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Sample records for differential cell capture

  1. Parametric control of collision rates and capture rates in geometrically enhanced differential immunocapture (GEDI) microfluidic devices for rare cell capture.

    Science.gov (United States)

    Smith, James P; Lannin, Timothy B; Syed, Yusef; Santana, Steven M; Kirby, Brian J

    2014-02-01

    The enrichment and isolation of rare cells from complex samples, such as circulating tumor cells (CTCs) from whole blood, is an important engineering problem with widespread clinical applications. One approach uses a microfluidic obstacle array with an antibody surface functionalization to both guide cells into contact with the capture surface and to facilitate adhesion; geometrically enhanced differential immunocapture is a design strategy in which the array is designed to promote target cell–obstacle contact and minimize other interactions (Gleghorn et al. 2010; Kirby et al. 2012). We present a simulation that uses capture experiments in a simple Hele-Shaw geometry (Santana et al. 2012) to inform a target-cell-specific capture model that can predict capture probability in immunocapture microdevices of any arbitrary complex geometry. We show that capture performance is strongly dependent on the array geometry, and that it is possible to select an obstacle array geometry that maximizes capture efficiency (by creating combinations of frequent target cell–obstacle collisions and shear stress low enough to support capture), while simultaneously enhancing purity by minimizing nonspecific adhesion of both smaller contaminant cells (with infrequent cell–obstacle collisions) and larger contaminant cells (by focusing those collisions into regions of high shear stress).

  2. Transcriptome differentiation along the dorso-ventral axis in laser-captured microdissected rat hippocampal granular cell layer

    DEFF Research Database (Denmark)

    Christensen, T.; Bisgaard, C.F.; Nielsen, Henrik Bjørn

    2010-01-01

    -related behaviors. Based on such findings our aim was to investigate the molecular differentiation along the dorso-ventral axis of the hippocampal granular cell layer of the rat dentate gyrus. Homogeneous isolation of this specific area was performed by laser-capture microdissection and Illumina microarray chips...

  3. RNA sequencing of laser-capture microdissected compartments of the maize kernel identifies regulatory modules associated with endosperm cell differentiation.

    Science.gov (United States)

    Zhan, Junpeng; Thakare, Dhiraj; Ma, Chuang; Lloyd, Alan; Nixon, Neesha M; Arakaki, Angela M; Burnett, William J; Logan, Kyle O; Wang, Dongfang; Wang, Xiangfeng; Drews, Gary N; Yadegari, Ramin

    2015-03-01

    Endosperm is an absorptive structure that supports embryo development or seedling germination in angiosperms. The endosperm of cereals is a main source of food, feed, and industrial raw materials worldwide. However, the genetic networks that regulate endosperm cell differentiation remain largely unclear. As a first step toward characterizing these networks, we profiled the mRNAs in five major cell types of the differentiating endosperm and in the embryo and four maternal compartments of the maize (Zea mays) kernel. Comparisons of these mRNA populations revealed the diverged gene expression programs between filial and maternal compartments and an unexpected close correlation between embryo and the aleurone layer of endosperm. Gene coexpression network analysis identified coexpression modules associated with single or multiple kernel compartments including modules for the endosperm cell types, some of which showed enrichment of previously identified temporally activated and/or imprinted genes. Detailed analyses of a coexpression module highly correlated with the basal endosperm transfer layer (BETL) identified a regulatory module activated by MRP-1, a regulator of BETL differentiation and function. These results provide a high-resolution atlas of gene activity in the compartments of the maize kernel and help to uncover the regulatory modules associated with the differentiation of the major endosperm cell types. © 2015 American Society of Plant Biologists. All rights reserved.

  4. Osteoblastic cells: differentiation and trans-differentiation

    DEFF Research Database (Denmark)

    Kassem, Moustapha; Abdallah, Basem; Saeed, Hamid

    2008-01-01

    The osteoblast is the bone forming cell and is derived from mesenchymal stem cells (MSC) present among the bone marrow stroma. MSC are capable of multi-lineage differentiation into mesoderm-type cells such as osteoblasts and adipocytes. Understanding the mechanisms underlying osteoblast...... differentiation from MSC is a central topic in bone biology that can provide insight into mechanisms of bone maintenance and also novel pharmacological targets to increase osteoblast differentiation and consequently bone formation....

  5. Ghrelin and cell differentiation

    Institute of Scientific and Technical Information of China (English)

    Geyang Xu; Yin Li; Wenjiao An; Weizhen Zhang

    2008-01-01

    Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, is a gastric hormone that has been found to have a wide variety of biological functions. This review summarizes our current understanding of the effects of ghrelin on cell differentiation and tissue development, with an emphasis on the lineage determination of mesenchymal stem cells.

  6. A microfluidic chip for highly efficient cell capturing and pairing.

    Science.gov (United States)

    Cui, Shaoyan; Liu, Yaoping; Wang, Wei; Sun, Yan; Fan, Yubo

    2011-09-01

    This paper examined the feasibility of a microfluidics chip for cell capturing and pairing with a high efficiency. The chip was fabricated by the polydimethylsiloxane-based soft-lithography technique and contained two suction duct arrays set in parallel on both sides of a main microchannel. Cells were captured and paired by activating two sets of suction ducts one by one with the help of syringe pumps along with switching the cell suspensions inside the main microchannel correspondingly. The effects of suction flow rate and the dimensions of suction channels on the cell capturing and pairing efficiency were characterized. The present chip was capable of creating 1024 pairs of two different cell populations in parallel. The preliminary experimental results showed that the cell capturing efficiency was 100% and the pairing one was 88% with an optimal suction rate of 5 μl/min in the chip in the 2 μm-sized suction duct chip. The cell viability after capture inside the microfluidic device was 90.0 ± 5.3%. With this cell capturing and pairing chip, interaction between cells in a single pair mode can be studied. The ability to create cell pairs has a number of biological applications for cell fusion, cell-cell interaction studies, and cell toxicity screening.

