonic development from single-cell analysis.

PubMed

Otte, Jörg; Wruck, Wasco; Adjaye, James

2017-08-01

Human preimplantation developmental studies are difficult to accomplish due to associated ethical and moral issues. Preimplantation cells are rare and exist only in transient cell states. From a single cell, it is very challenging to analyse the origination of the heterogeneity and complexity inherent to the human body. However, recent advances in single-cell technology and data analysis have provided new insights into the process of early human development and germ cell specification. In this Review, we examine the latest single-cell datasets of human preimplantation embryos and germ cell development, compare them to bulk cell analyses, and interpret their biological implications. © 2017 Federation of European Biochemical Societies.

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

PubMed Central

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

2013-01-01

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

Primary Cell Culture of Live Neurosurgically Resected Aged Adult Human Brain Cells and Single Cell Transcriptomics.

PubMed

Spaethling, Jennifer M; Na, Young-Ji; Lee, Jaehee; Ulyanova, Alexandra V; Baltuch, Gordon H; Bell, Thomas J; Brem, Steven; Chen, H Isaac; Dueck, Hannah; Fisher, Stephen A; Garcia, Marcela P; Khaladkar, Mugdha; Kung, David K; Lucas, Timothy H; O'Rourke, Donald M; Stefanik, Derek; Wang, Jinhui; Wolf, John A; Bartfai, Tamas; Grady, M Sean; Sul, Jai-Yoon; Kim, Junhyong; Eberwine, James H

2017-01-17

Investigation of human CNS disease and drug effects has been hampered by the lack of a system that enables single-cell analysis of live adult patient brain cells. We developed a culturing system, based on a papain-aided procedure, for resected adult human brain tissue removed during neurosurgery. We performed single-cell transcriptomics on over 300 cells, permitting identification of oligodendrocytes, microglia, neurons, endothelial cells, and astrocytes after 3 weeks in culture. Using deep sequencing, we detected over 12,000 expressed genes, including hundreds of cell-type-enriched mRNAs, lncRNAs and pri-miRNAs. We describe cell-type- and patient-specific transcriptional hierarchies. Single-cell transcriptomics on cultured live adult patient derived cells is a prime example of the promise of personalized precision medicine. Because these cells derive from subjects ranging in age into their sixties, this system permits human aging studies previously possible only in rodent systems. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Comparison of the Gene Expression Profiles of Human Hematopoietic Stem Cells between Humans and a Humanized Xenograft Model.

PubMed

Matsuzawa, Hideyuki; Matsushita, Hiromichi; Yahata, Takashi; Tanaka, Masayuki; Ando, Kiyoshi

2017-04-20

The aim of this study is to evaluate the feasibility of NOD/Shi-scid-IL2Rγ null (NOG) mice transplanted with human CD34 + /CD38 - /Lin -/low hematopoietic cells from cord blood (CB) as an experimental model of the gene expression in human hematopoiesis. We compared the gene expressions of human CD34 + /CD38 - /Lin -/low cells from human bone marrow (BM) and in xenograft models. The microarray data revealed that 25 KEGG pathways were extracted from the comparison of human CD34 + /CD38 - /Lin -/low HSCs between CB and BM, and that 17 of them--which were mostly related to cellular survival, RNA metabolism and lymphoid development--were shared with the xenograft model. When the probes that were commonly altered in CD34 + /CD38 - /Lin -/low cells from both human and xenograft BM were analyzed, most of them, including the genes related hypoxia, hematopoietic differentiation, epigenetic modification, translation initiation, and RNA degradation, were downregulated. These alterations of gene expression suggest a reduced differentiation capacity and likely include key alterations of gene expression for settlement of CB CD34 + /CD38 - /Lin -/low cells in BM. Our findings demonstrate that the xenograft model of human CB CD34 + /CD38 - /Lin -/low cells using NOG mice was useful, at least in part, for the evaluation of the gene expression profile of human hematopoietic stem cells.

HMGB1-mediated autophagy decreases sensitivity to oxymatrine in SW982 human synovial sarcoma cells

PubMed Central

Cai, Yongsong; Xu, Peng; Yang, Le; Xu, Ke; Zhu, Jialin; Wu, Xiaoqing; Jiang, Congshan; Yuan, Qiling; Wang, Bo; Li, Yuanbo; Qiu, Yusheng

2016-01-01

Oxymatrine (OMT) is a type of alkaloid extracted from a traditional Chinese medicinal herb, Sophora flavescens. Although the antitumor activities of OMT have been observed in various cancers, there are no reports regarding the effects of OMT on human synovial sarcoma. In the present study, we analyzed the antitumor activities of OMT in SW982 human synovial sarcoma cells and determine whether high mobility group box protein 1 (HMGB1)-mediated autophagy was associated with its therapeutic effects. We found that OMT exhibited antitumor activity in SW982 cells and facilitated increases in autophagy. Inhibition of autophagy by 3-MA or ATG7 siRNA increased the level of apoptosis, which indicated that OMT-induced autophagy protected cells from the cytotoxicity of OMT. Administration of OMT to SW982 cells increased the expression of HMGB1. When HMGB1 was inhibited via HMGB1-siRNA, OMT-induced autophagy was decreased, and apoptosis was increased. Furthermore, we found that HMGB1-siRNA significantly increased the expression of p-Akt and p-mTOR. OMT-induced autophagy may be mediated by the Akt/mTOR pathway, and HMGB1 plays a vital role in the regulation of autophagy. Therefore, we believe that combining OMT with an inhibitor of autophagy or HMGB1 may make OMT more effective in the treatment of human synovial sarcoma. PMID:27897164

Tuft (caveolated) cells in two human colon carcinoma cell lines.

PubMed

Barkla, D H; Whitehead, R H; Foster, H; Tutton, P J

1988-09-01

The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting from the apical surface. The microvilli are attached by a core of long microfilaments passing deep into the apical cytoplasm. Between the microvilli are parallel arrays of vesicles (caveoli) containing flocculent material. Two different but not mutually exclusive explanations for the presence of tuft cells are proposed. The first explanation is that tuft cells came from the resected tumour and have survived by mitotic division during subsequent passages. The second explanation suggests that tuft cells are the progeny of undifferentiated tumour cells. Descriptions of tuft cells in colon carcinomas are uncommon and possible reasons for this are presented. The morphology of tuft cells is consistent with that of a highly differentiated cell specialised for absorption, and these new models provide an opportunity to further investigate the structure and function of tuft cells.

Modeling Human Natural Killer Cell Development in the Era of Innate Lymphoid Cells

PubMed Central

Scoville, Steven D.; Freud, Aharon G.; Caligiuri, Michael A.

2017-01-01

Decades after the discovery of natural killer (NK) cells, their developmental pathways in mice and humans have not yet been completely deciphered. Accumulating evidence indicates that NK cells can develop in multiple tissues throughout the body. Moreover, detailed and comprehensive models of NK cell development were proposed soon after the turn of the century. However, with the recent identification and characterization of other subtypes of innate lymphoid cells (ILCs), which show some overlapping functional and phenotypic features with NK cell developmental intermediates, the distinct stages through which human NK cells develop from early hematopoietic progenitor cells remain unclear. Thus, there is a need to reassess and refine older models of NK cell development in the context of new data and in the era of ILCs. Our group has focused on elucidating the developmental pathway of human NK cells in secondary lymphoid tissues (SLTs), including tonsils and lymph nodes. Here, we provide an update of recent progress that has been made with regard to human NK cell development in SLTs, and we discuss these new findings in the context of contemporary models of ILC development. PMID:28396671

Modeling Human Natural Killer Cell Development in the Era of Innate Lymphoid Cells.

PubMed

Scoville, Steven D; Freud, Aharon G; Caligiuri, Michael A

2017-01-01

Decades after the discovery of natural killer (NK) cells, their developmental pathways in mice and humans have not yet been completely deciphered. Accumulating evidence indicates that NK cells can develop in multiple tissues throughout the body. Moreover, detailed and comprehensive models of NK cell development were proposed soon after the turn of the century. However, with the recent identification and characterization of other subtypes of innate lymphoid cells (ILCs), which show some overlapping functional and phenotypic features with NK cell developmental intermediates, the distinct stages through which human NK cells develop from early hematopoietic progenitor cells remain unclear. Thus, there is a need to reassess and refine older models of NK cell development in the context of new data and in the era of ILCs. Our group has focused on elucidating the developmental pathway of human NK cells in secondary lymphoid tissues (SLTs), including tonsils and lymph nodes. Here, we provide an update of recent progress that has been made with regard to human NK cell development in SLTs, and we discuss these new findings in the context of contemporary models of ILC development.

In situ induction of dendritic cell–based T cell tolerance in humanized mice and nonhuman primates

PubMed Central

Jung, Kyeong Cheon; Jeon, Yoon Kyung; Ban, Young Larn; Min, Hye Sook; Kim, Eun Ji; Kim, Ju Hyun; Kang, Byung Hyun; Bae, Youngmee; Yoon, Il-Hee; Kim, Yong-Hee; Lee, Jae-Il; Kim, Jung-Sik; Shin, Jun-Seop; Yang, Jaeseok; Kim, Sung Joo; Rostlund, Emily; Muller, William A.

2011-01-01

Induction of antigen-specific T cell tolerance would aid treatment of diverse immunological disorders and help prevent allograft rejection and graft versus host disease. In this study, we establish a method of inducing antigen-specific T cell tolerance in situ in diabetic humanized mice and Rhesus monkeys receiving porcine islet xenografts. Antigen-specific T cell tolerance is induced by administration of an antibody ligating a particular epitope on ICAM-1 (intercellular adhesion molecule 1). Antibody-mediated ligation of ICAM-1 on dendritic cells (DCs) led to the arrest of DCs in a semimature stage in vitro and in vivo. Ablation of DCs from mice completely abrogated anti–ICAM-1–induced antigen-specific T cell tolerance. T cell responses to unrelated antigens remained unaffected. In situ induction of DC-mediated T cell tolerance using this method may represent a potent therapeutic tool for preventing graft rejection. PMID:22025302

Preparative electrophoresis of cultured human cells: Effect of cell cycle phase

NASA Technical Reports Server (NTRS)

Kunze, M. E.; Todd, P. W.; Goolsby, C. L.; Walker, J. T.

1985-01-01

Human epithelioid T-1E cells were cultured in suspension and subjected to density gradient electrophoresis upward in a vertical column. It is indicated that the most rapidly migrating cells were at the beginning of the cell cycle and the most slowly migrating cells were at the end of the cell cycle. The fastest migrating cells divided 24 hr later than the slowest migrating cells. Colonies developing from slowly migrating cells had twice as many cells during exponential growth as did the most rapidly migrating cells, and the numbers of cells per colony at any time was inversely related to the electrophoretic migration rate. The DNA measurements by fluorescence flow cytometry indicates that the slowest migrating cell populations are enriched in cells that have twice as much DNA as the fastest migrating cells. It is concluded that electrophoretic mobility of these cultured human cells declines steadily through the cell cycle and that the mobility is lowest at the end of G sub 2 phase and highest at the beginning of G sub 1 phase.