  7. T Cells Capture Bacteria by Transinfection from Dendritic Cells.

    Science.gov (United States)

    Cruz-Adalia, Aranzazu; Ramírez-Santiago, Guillermo; Torres-Torresano, Mónica; Garcia-Ferreras, Raquel; Veiga Chacón, Esteban

    2016-01-13

    Recently, we have shown, contrary to what is described, that CD4(+) T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4(+) T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

  8. Differential cross sections for single-electron capture in He{sup 2+}-D collisions

    Energy Technology Data Exchange (ETDEWEB)

    Bordenave-Montesquieu, D.; Dagnac, R. [Centre National de la Recherche Scientifique (CNRS), 31 - Toulouse (France)]|[Toulouse-3 Univ., 31 (France)

    1995-06-14

    A translational energy spectroscopy technique was used to study single-electron capture into the He{sup +} (n = 2) and He{sup +} (n 3) states in He{sup 2+}-D collisions. Differential cross sections were determined at 4, 6 and 8 keV in the angular range 5`-1{sup o}30` (laboratory frame). As expected, single-electron capture into the n = 2 state was found to be the dominant process; total cross sections for capture into the He{sup +} (n = 3) state were compared to other experimental and theoretical results. (author).

  9. Dielectrophoretic capture and genetic analysis of single neuroblastoma tumor cells

    Directory of Open Access Journals (Sweden)

    Erica L Carpenter

    2014-07-01

    Full Text Available Our understanding of the diversity of cells that escape the primary tumor and seed micrometastases remains rudimentary, and approaches for studying circulating and disseminated tumor cells have been limited by low throughput and sensitivity, reliance on single parameter sorting, and a focus on enumeration rather than phenotypic and genetic characterization. Here we utilize a highly sensitive microfluidic and dielectrophoretic approach for the isolation and genetic analysis of individual tumor cells. We employed fluorescence labeling to isolate 208 single cells from spiking experiments conducted with 11 cell lines, including 8 neuroblastoma cell lines, and achieved a capture sensitivity of 1 tumor cell per 106 white blood cells. Sample fixation or freezing had no detectable effect on cell capture. Point mutations were accurately detected in the whole genome amplification product of captured single tumor cells but not in negative control white blood cells. We applied this approach to capture 144 single tumor cells from 10 bone marrow samples from patients suffering from neuroblastoma. In this pediatric malignancy, high-risk patients often exhibit wide-spread hematogenous metastasis, but access to primary tumor can be difficult or impossible. Here we used flow-based sorting to pre-enrich samples with tumor involvement below 0.02%. For all patients for whom a mutation in the Anaplastic Lymphoma Kinase gene had already been detected in their primary tumor, the same mutation was detected in single cells from their marrow. These findings demonstrate a novel, non-invasive, and adaptable method for the capture and genetic analysis of single tumor cells from cancer patients.

  10. Dynamic Covalent Hydrogels for Triggered Cell Capture and Release.

    Science.gov (United States)

    Karimi, Fatemeh; Collins, Joe; Heath, Daniel E; Connal, Luke A

    2017-09-20

    A dual-responsive, cell capture and release surface was prepared through the incorporation of phenylboronic acid (PBA) groups into an oxime-based polyethylene glycol (PEG) hydrogel. Owing to its PEG-like properties, the unfunctionalized hydrogel was nonfouling. The use of highly efficient oxime chemistry allows the incorporation of commercially available 3,5-diformylphenyl boronic acid into the hydrogel matrix. Thus, the surface properties of the hydrogel were modified to enable reversible cell capture and release. Boronic ester formation between PBA groups and cell surface carbohydrates enabled efficient cell capture at pH 6.8. An increase to pH 7.8 resulted in cell detachment. This capture-and-release procedure was performed on MCF-7 human breast cancer cells, NIH-3T3 fibroblast cells, and primary human umbilical vein endothelial cells (HUVECs) and could be cycled with negligible loss in activity. The facile preparation of PBA-functionalized surfaces presented here has applications in biomedical fields such as cell diagnostics and cell culture.

  11. Ionization and capture in water: a multi-differential cross sections study

    Science.gov (United States)

    Champion, Christophe; Galassi, Mariel E.; Weck, Philippe F.; Fojón, Omar; Hanssen, Jocelyn; Rivarola, Roberto D.

    2012-11-01

    Two quantum mechanical models (CB1 and CDW-EIS) are here presented to provide accurate multiple differential and total cross sections for describing the two most important ionizing processes, namely, ionization and capture induced by heavy charged particles in water. A detailed study of the influence of the target description on the cross section calculations is also provided.

  12. A microfluidic dual-well device for high-throughput single-cell capture and culture.

    Science.gov (United States)

    Lin, Ching-Hui; Hsiao, Yi-Hsing; Chang, Hao-Chen; Yeh, Chuan-Feng; He, Cheng-Kun; Salm, Eric M; Chen, Chihchen; Chiu, Ing-Ming; Hsu, Chia-Hsien

    2015-07-21

    In vitro culture of single cells facilitates biological studies by deconvoluting complications from cell population heterogeneity. However, there is still a lack of simple yet high-throughput methods to perform single cell culture experiments. In this paper, we report the development and application of a microfluidic device with a dual-well (DW) design concept for high-yield single-cell loading (~77%) in large microwells (285 and 485 μm in diameter) which allowed for cell spreading, proliferation and differentiation. The increased single-cell loading yield is achieved by using sets of small microwells termed "capture-wells" and big microwells termed "culture-wells" according to their utilities for single-cell capture and culture, respectively. This novel device architecture allows the size of the "culture" microwells to be flexibly adjusted without affecting the single-cell loading efficiency making it useful for cell culture applications as demonstrated by our experiments of KT98 mouse neural stem cell differentiation, A549 and MDA-MB-435 cancer cell proliferation, and single-cell colony formation assay with A549 cells in this paper.

  13. Nanostructured Substrates for Capturing Circulating Tumor Cells in Whole Blood

    Science.gov (United States)

    Tseng, Hsian-Rong

    2009-03-01

    Over the past decade, circulating tumor cells (CTCs) has become an emerging ``biomarker'' for detecting early-stage cancer metastasis, predicting patient prognosis, as well as monitoring disease progression and therapeutic outcomes. However, isolation of CTCs has been technically challenging due to the extremely low abundance (a few to hundreds per ml) of CTCs among a high number of hematologic cells (109 per mL) in the blood. Our joint research team at UCLA has developed a new cell capture technology for quantification of CTCs in whole blood samples. Similar to most of the existing approaches, epithelial cell adhesion molecule antibody (anti-EpCAM) was grafted onto the surfaces to distinguish CTCs from the surrounding hematologic cells. The uniqueness of our technology is the use of nanostructured surfaces, which facilitates local topographical interactions between CTCs and substrates at the very first cell/substrate contacting time point. We demonstrated the ability of these nanostructured substrates to capture CTCs in whole blood samples with significantly improved efficiency and selectivity. The successful demonstration of this cell capture technology using brain, breast and prostate cancer cell lines encouraged us to test this approach in clinical setting. We have been able to bond our first validation study with a commercialized technology based on the use of immunomagnetic nanoparticles. A group of clinically well-characterized prostate cancer patients at UCLA hospital have been recruited and tested in parallel by these two technologies.

  14. Dynamic imaging of cell-free and cell-associated viral capture in mature dendritic cells.

    Science.gov (United States)

    Izquierdo-Useros, Nuria; Esteban, Olga; Rodriguez-Plata, Maria T; Erkizia, Itziar; Prado, Julia G; Blanco, Julià; García-Parajo, Maria F; Martinez-Picado, Javier

    2011-12-01

    Dendritic cells (DCs) capture human immunodeficiency virus (HIV) through a non-fusogenic mechanism that enables viral transmission to CD4(+) T cells, contributing to in vivo viral dissemination. Although previous studies have provided important clues to cell-free viral capture by mature DCs (mDCs), dynamic and kinetic insight on this process is still missing. Here, we used three-dimensional video microscopy and single-particle tracking approaches to dynamically dissect both cell-free and cell-associated viral capture by living mDCs. We show that cell-free virus capture by mDCs operates through three sequential phases: virus binding through specific determinants expressed in the viral particle, polarized or directional movements toward concrete regions of the cell membrane and virus accumulation in a sac-like structure where trapped viral particles display a hindered diffusive behavior. Moreover, real-time imaging of cell-associated viral transfer to mDCs showed a similar dynamics to that exhibited by cell-free virus endocytosis leading to viral accumulation in compartments. However, cell-associated HIV type 1 transfer to mDCs was the most effective pathway, boosted throughout enhanced cellular contacts with infected CD4(+) T cells. Our results suggest that in lymphoid tissues, mDC viral uptake could occur either by encountering cell-free or cell-associated virus produced by infected cells generating the perfect scenario to promote HIV pathogenesis and impact disease progression.

  15. CAPTURING EXHAUST CO2 GAS USING MOLTEN CARBONATE FUEL CELLS

    Directory of Open Access Journals (Sweden)

    Prateek Dhawan

    2016-03-01

    Full Text Available Carbon dioxide is considered as one of the major contenders when the question of greenhouse effect arises. So for any industry or power plant it is of utmost importance to follow certain increasingly stringent environment protection rules and laws. So it is significant to keep eye on any possible methods to reduce carbon dioxide emissions in an efficient way. This paper reviews the available literature so as to try to provide an insight of the possibility of using Molten Carbonate Fuel Cells (MCFCs as the carbon capturing and segregating devices and the various factors that affect the performance of MCFCs during the process of CO2 capture.

  16. Carboxybetaine methacrylate oligomer modified nylon for circulating tumor cells capture.

    Science.gov (United States)

    Dong, Chaoqun; Wang, Huiyu; Zhang, Zhuo; Zhang, Tao; Liu, Baorui

    2014-10-15

    Circulating tumor cells (CTC) capture is one of the most effective approaches in diagnosis and treatment of cancers in the field of personalized cancer medicine. In our study, zwitterionic carboxybetaine methacrylate (CBMA) oligomers were grafted onto nylon via atomic transfer random polymerization (ATRP) which would serve as a novel material for the development of convenient CTC capture interventional medical devices. The chemical, physical and biological properties of pristine and modified nylon surfaces were assessed by Fourier transform infrared spectra, atomic force microscope, water contact angle measurements, X-ray photoelectron spectroscopy, protein adsorption, platelet adhesion, and plasma recalcification time (PRT) determinations, etc. The results, including the significant decrease of proteins adsorption and platelets adhesion, as well as prolonged PRTs demonstrated the extraordinary biocompatibility and blood compatibility of the modified surface. Furthermore, we showed that upon immobilization of anti-epithelial cell adhesion molecular (anti-EpCAM) antibody onto the CBMA moiety, the modified nylon surface can selectively capture EpCAM positive tumor cells from blood with high efficiency, indicating the potential of the modified nylon in the manufacture of convenient interventional CTC capture medical devices.

  17. Magnetizable stent-grafts enable endothelial cell capture

    Science.gov (United States)

    Tefft, Brandon J.; Uthamaraj, Susheil; Harburn, J. Jonathan; Hlinomaz, Ota; Lerman, Amir; Dragomir-Daescu, Dan; Sandhu, Gurpreet S.