Coenzyme Q10 Protects Human Endothelial Cells from β-Amyloid Uptake and Oxidative Stress-Induced Injury

PubMed Central

Durán-Prado, Mario; Frontiñán, Javier; Santiago-Mora, Raquel; Peinado, Juan Ramón; Parrado-Fernández, Cristina; Gómez-Almagro, María Victoria; Moreno, María; López-Domínguez, José Alberto; Villalba, José Manuel; Alcaín, Francisco J.

2014-01-01

Neuropathological symptoms of Alzheimer's disease appear in advances stages, once neuronal damage arises. Nevertheless, recent studies demonstrate that in early asymptomatic stages, ß-amyloid peptide damages the cerebral microvasculature through mechanisms that involve an increase in reactive oxygen species and calcium, which induces necrosis and apoptosis of endothelial cells, leading to cerebrovascular dysfunction. The goal of our work is to study the potential preventive effect of the lipophilic antioxidant coenzyme Q (CoQ) against ß-amyloid-induced damage on human endothelial cells. We analyzed the protective effect of CoQ against Aβ-induced injury in human umbilical vein endothelial cells (HUVECs) using fluorescence and confocal microscopy, biochemical techniques and RMN-based metabolomics. Our results show that CoQ pretreatment of HUVECs delayed Aβ incorporation into the plasma membrane and mitochondria. Moreover, CoQ reduced the influx of extracellular Ca2+, and Ca2+ release from mitochondria due to opening the mitochondrial transition pore after β-amyloid administration, in addition to decreasing O2 .− and H2O2 levels. Pretreatment with CoQ also prevented ß-amyloid-induced HUVECs necrosis and apoptosis, restored their ability to proliferate, migrate and form tube-like structures in vitro, which is mirrored by a restoration of the cell metabolic profile to control levels. CoQ protected endothelial cells from Aβ-induced injury at physiological concentrations in human plasma after oral CoQ supplementation and thus could be a promising molecule to protect endothelial cells against amyloid angiopathy. PMID:25272163

Generation of functional hepatocytes from human spermatogonial stem cells.

PubMed

Chen, Zheng; Sun, Min; Yuan, Qingqing; Niu, Minghui; Yao, Chencheng; Hou, Jingmei; Wang, Hong; Wen, Liping; Liu, Yun; Li, Zheng; He, Zuping

2016-02-23

To generate functional human hepatocytes from stem cells and/or extra-hepatic tissues could provide an important source of cells for treating liver diseases. Spermatogonial stem cells (SSCs) have an unlimited plasticity since they can dedifferentiate and transdifferentiate to other cell lineages. However, generation of mature and functional hepatocytes from human SSCs has not yet been achieved. Here we have for the first time reported direct transdifferentiation of human SSCs to mature and functional hepatocytes by three-step induction using the defined condition medium. Human SSCs were first transdifferentiated to hepatic stem cells, as evidenced by their morphology and biopotential nature of co-expressing hepatocyte and cholangiocyte markers but not hallmarks for embryonic stem cells. Hepatic stem cells were further induced to differentiate into mature hepatocytes identified by their morphological traits and strong expression of CK8, CK18, ALB, AAT, TF, TAT, and cytochrome enzymes rather than CK7 or CK19. Significantly, mature hepatocytes derived from human SSCs assumed functional attributes of human hepatocytes, because they could produce albumin, remove ammonia, and uptake and release indocyanine green. Moreover, expression of β-CATENIN, HNF4A, FOXA1 and GATA4 was upregulated during the transdifferentiation of human SSCs to mature hepatocytes. Collectively, human SSCs could directly transdifferentiate to mature and functional hepatocytes. This study could offer an invaluable source of human hepatocytes for curing liver disorders and drug toxicology screening and provide novel insights into mechanisms underlying human liver regeneration.

Generation of functional hepatocytes from human spermatogonial stem cells

PubMed Central

Chen, Zheng; Sun, Min; Yuan, Qingqing; Niu, Minghui; Yao, Chencheng; Hou, Jingmei; Wang, Hong; Wen, Liping; Liu, Yun; Li, Zheng; He, Zuping

2016-01-01

To generate functional human hepatocytes from stem cells and/or extra-hepatic tissues could provide an important source of cells for treating liver diseases. Spermatogonial stem cells (SSCs) have an unlimited plasticity since they can dedifferentiate and transdifferentiate to other cell lineages. However, generation of mature and functional hepatocytes from human SSCs has not yet been achieved. Here we have for the first time reported direct transdifferentiation of human SSCs to mature and functional hepatocytes by three-step induction using the defined condition medium. Human SSCs were first transdifferentiated to hepatic stem cells, as evidenced by their morphology and biopotential nature of co-expressing hepatocyte and cholangiocyte markers but not hallmarks for embryonic stem cells. Hepatic stem cells were further induced to differentiate into mature hepatocytes identified by their morphological traits and strong expression of CK8, CK18, ALB, AAT, TF, TAT, and cytochrome enzymes rather than CK7 or CK19. Significantly, mature hepatocytes derived from human SSCs assumed functional attributes of human hepatocytes, because they could produce albumin, remove ammonia, and uptake and release indocyanine green. Moreover, expression of β-CATENIN, HNF4A, FOXA1 and GATA4 was upregulated during the transdifferentiation of human SSCs to mature hepatocytes. Collectively, human SSCs could directly transdifferentiate to mature and functional hepatocytes. This study could offer an invaluable source of human hepatocytes for curing liver disorders and drug toxicology screening and provide novel insights into mechanisms underlying human liver regeneration. PMID:26840458

Bacoside A Induces Tumor Cell Death in Human Glioblastoma Cell Lines through Catastrophic Macropinocytosis

PubMed Central

John, Sebastian; Sivakumar, K. C.; Mishra, Rashmi

2017-01-01

Glioblastoma multiforme (GBM) is a highly aggressive type of brain tumor with an extremely poor prognosis. Recent evidences have shown that the “biomechanical imbalances” induced in GBM patient-derived glioblastoma cells (GC) and in vivo via the administration of synthetic small molecules, may effectively inhibit disease progression and prolong survival of GBM animal models. This novel concept associated with de novo anti-GBM drug development has however suffered obstacles in adequate clinical utility due to the appearance of unrelated toxicity in the prolonged therapeutic windows. Here, we took a “drug repurposing approach” to trigger similar physico-chemical disturbances in the GBM tumor cells, wherein, the candidate therapeutic agent has been previously well established for its neuro-protective roles, safety, efficacy, prolonged tolerance and excellent brain bioavailability in human subjects and mouse models. In this study, we show that the extracts of an Indian traditional medicinal plant Bacopa monnieri (BM) and its bioactive component Bacoside A can generate dosage associated tumor specific disturbances in the hydrostatic pressure balance of the cell via a mechanism involving excessive phosphorylation of calcium/calmodulin-dependent protein kinase IIA (CaMKIIA/CaMK2A) enzyme that is further involved in the release of calcium from the smooth endoplasmic reticular networks. High intracellular calcium stimulated massive macropinocytotic extracellular fluid intake causing cell hypertrophy in the initial stages, excessive macropinosome enlargement and fluid accumulation associated organellar congestion, cell swelling, cell rounding and membrane rupture of glioblastoma cells; with all these events culminating into a non-apoptotic, physical non-homeostasis associated glioblastoma tumor cell death. These results identify glioblastoma tumor cells to be a specific target of the tested herbal medicine and therefore can be exploited as a safe anti-GBM therapeutic

Bacoside A Induces Tumor Cell Death in Human Glioblastoma Cell Lines through Catastrophic Macropinocytosis.

PubMed

John, Sebastian; Sivakumar, K C; Mishra, Rashmi

2017-01-01

Glioblastoma multiforme (GBM) is a highly aggressive type of brain tumor with an extremely poor prognosis. Recent evidences have shown that the "biomechanical imbalances" induced in GBM patient-derived glioblastoma cells (GC) and in vivo via the administration of synthetic small molecules, may effectively inhibit disease progression and prolong survival of GBM animal models. This novel concept associated with de novo anti-GBM drug development has however suffered obstacles in adequate clinical utility due to the appearance of unrelated toxicity in the prolonged therapeutic windows. Here, we took a "drug repurposing approach" to trigger similar physico-chemical disturbances in the GBM tumor cells, wherein, the candidate therapeutic agent has been previously well established for its neuro-protective roles, safety, efficacy, prolonged tolerance and excellent brain bioavailability in human subjects and mouse models. In this study, we show that the extracts of an Indian traditional medicinal plant Bacopa monnieri (BM) and its bioactive component Bacoside A can generate dosage associated tumor specific disturbances in the hydrostatic pressure balance of the cell via a mechanism involving excessive phosphorylation of calcium/calmodulin-dependent protein kinase IIA (CaMKIIA/CaMK2A) enzyme that is further involved in the release of calcium from the smooth endoplasmic reticular networks. High intracellular calcium stimulated massive macropinocytotic extracellular fluid intake causing cell hypertrophy in the initial stages, excessive macropinosome enlargement and fluid accumulation associated organellar congestion, cell swelling, cell rounding and membrane rupture of glioblastoma cells; with all these events culminating into a non-apoptotic, physical non-homeostasis associated glioblastoma tumor cell death. These results identify glioblastoma tumor cells to be a specific target of the tested herbal medicine and therefore can be exploited as a safe anti-GBM therapeutic.

Characterization of human-induced pluripotent stem cell-derived cardiomyocytes: bioenergetics and utilization in safety screening.

PubMed

Rana, Payal; Anson, Blake; Engle, Sandra; Will, Yvonne

2012-11-01

Cardiotoxicity remains the number one reason for drug withdrawal from the market, and Food and Drug Administration issued black box warnings, thus demonstrating the need for more predictive preclinical safety screening, especially early in the drug discovery process when much chemical substrate is available. Whereas human-ether-a-go-go related gene screening has become routine to mitigate proarrhythmic risk, the development of in vitro assays predicting additional on- and off-target biochemical toxicities will benefit from cellular models exhibiting true cardiomyocyte characteristics such as native tissue-like mitochondrial activity. Human stem cell-derived tissue cells may provide such a model. This hypothesis was tested using a combination of flux analysis, gene and protein expression, and toxicity-profiling techniques to characterize mitochondrial function in induced pluripotent stem cell (iPSC) derived human cardiomyocytes in the presence of differing carbon sources over extended periods in cell culture. Functional analyses demonstrate that iPSC-derived cardiomyocytes are (1) capable of utilizing anaerobic or aerobic respiration depending upon the available carbon substrate and (2) bioenergetically closest to adult heart tissue cells when cultured in galactose or galactose supplemented with fatty acids. We utilized this model to test a variety of kinase inhibitors with known clinical cardiac liabilities for their potential toxicity toward these cells. We found that the kinase inhibitors showed a dose-dependent toxicity to iPSC cardiomyocytes grown in galactose and that oxygen consumption rates were significantly more affected than adenosine triphosphate production. Sorafenib was found to have the most effect, followed by sunitinib, dasatinib, imatinib, lapatinib, and nioltinib.