    2017-04-01

    Emerging nanotechnologies have enabled the use of magnetic forces to guide the movement of magnetically-labeled cells, drugs, and other therapeutic agents. Endothelial cells labeled with superparamagnetic iron oxide nanoparticles (SPION) have previously been captured on the surface of magnetizable 2205 duplex stainless steel stents in a porcine coronary implantation model. Recently, we have coated these stents with electrospun polyurethane nanofibers to fabricate prototype stent-grafts. Facilitated endothelialization may help improve the healing of arteries treated with stent-grafts, reduce the risk of thrombosis and restenosis, and enable small-caliber applications. When placed in a SPION-labeled endothelial cell suspension in the presence of an external magnetic field, magnetized stent-grafts successfully captured cells to the surface regions adjacent to the stent struts. Implantation within the coronary circulation of pigs (n=13) followed immediately by SPION-labeled autologous endothelial cell delivery resulted in widely patent devices with a thin, uniform neointima and no signs of thrombosis or inflammation at 7 days. Furthermore, the magnetized stent-grafts successfully captured and retained SPION-labeled endothelial cells to select regions adjacent to stent struts and between stent struts, whereas the non-magnetized control stent-grafts did not. Early results with these prototype devices are encouraging and further refinements will be necessary in order to achieve more uniform cell capture and complete endothelialization. Once optimized, this approach may lead to more rapid and complete healing of vascular stent-grafts with a concomitant improvement in long-term device performance.

  18. Microfluidic device for cell capture and impedance measurement.

    Science.gov (United States)

    Jang, Ling-Sheng; Wang, Min-How

    2007-10-01

    This work presents a microfluidic device to capture physically single cells within microstructures inside a channel and to measure the impedance of a single HeLa cell (human cervical epithelioid carcinoma) using impedance spectroscopy. The device includes a glass substrate with electrodes and a PDMS channel with micro pillars. The commercial software CFD-ACE+ is used to study the flow of the microstructures in the channel. According to simulation results, the probability of cell capture by three micro pillars is about 10%. An equivalent circuit model of the device is established and fits closely to the experimental results. The circuit can be modeled electrically as cell impedance in parallel with dielectric capacitance and in series with a pair of electrode resistors. The system is operated at low frequency between 1 and 100 kHz. In this study, experiments show that the HeLa cell is successfully captured by the micro pillars and its impedance is measured by impedance spectroscopy. The magnitude of the HeLa cell impedance declines at all operation voltages with frequency because the HeLa cell is capacitive. Additionally, increasing the operation voltage reduces the magnitude of the HeLa cell because a strong electric field may promote the exchange of ions between the cytoplasm and the isotonic solution. Below an operating voltage of 0.9 V, the system impedance response is characteristic of a parallel circuit at under 30 kHz and of a series circuit at between 30 and 100 kHz. The phase of the HeLa cell impedance is characteristic of a series circuit when the operation voltage exceeds 0.8 V because the cell impedance becomes significant.

  19. Regulators of Tfh cell differentiation

    Directory of Open Access Journals (Sweden)

    Gajendra Motiram Jogdand

    2016-11-01

    Full Text Available The follicular helper T (Tfh cells help is critical for activation of B cells, antibody class switching and germinal center formation. The Tfh cells are characterized by the expression of CXCR5, ICOS, PD-1, Bcl-6, and IL-21. They are involved in clearing infections and are adversely linked with autoimmune diseases and also have a role in viral replication as well as clearance. Tfh cells are generated from naïve CD4 T cells with sequential steps involving cytokine signaling (IL-21, IL-6, IL-12, activin A, migration and positioning in the germinal center by CXCR5, surface receptors (ICOS/ICOSL, SAP/SLAM as well as transcription factor (Bcl-6, c-Maf, STAT3 signaling and repressor miR155. On the other hand Tfh generation is negatively regulated at specific steps of Tfh generation by specific cytokine (IL-2, IL-7, surface receptor (PD-1, CTLA-4, transcription factors Blimp-1, STAT5, T-bet, KLF-2 signaling and repressor miR 146a. Interestingly, miR 17-92 and FOXO1 acts as a positive as well as a negative regulator of Tfh differentiation depending on the time of expression and disease specificity. Tfh cells are also generated from the conversion of other effector T cells as exemplified by Th1 cells converting into Tfh during viral infection. The mechanistic details of effector T cells conversion into Tfh are yet to be clear. To manipulate Tfh cells for therapeutic implication and or for effective vaccination strategies, it is important to know positive and negative regulators of Tfh generation. Hence, in this review we have highlighted and interlinked molecular signaling from cytokines, surface receptors, transcription factors, ubiquitin Ligase and miRNA as positive and negative regulators for Tfh differentiation.

  20. Transparent, biocompatible nanostructured surfaces for cancer cell capture and culture

    Directory of Open Access Journals (Sweden)

    Cheng BR

    2014-05-01

    Full Text Available Boran Cheng,1,* Zhaobo He,2,* Libo Zhao,2,* Yuan Fang,1 Yuanyuan Chen,1 Rongxiang He,2 Fangfang Chen,1 Haibin Song,1 Yuliang Deng,2 Xingzhong Zhao,2 Bin Xiong1 1Department of Oncology, Zhongnan Hospital of Wuhan University, Hubei Key Laboratory of Tumor Biological Behaviors, Hubei Cancer Clinical Study Center, Wuhan, Hubei, People’s Republic of China; 2Key Laboratory of Artificial Micro- and Nano-Structures of Ministry of Education, School of Physics and Technology, Wuhan University, Wuhan, Hubei, People’s Republic of China *These authors contributed equally to this work Abstract: Circulating tumor cells (CTCs in the blood which have detached from both the primary tumor and any metastases may be considered as a “liquid biopsy” and are expected to replace tumor biopsies in the monitoring of treatment response and determining patient prognosis. Here, we introduce a facile and efficient CTC detection material made of hydroxyapatite/chitosan (HA/CTS, which is beneficial because of its transparency and excellent biological compatibility. Atomic force microscopy images show that the roughness of the HA/CTS nanofilm (HA/CTSNF substrates can be controlled by changing the HA:CTS ratio. Enhanced local topographic interactions between nano-components on cancer cell membranes, and the antibody coated nanostructured substrate lead to improved CTC capture and separation. This remarkable nanostructured substrate has the potential for CTC culture in situ and merits further analysis. CTCs captured from artificial blood samples were observed in culture on HA/CTSNF substrates over a period of 14 days by using conventional staining methods (hematoxylin eosin and Wright’s stain. We conclude that these substrates are multifunctional materials capable of isolating and culturing CTCs for subsequent studies. Keywords: cell capture, cell culture, nanofilms, hydroxyapatite/chitosan

  1. Embryonic stem cell differentiation: A chromatin perspective

    OpenAIRE

    Rasmussen Theodore P

    2003-01-01

    Abstract Embryonic stem (ES) cells hold immense promise for the treatment of human degenerative disease. Because ES cells are pluripotent, they can be directed to differentiate into a number of alternative cell-types with potential therapeutic value. Such attempts at "rationally-directed ES cell differentiation" constitute attempts to recapitulate aspects of normal development in vitro. All differentiated cells retain identical DNA content, yet gene expression varies widely from cell-type to ...

  2. Studies for the application of boron neutron capture therapy to the treatment of differentiated thyroid cancer

    Energy Technology Data Exchange (ETDEWEB)

    Dagrosa, A., E-mail: dagrosa@cnea.gov.ar [Department of Radiobiology, National Atomic Energy Commission (CNEA), Av General Paz 1499, San Martin (1560) (Argentina)] [National Research Council of Argentina (CONICET), Rivadavia 1917, (1033) Buenos Aires (Argentina); Carpano, M. [Department of Radiobiology, National Atomic Energy Commission (CNEA), Av General Paz 1499, San Martin (1560) (Argentina); Perona, M. [Department of Radiobiology, National Atomic Energy Commission (CNEA), Av General Paz 1499, San Martin (1560) (Argentina)] [National Research Council of Argentina (CONICET), Rivadavia 1917, (1033) Buenos Aires (Argentina); Thomasz, L. [Department of Radiobiology, National Atomic Energy Commission (CNEA), Av General Paz 1499, San Martin (1560) (Argentina); Nievas, S. [Department of Chemistry, National Atomic Energy Commission (CNEA), Av General Paz 1499, San Martin (1560) (Argentina); Cabrini, R. [Department of Radiobiology, National Atomic Energy Commission (CNEA), Av General Paz 1499, San Martin (1560) (Argentina); Juvenal, G. [Department of Radiobiology, National Atomic Energy Commission (CNEA), Av General Paz 1499, San Martin (1560) (Argentina)] [National Research Council of Argentina (CONICET), Rivadavia 1917, (1033) Buenos Aires (Argentina); Pisarev, M. [Department of Radiobiology, National Atomic Energy Commission (CNEA), Av General Paz 1499, San Martin (1560) (Argentina)] [National Research Council of Argentina (CONICET), Rivadavia 1917, (1033) Buenos Aires (Argentina)] [Department of Biochemistry, UBA School of Medicine (Argentina)

    2011-12-15

    The aim of these studies was to evaluate the possibility of treating differentiated thyroid cancer by BNCT. These carcinomas are well controlled with surgery followed by therapy with {sup 131}I; however, some patients do not respond to this treatment. BPA uptake was analyzed both in vitro and in nude mice implanted with cell lines of differentiated thyroid carcinoma. The boron intracellular concentration in the different cell lines and the biodistribution studies showed the selectivity of the BPA uptake by this kind of tumor.

  3. Mesothelial cell differentiation into osteoblast- and adipocyte-like cells

    OpenAIRE

    Sally M Lansley; Searles, Richelle G.; Hoi, Aina; Thomas, Carla; Moneta, Helena; Herrick, Sarah E; Thompson, Philip J; Mark, Newman; Sterrett, Gregory F; Prêle, Cecilia M; Mutsaers, Steven E.

    2011-01-01

    Serosal pathologies including malignant mesothelioma (MM) can show features of osseous and/or cartilaginous differentiation although the mechanism for its formation is unknown. Mesothelial cells have the capacity to differentiate into cells with myofibroblast, smooth muscle and endothelial cell characteristics. Whether they can differentiate into other cell types is unclear. This study tests the hypothesis that mesothelial cells can differentiate into cell lineages of the embryonic mesoderm i...

  4. Nanobiotechnology for the capture and manipulation of circulating tumor cells.

    Science.gov (United States)

    Hughes, Andrew D; King, Michael R

    2012-01-01

    A necessary step in metastasis is the dissemination of malignant cells into the bloodstream, where cancer cells travel throughout the body as circulating tumor cells (CTC) in search of an opportunity to seed a secondary tumor. CTC represent a valuable diagnostic tool: evidence indicates that the quantity of CTC in the blood has been shown to relate to the severity of the illness, and samples are readily obtained through routine blood draws. As such, there has been a push toward developing technologies to reliably detect CTC using a variety of molecular and immunocytochemical techniques. In addition to their use in diagnostics, CTC detection systems that isolate CTC in such a way that the cells remain viable will allow for the performance of live-cell assays to facilitate the development of personalized cancer therapies. Moreover, techniques for the direct manipulation of CTC in circulation have been developed, intending to block metastasis in situ. We review a number of current and emerging micro- and nanobiotechnology approaches for the detection, capture, and manipulation of rare CTC aimed at advancing cancer treatment.

  5. Differentiation into Endoderm Lineage: Pancreatic differentiation from Embryonic Stem Cells

    OpenAIRE

    2011-01-01

    The endoderm gives rise to digestive and respiratory tracts, thyroid, liver, and pancreas. Representative disease of endoderm lineages is type 1 diabetes resulting from destruction of the insulin-producing β cells. Generation of functional β cells from human embryonic stem (ES) cells in vitro can be practical, renewable cell source for replacement cell therapy for type 1 diabetes. It has been achieved by progressive instructive differentiation through each of the developmental stages. In this...