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

PubMed

Takayama, Kazuo; Mizuguchi, Hiroyuki

2017-02-01

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

Human spleen and red blood cells

NASA Astrophysics Data System (ADS)

Pivkin, Igor; Peng, Zhangli; Karniadakis, George; Buffet, Pierre; Dao, Ming

2016-11-01

Spleen plays multiple roles in the human body. Among them is removal of old and altered red blood cells (RBCs), which is done by filtering cells through the endothelial slits, small micron-sized openings. There is currently no experimental technique available that allows us to observe RBC passage through the slits. It was previously noticed that people without a spleen have less deformable red blood cells, indicating that the spleen may play a role in defining the size and shape of red blood cells. We used detailed RBC model implemented within the Dissipative Particle Dynamics (DPD) simulation framework to study the filter function of the spleen. Our results demonstrate that spleen indeed plays major role in defining the size and shape of the healthy human red blood cells.

Formononetin suppresses the proliferation of human non-small cell lung cancer through induction of cell cycle arrest and apoptosis

PubMed Central

Yang, Yi; Zhao, Yi; Ai, Xinghao; Cheng, Baijun; Lu, Shun

2014-01-01

Formononetin is a novel herbal isoflavonoid isolated from Astragalus membranaceus and possesses antitumorigenic properties. In the present study, we investigated the anti-proliferative effects of formononetin on human non-small cell lung cancer (NSCLC), and further elucidated the molecular mechanism underlying the anti-tumor property. MTT assay showed that formononetin treatment significantly inhibited the proliferation of two NSCLC cell lines including A549 and NCI-H23 in a time- and dose-dependent manner. Flow cytometric analysis demonstrated that formononetin induced G1-phase cell cycle arrest and promoted cell apoptosis in NSCLC cells. On the molecular level, we observed that exposure to formononetin altered the expression levels of cell cycle arrest-associated proteins p21, cyclin A and cyclin D1. Meanwhile, the apoptosis-related proteins cleaved caspase-3, bax and bcl-2 were also changed following treatment with formononetin. In addition, the expression level of p53 was dose-dependently upregulated after administration with formononetin. We also found that formononetin treatment increased the phosphorylation of p53 at Ser15 and Ser20 and enhances its transcriptional activity in a dose-dependent manner. Collectively, these results demonstrated that formononetin might be a potential chemopreventive drug for lung cancer therapy through induction of cell cycle arrest and apoptosis in NSCLC cells. PMID:25674209

Formononetin suppresses the proliferation of human non-small cell lung cancer through induction of cell cycle arrest and apoptosis.

PubMed

Yang, Yi; Zhao, Yi; Ai, Xinghao; Cheng, Baijun; Lu, Shun

2014-01-01

Formononetin is a novel herbal isoflavonoid isolated from Astragalus membranaceus and possesses antitumorigenic properties. In the present study, we investigated the anti-proliferative effects of formononetin on human non-small cell lung cancer (NSCLC), and further elucidated the molecular mechanism underlying the anti-tumor property. MTT assay showed that formononetin treatment significantly inhibited the proliferation of two NSCLC cell lines including A549 and NCI-H23 in a time- and dose-dependent manner. Flow cytometric analysis demonstrated that formononetin induced G1-phase cell cycle arrest and promoted cell apoptosis in NSCLC cells. On the molecular level, we observed that exposure to formononetin altered the expression levels of cell cycle arrest-associated proteins p21, cyclin A and cyclin D1. Meanwhile, the apoptosis-related proteins cleaved caspase-3, bax and bcl-2 were also changed following treatment with formononetin. In addition, the expression level of p53 was dose-dependently upregulated after administration with formononetin. We also found that formononetin treatment increased the phosphorylation of p53 at Ser15 and Ser20 and enhances its transcriptional activity in a dose-dependent manner. Collectively, these results demonstrated that formononetin might be a potential chemopreventive drug for lung cancer therapy through induction of cell cycle arrest and apoptosis in NSCLC cells.

Symmetry breaking in human neuroblastoma cells

PubMed Central

Izumi, Hideki; Kaneko, Yasuhiko

2014-01-01

Asymmetric cell division (ACD) is a characteristic of cancer stem cells, which exhibit high malignant potential. However, the cellular mechanisms that regulate symmetric (self-renewal) and asymmetric cell divisions are mostly unknown. Using human neuroblastoma cells, we found that the oncosuppressor protein tripartite motif containing 32 (TRIM32) positively regulates ACD. PMID:27308367

Comparison of intracellular accumulation and cytotoxicity of free mTHPC and mTHPC-loaded PLGA nanoparticles in human colon carcinoma cells.

PubMed

Löw, Karin; Knobloch, Thomas; Wagner, Sylvia; Wiehe, Arno; Engel, Andrea; Langer, Klaus; von Briesen, Hagen

2011-06-17

The second generation photosensitizer mTHPC was approved by the European Medicines Agency (EMA) for the palliative treatment of advanced head and neck cancer in October 2001. It is known that mTHPC possesses a significant phototoxicity against a variety of human cancer cells in vitro but also exhibits dark toxicity and can cause adverse effects (especially skin photosensitization). Due to its poor water solubility, the administration of hydrophobic photosensitizer still presents several difficulties. To overcome the administration problems, the use of nanoparticles as drug carrier systems is much investigated. Nanoparticles based on poly(lactic-co-glycolic acid) (PLGA) have been extensively studied as delivery systems into tumours due to their biocompatibility and biodegradability. The goal of this study was the comparison of free mTHPC and mTHPC-loaded PLGA nanoparticles concerning cytotoxicity and intracellular accumulation in human colon carcinoma cells (HT29). The nanoparticles delivered the photosensitizer to the colon carcinoma cells and enabled drug release without losing its activity. The cytotoxicity assays showed a time- and concentration-dependent decrease in cell proliferation and viability after illumination. However, first and foremost mTHPC lost its dark toxic effects using the PLGA nanoparticles as a drug carrier system. Therefore, PLGA nanoparticles are a promising drug carrier system for the hydrophobic photosensitizer mTHPC.

Magnetic Resonance Imaging of Ferumoxytol-Labeled Human Mesenchymal Stem Cells in the Mouse Brain.

PubMed

Lee, Na Kyung; Kim, Hyeong Seop; Yoo, Dongkyeom; Hwang, Jung Won; Choi, Soo Jin; Oh, Wonil; Chang, Jong Wook; Na, Duk L

2017-02-01

The success of stem cell therapy is highly dependent on accurate delivery of stem cells to the target site of interest. Possible ways to track the distribution of MSCs in vivo include the use of reporter genes or nanoparticles. The U.S. Food and Drug Administration (FDA) has approved ferumoxytol (Feraheme® [USA], Rienso® [UK]) as a treatment for iron deficiency anemia. Ferumoxytol is an ultrasmall superparamagnetic iron oxide nanoparticle (USPIO) that has recently been used to track the fate of transplanted cells using magnetic resonance imaging (MRI). The major objectives of this study were to demonstrate the feasibility of labeling hUCB-MSCs with ferumoxytol and to observe, through MRI, the engraftment of ferumoxytol-labeled human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) delivered via stereotactic injection into the hippocampi of a transgenic mouse model of familial Alzheimer's disease (5XFAD). Ferumoxytol had no toxic effects on the viability or stemness of hUCB-MSCs when assessed in vitro. Through MRI, hypointense signals were discernible at the site where ferumoxytol-labeled human MSCs were injected. Iron-positive areas were also observed in the engrafted hippocampi. The results from this study support the use of nanoparticle labeling to monitor transplanted MSCs in real time as a follow-up for AD stem cell therapy in the clinical field.

Reporter gene expression in dendritic cells after gene gun administration of plasmid DNA.

PubMed

Watkins, Craig; Hopkins, John; Harkiss, Gordon

2005-07-21

Dendritic cells (DC) play an integral role in plasmid DNA vaccination. However, the interaction between plasmid DNA and DC in vivo is incompletely understood. In this report, we utilise the sheep pseudoafferent cannulation model to examine the interaction between plasmid DNA encoding enhanced green fluorescent protein (pEGFP) and afferent lymph DC (ALDC) following gene gun administration. The results show that peaks of fluorescent ALDC tended to appear around days 1-4 and 9-13, then erratically thereafter for up to 2 months. Phenotypic analysis showed that EGFP+ ALDC expressed MHC class II, WC6, CD1b, and SIRPalpha markers. Plasmid, detected by PCR, was found in lymph cells and cell-free plasma on a daily basis, and was present variably for up to 2 months. Plasmid was also detected in purified CD1b+ ALDC, but the presence of plasmid did not correlate with EGFP expression by ALDC. Free EGFP in afferent lymph plasma was detectable by luminometry only after three administrations of the plasmid. The results show that gene gun administered pEGFP persisted for extended periods after a single administration, leeching out of skin on a daily basis. The plasmid was associated with both the cellular and fluid components of afferent lymph. EGFP protein appeared in afferent lymph in a pulsatile manner, but associated only with ALDC.

The pharmacodynamics of thymomodulin in elderly humans.

PubMed

Calsini, P; Mocchegiani, E; Fabris, N

1985-01-01

It has been shown that, after oral administration of thymomodulin (Leucotrofina, Ellem Industria Farmaceutica SpA, Milan, Italy) nude mice show in their sera inductive activity on null cells, as measured by the Thy 1.2 antigen bioassay. The appearance of thymic hormone-like activity, as measured by the rosette inhibition assay, was investigated after oral administration of thymomodulin in elderly humans, who show no detectable levels of FTS ("facteur thymique sérique") in their serum. In human subjects over 70 years old, thymomodulin administration induced the appearance of FTS-like activity which reached maximum level at 2-6 h, was maintained for up to 12 h and disappeared by the 48th hour. By using different thymomodulin doses (80, 160, 320, 640, 800 mg) in a single administration, a dose-dependent effect appeared to exist: the higher the dose, the longer the maintenance of FTS levels. These data suggest that oral administration of thymomodulin induces, in humans, the appearance in the serum of substances with a modulating effect on the maturation of T-cells, and that the intestinal absorption of thymomodulin is efficient also in advanced age.

Effects of human interleukin-18 and interleukin-12 treatment on human lymphocyte engraftment in NOD-scid mouse

PubMed Central

Senpuku, Hidenobu; Asano, Toshihiko; Matin, Khairul; Salam, M Abdus; Tsuha, Yuzo; Horibata, Shigeo; Shimazu, Yoshihito; Soeno, Yuichi; Aoba, Takaaki; Sata, Tetsutaro; Hanada, Nobuhiro; Honda, Mitsuo

2002-01-01

NOD/LtSz-prkdcscid/prkdcscid (non-obese diabetic-severe combine immunodeficiency; NOD-scid) mice grafted with human peripheral blood lymphoid cells have been used as an in vivo humanized mouse model in various studies. However, cytotoxic human T cells are induced in this model during immune responses, which gives misleading results. To assist in grafting of human lymphocytes without the induction of cytotoxic human T cells, we investigated the effects of T helper type 1 (Th1) and Th2 cytokines on human lymphocyte grafting and migration, as well as the production of immunoglobulin deposited in glomeruli and human immunodeficiency virus-1 (HIV-1) infection using NOD-scid mice. Administration of interleukin-18 (IL-18) and IL-12 enhanced the grafting of human CD4+ and CD8+ T cells in the mice, whereas co-administration prevented grafting due to interferon-γ-dependent apoptosis. Immunoglobulin A (IgA) deposits were observed in mice treated with IL-18 alone, but not in those given phosphate-buffered saline, IL-12 alone, or IL-18 + IL-12. A high rate of HIV infection was also observed in the IL-18-treated group. Together, these results indicate that IL-18 may be effective for the grafting and migration of CD4+ and CD8+ T cells, except for the induction of apoptosis and regulation of class-switching IgA. IL-18-administered NOD-scid mice provide a useful small humanized model for the study of HIV infection and IgA nephropathy. PMID:12383203

Tuft (caveolated) cells in two human colon carcinoma cell lines.