  6. Regulating cell differentiation at different layers

    Institute of Scientific and Technical Information of China (English)

    Jiarui Wu

    2011-01-01

    Cell differentiation is a basic behavior in the developmental process of multi-cellular organisms,through which various cell types are generated from one embryonic cell for further building different tissues and organs of animals or plants.It is estimated that there are more than two hundred cell types in a human body.To understand the molecular mechanisms of cell differentiation,researchers usually focus on a question how particular genes are selectively expressed during the differentiation process.However,more and more evidence indicates that the regulation of cell differentiation is far beyond simply controlling the expression of genetic program,which is supported by the collection of four research articles in this issue that the regulation of cell differentiation involves various factors at different layers,including epigenetics,metabolism and cell-cell interaction.

  7. Nuclear Mechanics and Stem Cell Differentiation.

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    Mao, Xinjian; Gavara, Nuria; Song, Guanbin

    2015-12-01

    Stem cells are characterized by their self-renewal and multi-lineage differentiation potential. Stem cell differentiation is a prerequisite for the application of stem cells in regenerative medicine and clinical therapy. In addition to chemical stimulation, mechanical cues play a significant role in regulating stem cell differentiation. The integrity of mechanical sensors is necessary for the ability of cells to respond to mechanical signals. The nucleus, the largest and stiffest cellular organelle, interacts with the cytoskeleton as a key mediator of cell mechanics. Nuclear mechanics are involved in the complicated interactions of lamins, chromatin and nucleoskeleton-related proteins. Thus, stem cell differentiation is intimately associated with nuclear mechanics due to its indispensable role in mechanotransduction and mechanical response. This paper reviews several main contributions of nuclear mechanics, highlights the hallmarks of the nuclear mechanics of stem cells, and provides insight into the relationship between nuclear mechanics and stem cell differentiation, which may guide clinical applications in the future.

  8. Isologous diversification a theory of cell differentiation

    CERN Document Server

    Kaneko, K; Kaneko, Kunihiko; Yomo, Tetsuya

    1996-01-01

    Isologous diversification theory for cell differentiation is proposed, based on simulations of interacting cells with biochemical networks and cell division process following consumption of some chemicals. According to the simulations of the interaction-based dynamical systems model, the following scenario of the cell differentiation is proposed. (1) Up to some threshold number, divisions bring about almost identical cells with synchronized biochemical oscillations. (2)As the number is increased the oscillations lose the synchrony, leading to groups of cells with different phases of oscillations. (3)Amplitudes of oscillation and averaged chemical compositions start to differ by groups of cells. The differentiated behavior of states is transmitted to daughter cells. (4)Recursivity is formed so that the daughter cells keep the identical chemical character. This ``memory" is made possible through the transfer of initial conditions. (5) Successive differentiation proceeds. Mechanism of tumor cell formation, origi...

  9. Differential Proteomics in Malignant and Normal Liver Cell Lines

    Institute of Scientific and Technical Information of China (English)

    LIU Zhi-jun; WANG Bin; YAN Zhi-yong; QIAN Dong-meng; SONG Xu-xia; Ding Shou-yi; BAI Zhi-qiang

    2007-01-01

    Objective: To detect differential protein expression in malignant and normal liver cell lines in vitro using the SELDI ProteinChip platform, for investigating the pathogenesis of liver cancer. Methods: Two cell lines, human normal liver cell line L02 and hepatoma cell line SMMC-7721 were cultured routinely, harvested in good condition and lysed. After quantification, the supernatant of the lysate was tested by IMAC3 (Immobilized Mental Affinity Capture) and WCX2 (Weak Cation Exchange) chips on the SELDI-TOF-MS ProteinChip reader. Results: Protein expression differed between the malignant and normal liver cell lines. A total of 20 differentially expressed proteins were found, among which, 7 were captured by the IMAC3 chip and 14 by the WCX2 chip. Peaks at 5,419, 7,979 and 11,265 Da were higher and at 8,103, 8,492, 10,160 and 11,304 Da lower in SMMC-7721 cells by the IMAC3 chip; peaks at 7,517, 7,945 and 7,979 Da were higher and at 5,061, 5,551, 5,818, 7,439, 9,401,10,100, 10,312, 11,621, 11,662, 11,830 and 12,772 Da lower in SMMC-7721 cells by the WCX2 chip. Interestingly, both chips captured the 7,979 Da peak. In addition, the 11,081 Da peak corresponded precisely with the molecular mass of the calcium binding protein S100A10, which may participate in the formation of liver cancer in association with p36. Conclusion: Detecting differential protein expression in malignant and normal liver cell lines using the SELDI ProteinChip platform was simple, sensitive and repeatable. The results we obtained can serve as a basis for investigating the pathogenesis of liver cancer and aid the discovery of new therapeutic targets.

  10. Improved method for bacterial cell capture after flow cytometry cell sorting.

    Science.gov (United States)

    Guillebault, D; Laghdass, M; Catala, P; Obernosterer, I; Lebaron, P

    2010-11-01

    Fixed cells with different nucleic acid contents and scatter properties (low nucleic acid [LNA], high nucleic acid 1 [HNA1], and HNA2) were sorted by flow cytometry (FCM). For each sort, 10,000 cells were efficiently captured on poly-l-lysine-coated microplates, resulting in efficient and reproducible PCR amplification.

  11. Embryonic stem cell differentiation: a chromatin perspective.

    Science.gov (United States)

    Rasmussen, Theodore P

    2003-11-13

    Embryonic stem (ES) cells hold immense promise for the treatment of human degenerative disease. Because ES cells are pluripotent, they can be directed to differentiate into a number of alternative cell-types with potential therapeutic value. Such attempts at "rationally-directed ES cell differentiation" constitute attempts to recapitulate aspects of normal development in vitro. All differentiated cells retain identical DNA content, yet gene expression varies widely from cell-type to cell-type. Therefore, a potent epigenetic system has evolved to coordinate and maintain tissue-specific patterns of gene expression. Recent advances show that mechanisms that govern epigenetic regulation of gene expression are rooted in the details of chromatin dynamics. As embryonic cells differentiate, certain genes are activated while others are silenced. These activation and silencing events are exquisitely coordinated with the allocation of cell lineages. Remodeling of the chromatin of developmentally-regulated genes occurs in conjunction with lineage commitment. Oocytes, early embryos, and ES cells contain potent chromatin-remodeling activities, an observation that suggests that chromatin dynamics may be especially important for early lineage decisions. Chromatin dynamics are also involved in the differentiation of adult stem cells, where the assembly of specialized chromatin upon tissue-specific genes has been studied in fine detail. The next few years will likely yield striking advances in the understanding of stem cell differentiation and developmental biology from the perspective of chromatin dynamics.

  12. Embryonic stem cell differentiation: A chromatin perspective

    Directory of Open Access Journals (Sweden)

    Rasmussen Theodore P

    2003-11-01

    Full Text Available Abstract Embryonic stem (ES cells hold immense promise for the treatment of human degenerative disease. Because ES cells are pluripotent, they can be directed to differentiate into a number of alternative cell-types with potential therapeutic value. Such attempts at "rationally-directed ES cell differentiation" constitute attempts to recapitulate aspects of normal development in vitro. All differentiated cells retain identical DNA content, yet gene expression varies widely from cell-type to cell-type. Therefore, a potent epigenetic system has evolved to coordinate and maintain tissue-specific patterns of gene expression. Recent advances show that mechanisms that govern epigenetic regulation of gene expression are rooted in the details of chromatin dynamics. As embryonic cells differentiate, certain genes are activated while others are silenced. These activation and silencing events are exquisitely coordinated with the allocation of cell lineages. Remodeling of the chromatin of developmentally-regulated genes occurs in conjunction with lineage commitment. Oocytes, early embryos, and ES cells contain potent chromatin-remodeling activities, an observation that suggests that chromatin dynamics may be especially important for early lineage decisions. Chromatin dynamics are also involved in the differentiation of adult stem cells, where the assembly of specialized chromatin upon tissue-specific genes has been studied in fine detail. The next few years will likely yield striking advances in the understanding of stem cell differentiation and developmental biology from the perspective of chromatin dynamics.

  13. A photonic crystal hydrogel suspension array for the capture of blood cells from whole blood

    Science.gov (United States)

    Zhang, Bin; Cai, Yunlang; Shang, Luoran; Wang, Huan; Cheng, Yao; Rong, Fei; Gu, Zhongze; Zhao, Yuanjin

    2016-02-01

    Diagnosing hematological disorders based on the separation and detection of cells in the patient's blood is a significant challenge. We have developed a novel barcode particle-based suspension array that can simultaneously capture and detect multiple types of blood cells. The barcode particles are polyacrylamide (PAAm) hydrogel inverse opal microcarriers with characteristic reflection peak codes that remain stable during cell capture on their surfaces. The hydrophilic PAAm hydrogel scaffolds of the barcode particles can entrap various plasma proteins to capture different cells in the blood, with little damage to captured cells.Diagnosing hematological disorders based on the separation and detection of cells in the patient's blood is a significant challenge. We have developed a novel barcode particle-based suspension array that can simultaneously capture and detect multiple types of blood cells. The barcode particles are polyacrylamide (PAAm) hydrogel inverse opal microcarriers with characteristic reflection peak codes that remain stable during cell capture on their surfaces. The hydrophilic PAAm hydrogel scaffolds of the barcode particles can entrap various plasma proteins to capture different cells in the blood, with little damage to captured cells. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06368j

  14. Neurogenic differentiation of amniotic fluid stem cells.

    Science.gov (United States)

    Rosner, M; Mikula, M; Preitschopf, A; Feichtinger, M; Schipany, K; Hengstschläger, M

    2012-05-01

    In 2003, human amniotic fluid has been shown to contain stem cells expressing Oct-4, a marker for pluripotency. This finding initiated a rapidly growing and very promising new stem cell research field. Since then, amniotic fluid stem (AFS) cells have been demonstrated to harbour the potential to differentiate into any of the three germ layers and to form three-dimensional aggregates, so-called embryoid bodies, known as the principal step in the differentiation of pluripotent stem cells. Marker selection and minimal dilution approaches allow the establishment of monoclonal AFS cell lineages with high proliferation potential. AFS cells have a lower risk for tumour development and do not raise the ethical issues of embryonic stem cells. Compared to induced pluripotent stem cells, AFS cells do not need exogenic treatment to induce pluripotency, are chromosomal stable and do not harbour the epigenetic memory and accumulated somatic mutations of specific differentiated source cells. Compared to adult stem cells, AFS can be grown in larger quantities and show higher differentiation potential. Accordingly, in the recent past, AFS became increasingly accepted as an optimal tool for basic research and probably also for specific cell-based therapies. Here, we review the current knowledge on the neurogenic differentiation potential of AFS cells.

  15. Cell Division, Differentiation and Dynamic Clustering

    CERN Document Server

    Kaneko, K; Kaneko, Kunihiko; Yomo, Tetsuya

    1993-01-01

    A novel mechanism for cell differentiation is proposed, based on the dynamic clustering in a globally coupled chaotic system. A simple model with metabolic reaction, active transport of chemicals from media, and cell division is found to show three successive stages with the growth of the number of cells; coherent growth, dynamic clustering, and fixed cell differentiation. At the last stage, disparity in activities, germ line segregation, somatic cell differentiation, and homeochaotic stability against external perturbation are found. Our results, in consistency with the experiments of the preceding paper, imply that cell differentiation can occur without a spatial pattern. From dynamical systems viewpoint, the new concept of ``open chaos" is proposed, as a novel and general scenario for systems with growing numbers of elements, also seen in economics and sociology.A

  16. Transcriptome changes during intestinal cell differentiation

    DEFF Research Database (Denmark)

    Tadjali, Mehrdad; Seidelin, Jakob B; Olsen, Jørgen Lillelund

    2002-01-01

    The expression of 18149 genes have been analysed during the differentiation of the human intestinal cell line Caco-2. cDNA probes from undifferentiated and differentiated Caco-2 cells were separately hybridised to EST DNAs spotted in an array on a nylon membrane. A remarkable change...... in the transcriptome was observed during the differentiation of the Caco-2 cells. 8762 of the 18149 genes analysed were expressed above background level in the undifferentiated Caco-2 cells, whereas only 5767 genes were expressed above background in differentiated Caco-2 cells. This pattern of expression was caused...... by a general down-regulation of genes in the low abundance class. Similar results were found using mouse small intestinal crypt and villus cells, suggesting that the phenomenon also occurs in the intestine in vivo. The expression data were subsequently used in a search for markers for subsets of epithelial...