PubMed Central

Barkla, D. H.; Whitehead, R. H.; Foster, H.; Tutton, P. J.

1988-01-01

The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting from the apical surface. The microvilli are attached by a core of long microfilaments passing deep into the apical cytoplasm. Between the microvilli are parallel arrays of vesicles (caveoli) containing flocculent material. Two different but not mutually exclusive explanations for the presence of tuft cells are proposed. The first explanation is that tuft cells came from the resected tumour and have survived by mitotic division during subsequent passages. The second explanation suggests that tuft cells are the progeny of undifferentiated tumour cells. Descriptions of tuft cells in colon carcinomas are uncommon and possible reasons for this are presented. The morphology of tuft cells is consistent with that of a highly differentiated cell specialised for absorption, and these new models provide an opportunity to further investigate the structure and function of tuft cells. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:3414781

Human β-cell Precursors Mature Into Functional Insulin-producing Cells in an Immunoisolation Device: Implications for Diabetes Cell Therapies

PubMed Central

Lee, Seung-Hee; Hao, Ergeng; Savinov, Alexei Y.; Geron, Ifat; Strongin, Alex Y.; Itkin-Ansari, Pamela

2009-01-01

Background Islet transplantation is limited by the need for chronic immunosuppression and the paucity of donor tissue. As new sources of human β-cells are developed (e.g., stem cell-derived tissue), transplanting them in a durable device could obviate the need for immunosuppression, while also protecting the patient from any risk of tumorigenicity. Here, we studied (1) the survival and function of encapsulated human β-cells and their progenitors and (2) the engraftment of encapsulated murine β-cells in allo- and autoimmune settings. Methods Human islets and human fetal pancreatic islet-like cell clusters were encapsulated in polytetrafluorethylene devices (TheraCyte) and transplanted into immunodeficient mice. Graft survival and function was measured by immunohistochemistry, circulating human C-peptide levels, and blood glucose levels. Bioluminescent imaging was used to monitor encapsulated neonatal murine islets. Results Encapsulated human islet-like cell clusters survived, replicated, and acquired a level of glucose responsive insulin secretion sufficient to ameliorate hyperglycemia in diabetic mice. Bioluminescent imaging of encapsulated murine neonatal islets revealed a dynamic process of cell death followed by regrowth, resulting in robust long-term allograft survival. Further, in the non-obese diabetic (NOD) mouse model of type I diabetes, encapsulated primary β-cells ameliorated diabetes without stimulating a detectable T-cell response. Conclusions We demonstrate for the first time that human β-cells function is compatible with encapsulation in a durable, immunoprotective device. Moreover, our study suggests that encapsulation of β-cells before terminal differentiation will be a successful approach for new cell-based therapies for diabetes, such as those derived from stem cells. PMID:19352116

Human beta-cell precursors mature into functional insulin-producing cells in an immunoisolation device: implications for diabetes cell therapies.

PubMed

Lee, Seung-Hee; Hao, Ergeng; Savinov, Alexei Y; Geron, Ifat; Strongin, Alex Y; Itkin-Ansari, Pamela

2009-04-15

Islet transplantation is limited by the need for chronic immunosuppression and the paucity of donor tissue. As new sources of human beta-cells are developed (e.g., stem cell-derived tissue), transplanting them in a durable device could obviate the need for immunosuppression, while also protecting the patient from any risk of tumorigenicity. Here, we studied (1) the survival and function of encapsulated human beta-cells and their progenitors and (2) the engraftment of encapsulated murine beta-cells in allo- and autoimmune settings. Human islets and human fetal pancreatic islet-like cell clusters were encapsulated in polytetrafluorethylene devices (TheraCyte) and transplanted into immunodeficient mice. Graft survival and function was measured by immunohistochemistry, circulating human C-peptide levels, and blood glucose levels. Bioluminescent imaging was used to monitor encapsulated neonatal murine islets. Encapsulated human islet-like cell clusters survived, replicated, and acquired a level of glucose responsive insulin secretion sufficient to ameliorate hyperglycemia in diabetic mice. Bioluminescent imaging of encapsulated murine neonatal islets revealed a dynamic process of cell death followed by regrowth, resulting in robust long-term allograft survival. Further, in the non-obese diabetic (NOD) mouse model of type I diabetes, encapsulated primary beta-cells ameliorated diabetes without stimulating a detectable T-cell response. We demonstrate for the first time that human beta-cells function is compatible with encapsulation in a durable, immunoprotective device. Moreover, our study suggests that encapsulation of beta-cells before terminal differentiation will be a successful approach for new cell-based therapies for diabetes, such as those derived from stem cells.

Xanthosine administration does not affect the proportion of epithelial stem cells in bovine mammary tissue, but has a latent negative effect on cell proliferation.

PubMed

Rauner, Gat; Barash, Itamar

2014-10-15

The challenge in manipulating the proportion of somatic stem cells lies in having to override tissue homeostasis. Xanthosine infusion via the teat canal has been reported to augment the number of label-retaining cells in the mammary gland of 3-month-old bovine calves. To further delineate xanthosine׳s effect on defined stem cells in the mammary gland of heifers-which are candidates for increased prospective milk production following such manipulation-bovine mammary parenchymal tissue was transplanted and integrated into the cleared mammary fat pad of immunodeficient mice. Xanthosine administration for 14 days did not affect the number of label-retaining cells after 10- and 11-week chases. No change in stem cell proportion, analyzed according to CD49f and CD24 expression, was noted. Clone formation and propagation rate of cultured cells, as well as expression of stem cell markers, were also unaffected. In contrast, a latent 50% decrease in bovine mammary cell proliferation rate was observed 11 weeks after xanthosine administration. Tumor development in mice was also limited by xanthosine administration. These effects may have resulted from an initial decrease in expression of the rate-limiting enzyme in guanine synthesis, IMPDH. The data indicate that caution should be exerted when considering xanthosine for stem cell manipulation. Copyright © 2014 Elsevier Inc. All rights reserved.

Standardized cryopreservation of human primary cells.

PubMed

Ramos, Thomas V; Mathew, Aby J; Thompson, Maria L; Ehrhardt, Rolf O

2014-09-02

Cryopreservation is the use of low temperatures to preserve structurally intact living cells. The cells that survive the thermodynamic journey from the 37 °C incubator to the -196 °C liquid nitrogen storage tank are free from the influences of time. Thus, cryopreservation is a critical component of cell culture and cell manufacturing protocols. Successful cryopreservation of human cells requires that the cells be derived from patient samples that are collected in a standardized manner, and carefully handled from blood draw through cell isolation. Furthermore, proper equipment must be in place to ensure consistency, reproducibility, and sterility. In addition, the correct choice and amount of cryoprotectant agent must be added at the correct temperature, and a controlled rate of freezing (most commonly 1 °C/min) must be applied prior to a standardized method of cryogenic storage. This appendix describes how human primary cells can be frozen for long-term storage and thawed for growth in a tissue culture vessel. Copyright © 2014 John Wiley & Sons, Inc.

Gene therapy of spontaneous canine melanoma and feline fibrosarcoma by intratumoral administration of histoincompatible cells expressing human interleukin-2.

PubMed

Quintin-Colonna, F; Devauchelle, P; Fradelizi, D; Mourot, B; Faure, T; Kourilsky, P; Roth, C; Mehtali, M

1996-12-01

The production of human interleukin-2 (hIL-2) local to the tumor site by engineered histoincompatible cells has been shown in various murine models to promote a strong immune response leading to tumor growth inhibition or rejection. To assess whether this strategy would be similarly applicable for treatment of primary neoplastic cells, two naturally occurring tumors were used as preclinical models; the highly metastatic melanoma of the dog and the low metastatic fibrosarcoma of the cat. We demonstrate that both cats and dogs when treated by tumor surgery, radiotherapy and repeated local injections of xenogeneic Vero cells secreting high levels of hIL-2 relapse less frequently and survive longer than control animals treated by surgery and radiotherapy alone. Local secretion of hIL-2 by the xenogeneic cells is shown to be necessary for the induction of an optimal antitumor effect. Moreover, the safety of the procedure was demonstrated in both animal models and through extensive toxicological analysis performed in rats. These results confirm for the first time to our knowledge the safety and therapeutic potential of a gene transfer strategy in animals with spontaneous metastatic and nonmetastatic tumors.

Genome Editing in Human Pluripotent Stem Cells.

PubMed

Carlson-Stevermer, Jared; Saha, Krishanu

2017-01-01

Genome editing in human pluripotent stem cells (hPSCs) enables the generation of reporter lines and knockout cell lines. Zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9 technology have recently increased the efficiency of proper gene editing by creating double strand breaks (DSB) at defined sequences in the human genome. These systems typically use plasmids to transiently transcribe nucleases within the cell. Here, we describe the process for preparing hPSCs for transient expression of nucleases via electroporation and subsequent analysis to create genetically modified stem cell lines.

Molecular cloning of a human Ca2+-dependent cell-cell adhesion molecule homologous to mouse placental cadherin: its low expression in human placental tissues

PubMed Central

1989-01-01

P-cadherin is a subclass of Ca2+-dependent cell-cell adhesion molecules present in mouse placenta, where its localization suggests a function of connecting the embryo to the uterus (Nose, A., and M. Takeichi. 1986. J. Cell Biol. 103:2649-2658). We recently identified a human cadherin detected by an mAb capable of disrupting cell-cell adhesion of A-431 cells, and found that it was closely related immunochemically to mouse P-cadherin. Curiously, this cadherin was undetectable in human placenta by immunohistochemical examination (Shimoyama, Y., S. Hirohashi, S. Hirano, M. Noguchi, Y. Shimosato, M. Takeichi, and O. Abe. 1989. Cancer Res. 49:2128-2133). We here report the cloning and sequencing of cDNA clone encoding the human homologue of mouse P- cadherin. The deduced amino acid sequence of the human P-cadherin consists of 829 amino acid and shows striking homology with mouse P- cadherin. On Northern blot analysis, human P-cadherin was scarcely expressed in human placenta in contrast to mouse P-cadherin, which was abundantly expressed in mouse placenta throughout pregnancy, and it was shown that E-cadherin, but not P-cadherin, was the major cadherin molecule in human placenta. Moreover, NIH3T3 cells transfected with human P-cadherin cDNA expressed the functional cadherin molecule, which was identical to the cadherin we had previously identified using the mAb, showing that this molecule really does mediate cell-cell adhesion and that the cadherin we detected immunochemically is undoubtedly human P-cadherin. The results obtained in this study support the idea that P- cadherin plays little role, if any, in Ca2+-dependent cell-cell binding in human placental tissue at least after several weeks of pregnancy. PMID:2793940

Human Urinary Epithelial Cells as a Source of Engraftable Hepatocyte-Like Cells Using Stem Cell Technology.

PubMed

Sauer, Vanessa; Tchaikovskaya, Tatyana; Wang, Xia; Li, Yanfeng; Zhang, Wei; Tar, Krisztina; Polgar, Zsuzsanna; Ding, Jianqiang; Guha, Chandan; Fox, Ira J; Roy-Chowdhury, Namita; Roy-Chowdhury, Jayanta

2016-12-13

Although several types of somatic cells have been reprogrammed into induced pluripotent stem cells (iPSCs) and then differentiated to hepatocyte-like cells (iHeps), the method for generating such cells from renal tubular epithelial cells shed in human urine and transplanting them into animal livers has not been described systematically. We report reprogramming of human urinary epithelial cells into iPSCs and subsequent hepatic differentiation, followed by a detailed characterization of the newly generated iHeps. The epithelial cells were reprogrammed into iPSCs by delivering the pluripotency factors OCT3/4, SOX2, KLF4, and MYC using methods that do not involve transgene integration, such as nucleofection of episomal (oriP/EBNA-1) plasmids or infection with recombinant Sendai viruses. After characterization of stable iPSC lines, a three-step differentiation toward hepatocytes was performed. The iHeps expressed a large number of hepatocyte-preferred genes, including nuclear receptors that regulate genes involved in cholesterol homeostasis, bile acid transport, and detoxification. MicroRNA profile of the iHeps largely paralleled that of primary human hepatocytes. The iHeps engrafted into the livers of Scid mice transgenic for mutant human SERPINA1 after intrasplenic injection. Thus, urine is a readily available source for generating human iHeps that could be potentially useful for disease modeling, pharmacological development, and regenerative medicine.

Suitability of human Tenon's fibroblasts as feeder cells for culturing human limbal epithelial stem cells.

PubMed

Scafetta, Gaia; Tricoli, Eleonora; Siciliano, Camilla; Napoletano, Chiara; Puca, Rosa; Vingolo, Enzo Maria; Cavallaro, Giuseppe; Polistena, Andrea; Frati, Giacomo; De Falco, Elena

2013-12-01

Corneal epithelial regeneration through ex vivo expansion of limbal stem cells (LSCs) on 3T3-J2 fibroblasts has revealed some limitations mainly due to the corneal microenvironment not being properly replicated, thus affecting long term results. Insights into the feeder cells that are used to expand LSCs and the mechanisms underlying the effects of human feeder cells have yet to be fully elucidated. We recently developed a standardized methodology to expand human Tenon's fibroblasts (TFs). Here we aimed to investigate whether TFs can be employed as feeder cells for LSCs, characterizing the phenotype of the co-cultures and assessing what human soluble factors are secreted. The hypothesis that TFs could be employed as alternative human feeder layer has not been explored yet. LSCs were isolated from superior limbus biopsies, co-cultured on TFs, 3T3-J2 or dermal fibroblasts (DFs), then analyzed by immunofluorescence (p63α), colony-forming efficiency (CFE) assay and qPCR for a panel of putative stem cell and epithelial corneal differentiation markers (KRT3). Co-cultures supernatants were screened for a set of soluble factors. Results showed that the percentage of p63α(+)LSCs co-cultured onto TFs was significantly higher than those on DFs (p = 0.032) and 3T3-J2 (p = 0.047). Interestingly, LSCs co-cultures on TFs exhibited both significantly higher CFE and mRNA expression levels of ΔNp63α than on 3T3-J2 and DFs (p < 0.0001), showing also significantly greater levels of soluble factors (IL-6, HGF, b-FGF, G-CSF, TGF-β3) than LSCs on DFs. Therefore, TFs could represent an alternative feeder layer to both 3T3-J2 and DFs, potentially providing a suitable microenvironment for LSCs culture.

Genetically-modified pig mesenchymal stromal cells: xenoantigenicity and effect on human T-cell xenoresponses.

PubMed

Ezzelarab, Mohamed; Ezzelarab, Corin; Wilhite, Tyler; Kumar, Goutham; Hara, Hidetaka; Ayares, David; Cooper, David K C

2011-01-01

Mesenchymal stromal cells (MSC) are being investigated as immunomodulatory therapy in the field of transplantation, particularly islet transplantation. While MSC can regenerate across species barriers, the immunoregulatory influence of genetically modified pig MSC (pMSC) on the human and non-human primate T-cell responses has not been studied. Mesenchymal stromal cells from wild-type (WT), α1,3-galactosyltransferase gene knockout (GTKO) and GTKO pigs transgenic for the human complement-regulatory protein CD46 (GTKO/CD46) were isolated and tested for differentiation. Antibody binding and T-cell responses to WT and GTKO pMSC in comparison with GTKO pig aortic endothelial cells (pAEC) were investigated. The expression of swine leukocyte antigen (SLA) class II (SLA II) was tested. Costimulatory molecules CD80 and CD86 mRNA levels were measured. Human T-cell proliferation and the production of pro-inflammatory cytokines in response to GTKO and GTKO/CD46 pMSC in comparison with human MSC (hMSC) were evaluated. α1,3-galactosyltransferase gene knockout and GTKO/CD46 pMSC isolation and differentiation were achieved in vitro. Binding of human antibodies and T-cell responses were lower to GTKO than those to WT pMSC. Human and baboon (naïve and sensitized) antibody binding were significantly lower to GTKO pMSC than to GTKO pAEC. Before activation, <1% of GTKO pMSC expressed SLA II, compared with 2.5% of GTKO pAEC. After pig interferon-gamma (pIFN-γ) activation, 99% of GTKO pAEC upregulated SLA II expression, compared with 49% of GTKO pMSC. Only 3% of GTKO pMSC expressed CD80 compared with 80% of GTKO pAEC without activation. After pIFN-γ activation, GTKO pAEC upregulated CD86 mRNA level stronger than GTKO pMSC. The human CD4(+) T-cell response to GTKO pMSC was significantly weaker than that to GTKO pAEC, even after pIFN-γ activation. More than 99% of GTKO/CD46 pMSC expressed hCD46. Human peripheral blood mononuclear cells and CD4(+) T-cell responses to GTKO and GTKO/CD46

Generation of folliculogenic human epithelial stem cells from induced pluripotent stem cells

NASA Astrophysics Data System (ADS)

Yang, Ruifeng; Zheng, Ying; Burrows, Michelle; Liu, Shujing; Wei, Zhi; Nace, Arben; Guo, Wei; Kumar, Suresh; Cotsarelis, George; Xu, Xiaowei

2014-01-01

Epithelial stem cells (EpSCs) in the hair follicle bulge are required for hair follicle growth and cycling. The isolation and propagation of human EpSCs for tissue engineering purposes remains a challenge. Here we develop a strategy to differentiate human iPSCs (hiPSCs) into CD200+/ITGA6+ EpSCs that can reconstitute the epithelial components of the hair follicle and interfollicular epidermis. The hiPSC-derived CD200+/ITGA6+ cells show a similar gene expression signature as EpSCs directly isolated from human hair follicles. Human iPSC-derived CD200+/ITGA6+ cells are capable of generating all hair follicle lineages including the hair shaft, and the inner and outer root sheaths in skin reconstitution assays. The regenerated hair follicles possess a KRT15+ stem cell population and produce hair shafts expressing hair-specific keratins. These results suggest an approach for generating large numbers of human EpSCs for tissue engineering and new treatments for hair loss, wound healing and other degenerative skin disorders.

Biokinetics of 13C in the human body after oral administration of 13C-labeled glucose as an index for the biokinetics of 14C.

PubMed

Masuda, Tsuyoshi; Tako, Yasuhiro; Matsushita, Kensaku; Takeda, Hiroshi; Endo, Masahiro; Nakamura, Yuji; Hisamatsu, Shun'ichi

2016-09-01

The retention of 13 C in the human body after oral administration of 13 C-labeled glucose was studied in three healthy volunteer subjects to estimate the 50 year cumulative body burden for 13 C as an index of the committed dose of the radioisotope 14 C. After administration of 13 C-labeled glucose, the volunteers ingested controlled diets with a fixed number of calories for 112 d. Samples of breath and urine were collected up to 112 d after administration. Samples of feces were collected up to 14 d after administration. Hair samples were obtained at 119 d after administration and analyzed as a representative index of the rate of excretion of organic 13 C via pathways such as skin cell exfoliation and mucus secretion. All samples were analyzed for 13 C/ 12 C atomic ratio to determine the rate of excretion via each pathway. We then constructed a metabolic model with a total of four pathways (breath, urine, feces, and other) comprising seven compartments. We determined the values of the biokinetic parameters in the model by using the obtained excretion data. From 74% to 94% of the 13 C administered was excreted in breath, whereas  <2% was excreted in urine and feces. In the other pathway, the excretion rate constant in the compartment with the longest residence time stretched to hundreds of days but the rate constant for each subject was not statistically significant (P value  >  0.1). In addition, the dataset for one of the three subjects was markedly different from those of the other two. When we estimated the 50 year cumulative body burden for 13 C by using our model and we included non-statistically significant parameters, a considerable cumulative body burden was found in the compartments excreting to the other pathway. Although our results on the cumulative body burden of 13 C from orally administered carbon as glucose were inconclusive, we found that the compartments excreting to the other pathway had a markedly long residence time and therefore

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.

Interplay between mast cells, enterochromaffin cells, and sensory signaling in the aging human bowel.

PubMed

Yu, Y; Daly, D M; Adam, I J; Kitsanta, P; Hill, C J; Wild, J; Shorthouse, A; Grundy, D; Jiang, W

2016-10-01

Advanced age is associated with a reduction in clinical visceral pain perception. However, the underlying mechanisms remain largely unknown. Previous studies have suggested that an abnormal interplay between mast cells, enterochromaffin (EC) cells, and afferent nerves contribute to nociception in gastrointestinal disorders. The aim of this study was to investigate how aging affects afferent sensitivity and neuro-immune association in the human bowel. Mechanical and chemical sensitivity of human bowel afferents were examined by ex vivo afferent nerve recordings. Age-related changes in the density of mast cells, EC cells, sensory nerve terminals, and mast cell-nerve micro-anatomical association were investigated by histological and immune staining. Human afferents could be broadly classified into subpopulations displaying mechanical and chemical sensitivity, adaptation, chemo-sensitization, and recruitment. Interestingly human bowel afferent nerve sensitivity was attenuated with age. The density of substance P-immunoreactive (SP-IR) nerve varicosities was also reduced with age. In contrast, the density of ileal and colonic mucosal mast cells was increased with age, as was ileal EC cell number. An increased proportion of mast cells was found in close apposition to SP-IR nerves. Afferent sensitivity in human bowel was reduced with advancing age. Augmentation of mast cells and EC cell numbers and the mast cell-nerve association suggest a compensatory mechanism for sensory neurodegeneration. © 2016 The Authors. Neurogastroenterology & Motility Published by John Wiley & Sons Ltd.

Basic FGF Support of Human Embryonic Stem Cell Self-Renewal

PubMed Central

Levenstein, Mark E.; Ludwig, Tenneille E.; Xu, Ren-He; Llanas, Rachel A.; VanDenHeuvel-Kramer, Kaitlyn; Manning, Daisy; Thomson, James A.

2015-01-01

Human embryonic stem (ES) cells have most commonly been cultured in the presence of basic FGF (FGF2) either on fibroblast feeder layers or in fibroblast-conditioned medium. Recently, it has been reported that elevated concentrations of FGF2 permit the culture of human ES cells in the absence of fibroblasts or fibroblast-conditioned medium. Here we compare the ability of unconditioned medium (UM) supplemented with 4, 24, 40, 80, 100 and 250 ng/ml FGF2 to sustain low-density human ES cell cultures through multiple passages. In these stringent culture conditions, 4, 24, and 40 ng/ml FGF2 failed to sustain human ES cells through three passages, but 100 ng/ml sustained human ES cells with an effectiveness comparable to conditioned medium (CM). Two human ES cell lines (H1 and H9) were maintained for up to 164 population doublings (7 and 4 months) in UM supplemented with 100 ng/ml FGF2. After prolonged culture the cells formed teratomas when injected into SCID-beige mice, and expressed markers characteristic of undifferentiated human ES cells. We also demonstrate that FGF2 is degraded more rapidly in UM than in CM, partly explaining the need for higher concentrations of FGF2 in UM. These results further facilitate the large-scale, routine culture of human ES cells, and suggest that fibroblasts and fibroblast-conditioned medium sustain human ES cells in part by stabilizing FGF signaling above a critical threshold. PMID:16282444

Rho GTPases and Regulation of Cell Migration and Polarization in Human Corneal Epithelial Cells

PubMed Central

Hou, Aihua; Toh, Li Xian; Gan, Kah Hui; Lee, Khee Jin Ryan; Manser, Edward; Tong, Louis

2013-01-01

Purpose Epithelial cell migration is required for regeneration of tissues and can be defective in a number of ocular surface diseases. This study aimed to determine the expression pattern of Rho family small G-proteins in human corneal epithelial cells to test their requirement in directional cell migration. Methods Rho family small G-protein expression was assessed by reverse transcription-polymerase chain reaction. Dominant-inhibitory constructs encoding Rho proteins or Rho protein targeting small interfering RNA were transfected into human corneal epithelial large T antigen cells, and wound closure rate were evaluated by scratch wounding assay, and a complementary non-traumatic cell migration assay. Immunofluorescence staining was performed to study cell polarization and to assess Cdc42 downstream effector. Results Cdc42, Chp, Rac1, RhoA, TC10 and TCL were expressed in human corneal epithelial cells. Among them, Cdc42 and TCL were found to significantly affect cell migration in monolayer scratch assays. These results were confirmed through the use of validated siRNAs directed to Cdc42 and TCL. Scramble siRNA transfected cells had high percentage of polarized cells than Cdc42 or TCL siRNA transfected cells at the wound edge. We showed that the Cdc42-specific effector p21-activated kinase 4 localized predominantly to cell-cell junctions in cell monolayers, but failed to translocate to the leading edge in Cdc42 siRNA transfected cells after monolayer wounding. Conclusion Rho proteins expressed in cultured human corneal epithelial cells, and Cdc42, TCL facilitate two-dimensional cell migration in-vitro. Although silencing of Cdc42 and TCL did not noticeably affect the appearance of cell adhesions at the leading edge, the slower migration of these cells indicates both GTP-binding proteins play important roles in promoting cell movement of human corneal epithelial cells. PMID:24130842

The use of human cells in biomedical research and testing.

PubMed

Combes, Robert D

2004-06-01

The ability to use human cells in biomedical research and testing has the obvious advantage over the use of laboratory animals that the need for species extrapolation is obviated, due to the presence of more-relevant morphological, physiological and biochemical properties, including receptors. Moreover, human cells exhibit the same advantages as animal cells in culture in that different cell types can be used, from different tissues, with a wide range of techniques, to investigate a wide variety of biological phenomena in tissue culture. Human cells can also be grown as organotypic cultures to facilitate the extrapolation from cells to whole organisms. Human cell lines have been available for many years on an ad hoc basis from individual researchers, and also from recognised sources, such as the European Collection of Animal Cell Cultures (ECACC) and, in the USA, the Human Cell Culture Centre (HCCC). Such cells have usually been derived from tumours and this has restricted the variety of types of cells available. This problem has been addressed by using primary human cells that can be obtained from a variety of sources, such as cadavers, diseased tissue, skin strips, peripheral blood, buccal cavity smears, hair follicles and surgical waste from biopsy material that is unsuitable for transplantation purposes. However, primary human cells need to be obtained, processed, distributed and handled in a safe and ethical manner. They also have to be made available at the correct time to researchers very shortly after they become available. It is only comparatively recently that the safe and controlled acquisition of surgical waste and non-transplantable human tissues has become feasible with the establishment of several human tissue banks. Recently, the formation of a UK and European centralised network for human tissue supply has been initiated. The problems of short longevity and loss of specialisation in culture are being approached by: a) cell immortalisation to

HIV-1 infection, response to treatment and establishment of viral latency in a novel humanized T cell-only mouse (TOM) model.

PubMed

Honeycutt, Jenna B; Wahl, Angela; Archin, Nancie; Choudhary, Shailesh; Margolis, David; Garcia, J Victor

2013-10-24

The major targets of HIV infection in humans are CD4⁺ T cells. CD4⁺ T cell depletion is a hallmark of AIDS. Previously, the SCID-hu thy/liv model was used to study the effect of HIV on thymopoeisis in vivo. However, these mice did not develop high levels of peripheral T cell reconstitution and required invasive surgery for infection and analysis. Here, we describe a novel variant of this model in which thy/liv implantation results in systemic reconstitution with human T cells in the absence of any other human hematopoietic lineages. NOD/SCID-hu thy/liv and NSG-hu thy/liv mice were created by implanting human fetal thymus and liver tissues under the kidney capsule of either NOD/SCID or NSG mice. In contrast to NOD/SCID-hu thy/liv mice that show little or no human cells in peripheral blood or tissues, substantial systemic human reconstitution occurs in NSG-hu thy/liv. These mice are exclusively reconstituted with human T cells (i.e. T-cell only mice or TOM). Despite substantial levels of human T cells no signs of graft-versus-host disease (GVHD) were noted in these mice over a period of 14 months. TOM are readily infected after parenteral exposure to HIV-1. HIV replication is sustained in peripheral blood at high levels and results in modest reduction of CD4⁺ T cells. HIV-1 replication in TOM responds to daily administration of combination antiretroviral therapy (ART) resulting in strong suppression of virus replication as determined by undetectable viral load in plasma. Latently HIV infected resting CD4⁺ T cells can be isolated from suppressed mice that can be induced to express HIV ex-vivo upon activation demonstrating the establishment of latency in vivo. NSG-hu thy/liv mice are systemically reconstituted with human T cells. No other human lymphoid lineages are present in these mice (i.e. monocytes/macrophages, B cells and DC are all absent). These T cell only mice do not develop GVHD, are susceptible to HIV-1 infection and can efficiently maintain virus

Differential effect on cell-mediated immunity in human volunteers after intake of different lactobacilli

PubMed Central

Rask, C; Adlerberth, I; Berggren, A; Ahrén, I L; Wold, A E

2013-01-01

Probiotics are live microorganisms which have beneficial effects on the host when ingested in adequate amounts. Probiotic bacteria may stimulate immune effector functions in a strain-specific manner. In this blind placebo-controlled trial, we investigated the effects on the immune system following daily intake of six different strains of lactobacilli or the Gram-negative bacterium Pseudomonas lundensis for 2 or 5 weeks. Blood lymphocyte subsets were quantified by fluorescence activated cell sorter and the expression of activation and memory markers was determined. The bacterial strains were also examined for their capacity to adhere to human intestinal cells and to be phagocytosed by human peripheral blood mononuclear cells. Intake of Lactobacillus plantarum strain 299v increased the expression of the activation marker CD25 (P = 0·01) on CD8+ T cells and the memory cell marker CD45RO on CD4+ T cells (P = 0·03), whereas intake of L. paracasei tended to expand the natural killer T (NK T) cell population (P = 0·06). The phagocytic activity of granulocytes was increased following intake of L. plantarum 299v, L. plantarum HEAL, L. paracasei or L. fermentum. In contrast, ingestion of L. rhamnosus decreased the expression of CD25 and CD45RO significantly within the CD4+ cell population. The observed immune effects after in-vivo administration of the probiotic bacteria could not be predicted by either their adherence capacity or the in-vitro-induced cytokine production. The stimulation of CD8+ T cells and NK T cells suggests that intake of probiotic bacteria may enhance the immune defence against, e.g. viral infections or tumours. PMID:23574328

Pathogenesis of Human Enterovirulent Bacteria: Lessons from Cultured, Fully Differentiated Human Colon Cancer Cell Lines

PubMed Central

Liévin-Le Moal, Vanessa

2013-01-01

SUMMARY Hosts are protected from attack by potentially harmful enteric microorganisms, viruses, and parasites by the polarized fully differentiated epithelial cells that make up the epithelium, providing a physical and functional barrier. Enterovirulent bacteria interact with the epithelial polarized cells lining the intestinal barrier, and some invade the cells. A better understanding of the cross talk between enterovirulent bacteria and the polarized intestinal cells has resulted in the identification of essential enterovirulent bacterial structures and virulence gene products playing pivotal roles in pathogenesis. Cultured animal cell lines and cultured human nonintestinal, undifferentiated epithelial cells have been extensively used for understanding the mechanisms by which some human enterovirulent bacteria induce intestinal disorders. Human colon carcinoma cell lines which are able to express in culture the functional and structural characteristics of mature enterocytes and goblet cells have been established, mimicking structurally and functionally an intestinal epithelial barrier. Moreover, Caco-2-derived M-like cells have been established, mimicking the bacterial capture property of M cells of Peyer's patches. This review intends to analyze the cellular and molecular mechanisms of pathogenesis of human enterovirulent bacteria observed in infected cultured human colon carcinoma enterocyte-like HT-29 subpopulations, enterocyte-like Caco-2 and clone cells, the colonic T84 cell line, HT-29 mucus-secreting cell subpopulations, and Caco-2-derived M-like cells, including cell association, cell entry, intracellular lifestyle, structural lesions at the brush border, functional lesions in enterocytes and goblet cells, functional and structural lesions at the junctional domain, and host cellular defense responses. PMID:24006470

Tissuelike 3D Assemblies of Human Broncho-Epithelial Cells

NASA Technical Reports Server (NTRS)

Goodwin, Thomas J.

2010-01-01

Three-dimensional (3D) tissuelike assemblies (TLAs) of human broncho-epithelial (HBE) cells have been developed for use in in vitro research on infection of humans by respiratory viruses. The 2D monolayer HBE cell cultures heretofore used in such research lack the complex cell structures and interactions characteristic of in vivo tissues and, consequently, do not adequately emulate the infection dynamics of in-vivo microbial adhesion and invasion. In contrast, the 3D HBE TLAs are characterized by more-realistic reproductions of the geometrical and functional complexity, differentiation of cells, cell-to-cell interactions, and cell-to-matrix interactions characteristic of human respiratory epithelia. Hence, the 3D HBE TLAs are expected to make it possible to perform at least some of the research in vitro under more-realistic conditions, without need to infect human subjects. The TLAs are grown on collagen-coated cyclodextran microbeads under controlled conditions in a nutrient liquid in the simulated microgravitational environment of a bioreactor of the rotating- wall-vessel type. Primary human mesenchymal bronchial-tracheal cells are used as a foundation matrix, while adult human bronchial epithelial immortalized cells are used as the overlying component. The beads become coated with cells, and cells on adjacent beads coalesce into 3D masses. The resulting TLAs have been found to share significant characteristics with in vivo human respiratory epithelia including polarization, tight junctions, desmosomes, and microvilli. The differentiation of the cells in these TLAs into tissues functionally similar to in vivo tissues is confirmed by the presence of compounds, including villin, keratins, and specific lung epithelium marker compounds, and by the production of tissue mucin. In a series of initial infection tests, TLA cultures were inoculated with human respiratory syncytial viruses and parainfluenza type 3 viruses. Infection was confirmed by photomicrographs that

Glycyrrhizic acid attenuates stem cell-like phenotypes of human dermal papilla cells.

PubMed

Kiratipaiboon, Chayanin; Tengamnuay, Parkpoom; Chanvorachote, Pithi

2015-12-15

Although the growth of unwanted hair or hirsutism is a harmless condition, many people find it bothersome and embarrassing. Maintaining stem cell features of dermal papilla cells is a critical biological process that keeps the high rate of hair growth. Glycyrrhizic acid has been reported to impair hair growth in some studies; however, its underlying mechanism has not yet been investigated. This study aimed to explore the effect and underlying mechanism of glycyrrhizic acid on stemness of human dermal papilla cells. The stem cell molecular markers, epithelial to mesenchymal markers and Wnt/β-catenin-associated proteins of human dermal papilla cell line and primary human dermal papilla cells were analysed by western blot analysis and immunocytochemistry. The present study demonstrated that glycyrrhizic acid significantly depressed the stemness of dermal papilla cells in dose- and time-dependent manners. Clonogenicity and stem cell markers in the glycyrrhizic acid-treated cells were found to gradually decrease in the culture in a time-dependent manner. Our results demonstrated that glycyrrhizic acid exerted the stem cell suppressing effects through the interruption of ATP-dependent tyrosine kinase/glycogen synthase kinase3β-dependent mechanism which in turn down-regulated the β-catenin signalling pathway, coupled with decreased its down-stream epithelial-mesenchymal transition and self-renewal transcription factors, namely, Oct-4, Nanog, Sox2, ZEB1 and Snail. The effect of glycyrrhizic acid on the reduction of stem cell features was also observed in the primary dermal papilla cells directly obtained from human hair follicles. These results revealed a novel molecular mechanism of glycyrrhizic acid in regulation of dermal papilla cells and provided the evidence supporting the use of this compound in suppressing the growth of unwanted hair. Copyright © 2015 Elsevier GmbH. All rights reserved.

Generation of mature T cells from human hematopoietic stem/progenitor cells in artificial thymic organoids

PubMed Central

Seet, Christopher S.; He, Chongbin; Bethune, Michael T.; Li, Suwen; Chick, Brent; Gschweng, Eric H.; Zhu, Yuhua; Kim, Kenneth; Kohn, Donald B.; Baltimore, David; Crooks, Gay M.; Montel-Hagen, Amélie

2017-01-01

Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCRab+ single positive (SP) CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports highly efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naïve phenotypes, a diverse TCR repertoire, and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen specific cytotoxicity and near complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ loci. ATOs provide a robust tool for studying human T cell development and stem cell based approaches to engineered T cell therapies. PMID:28369043

Productive Infection of Human Embryonic Stem Cell-Derived NKX2.1+ Respiratory Progenitors with Human Rhinovirus.

PubMed

Jenny, Robert A; Hirst, Claire; Lim, Sue Mei; Goulburn, Adam L; Micallef, Suzanne J; Labonne, Tanya; Kicic, Anthony; Ling, Kak-Ming; Stick, Stephen M; Ng, Elizabeth S; Trounson, Alan; Giudice, Antonietta; Elefanty, Andrew G; Stanley, Edouard G

2015-06-01

Airway epithelial cells generated from pluripotent stem cells (PSCs) represent a resource for research into a variety of human respiratory conditions, including those resulting from infection with common human pathogens. Using an NKX2.1-GFP reporter human embryonic stem cell line, we developed a serum-free protocol for the generation of NKX2.1(+) endoderm that, when transplanted into immunodeficient mice, matured into respiratory cell types identified by expression of CC10, MUC5AC, and surfactant proteins. Gene profiling experiments indicated that day 10 NKX2.1(+) endoderm expressed markers indicative of early foregut but lacked genes associated with later stages of respiratory epithelial cell differentiation. Nevertheless, NKX2.1(+) endoderm supported the infection and replication of the common respiratory pathogen human rhinovirus HRV1b. Moreover, NKX2.1(+) endoderm upregulated expression of IL-6, IL-8, and IL-1B in response to infection, a characteristic of human airway epithelial cells. Our experiments provide proof of principle for the use of PSC-derived respiratory epithelial cells in the study of cell-virus interactions. This report provides proof-of-principle experiments demonstrating, for the first time, that human respiratory progenitor cells derived from stem cells in the laboratory can be productively infected with human rhinovirus, the predominant cause of the common cold. ©AlphaMed Press.

Mesenchymal Stem Cell Levels of Human Spinal Tissues.

PubMed

Harris, Liam; Vangsness, C Thomas

2018-05-01

Systematic review. The aim of this study was to investigate, quantify, compare, and compile the various mesenchymal stem cell (MSC) tissue sources within human spinal tissues to act as a compendium for clinical and research application. Recent years have seen a dramatic increase in academic and clinical understanding of human MSCs. Previously limited to cells isolated from bone marrow, the past decade has illicited the characterization and isolation of human MSCs from adipose, bone marrow, synovium, muscle, periosteum, peripheral blood, umbilical cord, placenta, and numerous other tissues. As researchers explore practical applications of cells in these tissues, the absolute levels of MSCs in specific spinal tissue will be critical to guide future research. The PubMED, MEDLINE, EMBASE, and Cochrane databases were searched for articles relating to the harvest, characterization, isolation, and quantification of human MSCs from spinal tissues. Selected articles were examined for relevant data, categorized according to type of spinal tissue, and when possible, standardized to facilitate comparisons between sites. Human MSC levels varied widely between spinal tissues. Yields for intervertebral disc demonstrated roughly 5% of viable cells to be positive for MSC surface markers. Cartilage endplate cells yielded 18,500 to 61,875 cells/0.8 mm thick sample of cartilage end plate. Ligamentum flavum yielded 250,000 to 500,000 cells/g of tissue. Annulus fibrosus fluorescence activated cell sorting treatment found 29% of cells positive for MSC marker Stro-1. Nucleus pulposus yielded mean tissue samples of 40,584 to 234,137 MSCs per gram of tissue. Numerous tissues within and surrounding the spine represent a consistent and reliable source for the harvest and isolation of human MSCs. Among the tissues of the spine, the annulus fibrosus and ligamentum flavum each offer considerable levels of MSCs, and may prove comparable to that of bone marrow. 5.

I.V. infusion of brain-derived neurotrophic factor gene-modified human mesenchymal stem cells protects against injury in a cerebral ischemia model in adult rat.

PubMed

Nomura, T; Honmou, O; Harada, K; Houkin, K; Hamada, H; Kocsis, J D

2005-01-01

I.V. delivery of mesenchymal stem cells prepared from adult bone marrow reduces infarction size and ameliorates functional deficits in rat cerebral ischemia models. Administration of the brain-derived neurotrophic factor to the infarction site has also been demonstrated to be neuroprotective. To test the hypothesis that brain-derived neurotrophic factor contributes to the therapeutic benefits of mesenchymal stem cell delivery, we compared the efficacy of systemic delivery of human mesenchymal stem cells and human mesenchymal stem cells transfected with a fiber-mutant F/RGD adenovirus vector with a brain-derived neurotrophic factor gene (brain-derived neurotrophic factor-human mesenchymal stem cells). A permanent middle cerebral artery occlusion was induced by intraluminal vascular occlusion with a microfilament. Human mesenchymal stem cells and brain-derived neurotrophic factor-human mesenchymal stem cells were i.v. injected into the rats 6 h after middle cerebral artery occlusion. Lesion size was assessed at 6 h, 1, 3 and 7 days using MR imaging, and histological methods. Functional outcome was assessed using the treadmill stress test. Both human mesenchymal stem cells and brain-derived neurotrophic factor-human mesenchymal stem cells reduced lesion volume and elicited functional improvement compared with the control sham group, but the effect was greater in the brain-derived neurotrophic factor-human mesenchymal stem cell group. ELISA analysis of the infarcted hemisphere revealed an increase in brain-derived neurotrophic factor in the human mesenchymal stem cell groups, but a greater increase in the brain-derived neurotrophic factor-human mesenchymal stem cell group. These data support the hypothesis that brain-derived neurotrophic factor contributes to neuroprotection in cerebral ischemia and cellular delivery of brain-derived neurotrophic factor can be achieved by i.v. delivery of human mesenchymal stem cells.

Generation of a Chinese Hamster Ovary Cell Line Producing Recombinant Human Glucocerebrosidase

PubMed Central

Novo, Juliana Branco; Morganti, Ligia; Moro, Ana Maria; Paes Leme, Adriana Franco; Serrano, Solange Maria de Toledo; Raw, Isaias; Ho, Paulo Lee

2012-01-01

Impaired activity of the lysosomal enzyme glucocerebrosidase (GCR) results in the inherited metabolic disorder known as Gaucher disease. Current treatment consists of enzyme replacement therapy by administration of exogenous GCR. Although effective, it is exceptionally expensive, and patients worldwide have a limited access to this medicine. In Brazil, the public healthcare system provides the drug free of charge for all Gaucher's patients, which reaches the order of $ 84 million per year. However, the production of GCR by public institutions in Brazil would reduce significantly the therapy costs. Here, we describe a robust protocol for the generation of a cell line producing recombinant human GCR. The protein was expressed in CHO-DXB11 (dhfr−) cells after stable transfection and gene amplification with methotrexate. As expected, glycosylated GCR was detected by immunoblotting assay both as cell-associated (~64 and 59 kDa) and secreted (63–69 kDa) form. Analysis of subclones allowed the selection of stable CHO cells producing a secreted functional enzyme, with a calculated productivity of 5.14 pg/cell/day for the highest producer. Although being laborious, traditional methods of screening high-producing recombinant cells may represent a valuable alternative to generate expensive biopharmaceuticals in countries with limited resources. PMID:23091360

Human induced pluripotent stem cells: A disruptive innovation.

PubMed

De Vos, J; Bouckenheimer, J; Sansac, C; Lemaître, J-M; Assou, S

2016-01-01

This year (2016) will mark the 10th anniversary of the discovery of induced pluripotent stem cells (iPSCs). The finding that the transient expression of four transcription factors can radically remodel the epigenome, transcriptome and metabolome of differentiated cells and reprogram them into pluripotent stem cells has been a major and groundbreaking technological innovation. In this review, we discuss the major applications of this technology that we have grouped in nine categories: a model to study cell fate control; a model to study pluripotency; a model to study human development; a model to study human tissue and organ physiology; a model to study genetic diseases in a dish; a tool for cell rejuvenation; a source of cells for drug screening; a source of cells for regenerative medicine; a tool for the production of human organs in animals. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Diploid, but not haploid, human embryonic stem cells can be derived from microsurgically repaired tripronuclear human zygotes

PubMed Central

Fan, Yong; Li, Rong; Huang, Jin; Yu, Yang; Qiao, Jie

2013-01-01

Human embryonic stem cells have shown tremendous potential in regenerative medicine, and the recent progress in haploid embryonic stem cells provides new insights for future applications of embryonic stem cells. Disruption of normal fertilized embryos remains controversial; thus, the development of a new source for human embryonic stem cells is important for their usefulness. Here, we investigated the feasibility of haploid and diploid embryo reconstruction and embryonic stem cell derivation using microsurgically repaired tripronuclear human zygotes. Diploid and haploid zygotes were successfully reconstructed, but a large proportion of them still had a tripolar spindle assembly. The reconstructed embryos developed to the blastocyst stage, although the loss of chromosomes was observed in these zygotes. Finally, triploid and diploid human embryonic stem cells were derived from tripronuclear and reconstructed zygotes (from which only one pronucleus was removed), but haploid human embryonic stem cells were not successfully derived from the reconstructed zygotes when two pronuclei were removed. Both triploid and diploid human embryonic stem cells showed the general characteristics of human embryonic stem cells. These results indicate that the lower embryo quality resulting from abnormal spindle assembly contributed to the failure of the haploid embryonic stem cell derivation. However, the successful derivation of diploid embryonic stem cells demonstrated that microsurgical tripronuclear zygotes are an alternative source of human embryonic stem cells. In the future, improving spindle assembly will facilitate the application of triploid zygotes to the field of haploid embryonic stem cells. PMID:23255130

A novel matrine derivate inhibits differentiated human hepatoma cells and hepatic cancer stem-like cells by suppressing PI3K/AKT signaling pathways

PubMed Central

Liu, Ying; Qi, Yang; Bai, Zhi-hui; Ni, Chen-xu; Ren, Qi-hui; Xu, Wei-heng; Xu, Jing; Hu, Hong-gang; Qiu, Lei; Li, Jian-zhong; He, Zhi-gao; Zhang, Jun-ping

2017-01-01

Matrine is an alkaloid extracted from a Chinese herb Sophora flavescens Ait, which has shown chemopreventive potential against various cancers. In this study, we evaluated the anticancer efficacy of a novel derivative of matrine, (6aS, 10S, 11aR, 11bR, 11cS)-10- methylamino-dodecahydro- 3a,7a-diazabenzo (de) (MASM), against human hepatocellular carcinoma (HCC) cells and their corresponding sphere cells in vitro and in vivo. Human HCC cell lines (Hep3B and Huh7) were treated with MASM. Cell proliferation was assessed using CCK8 and colony assays; cell apoptosis and cell cycle distributions were examined with flow cytometry. The expression of cell markers and signaling molecules was detected using Western blot and qRT-PCR analyses. A sphere culture technique was used to enrich cancer stem cells (CSC) in Hep3B and Huh7 cells. The in vivo antitumor efficacy of MASM was evaluated in Huh7 cell xenograft model in BALB/c nude mice, which were administered MASM (10 mg·kg−1·d−1, ig) for 3 weeks. After the treatment was completed, tumor were excised and weighed. A portion of tumor tissue was enzymatically dissociated to obtain a single cell suspension for the spheroid formation assays. MASM (2, 10, 20 μmol/L) dose-dependently inhibited the proliferation of HCC cells, and induced apoptosis, which correlated with a reduction in Bcl-2 expression and an increase in PARP cleavage. MASM also induced cell cycle arrest in G0/G1 phase, which was accompanied by increased p27 and decreased Cyclin D1 expression. Interestingly, MASM (2, 10, and 20 μmol/L) drastically reduced the EpCAM+/CD133+ cell numbers, suppressed the sphere formation, inhibited the expression of stem cell marker genes and promoted the expression of mature hepatocyte markers in the Hep3B and Huh7 spheroids. Additionally, MASM dose-dependently suppressed the PI3K/AKT/mTOR and AKT/GSK3β/β-catenin signaling pathways in Hep3B and Huh7 cells. In Huh7 xenograft bearing nude mice, MASM administration significantly

Gossypol inhibition of mitosis, cyclin D1 and Rb protein in human mammary cancer cells and cyclin-D1 transfected human fibrosarcoma cells.

PubMed Central

Ligueros, M.; Jeoung, D.; Tang, B.; Hochhauser, D.; Reidenberg, M. M.; Sonenberg, M.

1997-01-01

The antiproliferative effects of gossypol on human MCF-7 mammary cancer cells and cyclin D1-transfected HT-1060 human fibrosarcoma cells were investigated by cell cycle analysis and effects on the cell cycle regulatory proteins Rb and cyclin D1. Flow cytometry of MCF-7 cells at 24 h indicated that 10 microM gossypol inhibited DNA synthesis by producing a G1/S block. Western blot analysis using anti-human Rb antibodies and anti-human cyclin D1 antibodies in MCF-7 cells and high- and low-expression cyclin D1-transfected fibrosarcoma cells indicated that, after 6 h exposure, gossypol decreased the expression levels of these proteins in a dose-dependent manner. Gossypol also decreased the ratio of phosphorylated to unphosphorylated Rb protein in human mammary cancer and fibrosarcoma cell lines. Gossypol (10 microM) treated also decreased cyclin D1-associated kinase activity on histone H1 used as a substrate in MCF-7 cells. These results suggest that gossypol might suppress growth by modulating the expression of cell cycle regulatory proteins Rb and cyclin D1 and the phosphorylation of Rb protein. Images Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 PMID:9218727

Generation of Alveolar Epithelial Spheroids via Isolated Progenitor Cells from Human Pluripotent Stem Cells

PubMed Central

Gotoh, Shimpei; Ito, Isao; Nagasaki, Tadao; Yamamoto, Yuki; Konishi, Satoshi; Korogi, Yohei; Matsumoto, Hisako; Muro, Shigeo; Hirai, Toyohiro; Funato, Michinori; Mae, Shin-Ichi; Toyoda, Taro; Sato-Otsubo, Aiko; Ogawa, Seishi; Osafune, Kenji; Mishima, Michiaki

2014-01-01

Summary No methods for isolating induced alveolar epithelial progenitor cells (AEPCs) from human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) have been reported. Based on a study of the stepwise induction of alveolar epithelial cells (AECs), we identified carboxypeptidase M (CPM) as a surface marker of NKX2-1+ “ventralized” anterior foregut endoderm cells (VAFECs) in vitro and in fetal human and murine lungs. Using SFTPC-GFP reporter hPSCs and a 3D coculture system with fetal human lung fibroblasts, we showed that CPM+ cells isolated from VAFECs differentiate into AECs, demonstrating that CPM is a marker of AEPCs. Moreover, 3D coculture differentiation of CPM+ cells formed spheroids with lamellar-body-like structures and an increased expression of surfactant proteins compared with 2D differentiation. Methods to induce and isolate AEPCs using CPM and consequently generate alveolar epithelial spheroids would aid human pulmonary disease modeling and regenerative medicine. PMID:25241738

Cell Type-Specific Chromatin Signatures Underline Regulatory DNA Elements in Human Induced Pluripotent Stem Cells and Somatic Cells.

PubMed

Zhao, Ming-Tao; Shao, Ning-Yi; Hu, Shijun; Ma, Ning; Srinivasan, Rajini; Jahanbani, Fereshteh; Lee, Jaecheol; Zhang, Sophia L; Snyder, Michael P; Wu, Joseph C

2017-11-10

Regulatory DNA elements in the human genome play important roles in determining the transcriptional abundance and spatiotemporal gene expression during embryonic heart development and somatic cell reprogramming. It is not well known how chromatin marks in regulatory DNA elements are modulated to establish cell type-specific gene expression in the human heart. We aimed to decipher the cell type-specific epigenetic signatures in regulatory DNA elements and how they modulate heart-specific gene expression. We profiled genome-wide transcriptional activity and a variety of epigenetic marks in the regulatory DNA elements using massive RNA-seq (n=12) and ChIP-seq (chromatin immunoprecipitation combined with high-throughput sequencing; n=84) in human endothelial cells (CD31 + CD144 + ), cardiac progenitor cells (Sca-1 + ), fibroblasts (DDR2 + ), and their respective induced pluripotent stem cells. We uncovered 2 classes of regulatory DNA elements: class I was identified with ubiquitous enhancer (H3K4me1) and promoter (H3K4me3) marks in all cell types, whereas class II was enriched with H3K4me1 and H3K4me3 in a cell type-specific manner. Both class I and class II regulatory elements exhibited stimulatory roles in nearby gene expression in a given cell type. However, class I promoters displayed more dominant regulatory effects on transcriptional abundance regardless of distal enhancers. Transcription factor network analysis indicated that human induced pluripotent stem cells and somatic cells from the heart selected their preferential regulatory elements to maintain cell type-specific gene expression. In addition, we validated the function of these enhancer elements in transgenic mouse embryos and human cells and identified a few enhancers that could possibly regulate the cardiac-specific gene expression. Given that a large number of genetic variants associated with human diseases are located in regulatory DNA elements, our study provides valuable resources for deciphering

Hexavalent chromium induces chromosome instability in human urothelial cells

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

Wise, Sandra S.; Holmes, Amie L.; Department of Radiation Oncology, Dana Farber Cancer Institute, 450 Brookline Ave., Boston, MA 02215

Numerous metals are well-known human bladder carcinogens. Despite the significant occupational and public health concern of metals and bladder cancer, the carcinogenic mechanisms remain largely unknown. Chromium, in particular, is a metal of concern as incidences of bladder cancer have been found elevated in chromate workers, and there is an increasing concern for patients with metal hip implants. However, the impact of hexavalent chromium (Cr(VI)) on bladder cells has not been studied. We compared chromate toxicity in two bladder cell lines; primary human urothelial cells and hTERT-immortalized human urothelial cells. Cr(VI) induced a concentration- and time-dependent increase in chromosome damagemore » in both cell lines, with the hTERT-immortalized cells exhibiting more chromosome damage than the primary cells. Chronic exposure to Cr(VI) also induced a concentration-dependent increase in aneuploid metaphases in both cell lines which was not observed after a 24 h exposure. Aneuploidy induction was higher in the hTERT-immortalized cells. When we correct for uptake, Cr(VI) induces a similar amount of chromosome damage and aneuploidy suggesting that the differences in Cr(VI) sensitivity between the two cells lines were due to differences in uptake. The increase in chromosome instability after chronic chromate treatment suggests this may be a mechanism for chromate-induced bladder cancer, specifically, and may be a mechanism for metal-induced bladder cancer, in general. - Highlights: • Hexavalent chromium is genotoxic to human urothelial cells. • Hexavalent chromium induces aneuploidy in human urothelial cells. • hTERT-immortalized human urothelial cells model the effects seen in primary urothelial cells. • Hexavalent chromium has a strong likelihood of being carcinogenic for bladder tissue.« less