  17. Stem cell differentiation and human liver disease

    Institute of Scientific and Technical Information of China (English)

    Wen-Li Zhou; Claire N Medine; Liang Zhu; David C Hay

    2012-01-01

    Human stem cells are scalable cell populations capable of cellular differentiation.This makes them a very attractive in vitro cellular resource and in theory provides unlimited amounts of primary cells.Such an approach has the potential to improve our understanding of human biology and treating disease.In the future it may be possible to deploy novel stem cell-based approaches to treat human liver diseases.In recent years,efficient hepatic differentiation from human stem cells has been achieved by several research groups including our own.In this review we provide an overview of the field and discuss the future potential and limitations of stem cell technology.

  18. Simulated Microgravity Modulates Differentiation Processes of Embryonic Stem Cells

    Directory of Open Access Journals (Sweden)

    Vaibhav Shinde

    2016-04-01

    Full Text Available Background/Aims: Embryonic developmental studies under microgravity conditions in space are very limited. To study the effects of altered gravity on the embryonic development processes we established an in vitro methodology allowing differentiation of mouse embryonic stem cells (mESCs under simulated microgravity within a fast-rotating clinostat (clinorotation and capture of microarray-based gene signatures. Methods: The differentiating mESCs were cultured in a 2D pipette clinostat. The microarray and bioinformatics tools were used to capture genes that are deregulated by simulated microgravity and their impact on developmental biological processes. Results: The data analysis demonstrated that differentiation of mESCs in pipettes for 3 days resultet to early germ layer differentiation and then to the different somatic cell types after further 7 days of differentiation in the Petri dishes. Clinorotation influences differentiation as well as non-differentiation related biological processes like cytoskeleton related 19 genes were modulated. Notably, simulated microgravity deregulated genes Cyr61, Thbs1, Parva, Dhrs3, Jun, Tpm1, Fzd2 and Dll1 are involved in heart morphogenesis as an acute response on day 3. If the stem cells were further cultivated under normal gravity conditions (1 g after clinorotation, the expression of cardiomyocytes specific genes such as Tnnt2, Rbp4, Tnni1, Csrp3, Nppb and Mybpc3 on day 10 was inhibited. This correlated well with a decreasing beating activity of the 10-days old embryoid bodies (EBs. Finally, we captured Gadd45g, Jun, Thbs1, Cyr61and Dll1 genes whose expressions were modulated by simulated microgravity and by real microgravity in various reported studies. Simulated microgravity also deregulated genes belonging to the MAP kinase and focal dhesion signal transduction pathways. Conclusion: One of the most prominent biological processes affected by simulated microgravity was the process of cardiomyogenesis. The

  19. Cell death and renewal during prey capture and digestion in the carnivorous sponge Asbestopluma hypogea (Porifera: Poecilosclerida).

    Science.gov (United States)

    Martinand-Mari, Camille; Vacelet, Jean; Nickel, Michael; Wörheide, Gert; Mangeat, Paul; Baghdiguian, Stephen

    2012-11-15

    The sponge Asbestopluma hypogea is unusual among sponges due to its peculiar carnivorous feeding habit. During various stages of its nutrition cycle, the sponge is subjected to spectacular morphological modifications. Starved animals are characterized by many elongated filaments, which are crucial for the capture of prey. After capture, and during the digestion process, these filaments actively regress before being regenerated during a subsequent period of starvation. Here, we demonstrate that these morphological events rely on a highly dynamic cellular turnover, implying a coordinated sequence of programmed cell death (apoptosis and autophagy), cell proliferation and cell migration. A candidate niche for cell renewal by stem cell proliferation and differentiation was identified at the base of the sponge peduncle, characterized by higher levels of BrdU/EdU incorporation. Therefore, BrdU/EdU-positive cells of the peduncle base are candidate motile cells responsible for the regeneration of the prey-capturing main sponge body, i.e. the dynamic filaments. Altogether, our results demonstrate that dynamics of cell renewal in sponge appear to be regulated by cellular mechanisms as multiple and complex as those already identified in bilaterian metazoans.

  20. EpCAM-independent capture of circulating tumor cells with a 'universal CTC-chip'.

    Science.gov (United States)

    Chikaishi, Yasuhiro; Yoneda, Kazue; Ohnaga, Takashi; Tanaka, Fumihiro

    2017-01-01

    Capture of circulating tumor cells (CTCs), which are shed from the primary tumor site and circulate in the blood, remains a technical challenge. CellSearch® is the only clinically approved CTC detection system, but has provided only modest sensitivity in detecting CTCs mainly because epithelial cell adhesion molecule (EpCAM)-negative tumor cells may not be captured. To achieve more sensitive CTC‑capture, we have developed a novel microfluidic platform, a 'CTC-chip' comprised of light-curable resins that has a unique advantage in that any capture antibody is easily conjugated. In the present study, we showed that EpCAM-negative tumor cells as well as EpCAM-positive cells were captured with the novel 'universal CTC-chip' as follows: i) human lung cancer cells (PC-9), with strong EpCAM expression, were efficiently captured with the CTC-chip coated with an anti-EpCAM antibody (with an average capture efficiency of 101% when tumor cells were spiked in phosphate‑buffered saline (PBS) and 88% when spiked in blood); ii) human mesothelioma cells (ACC-MESO-4), with no EpCAM expression but with podoplanin expression, were captured with the CTC-chip coated with an anti-podoplanin antibody (average capture efficiency of 78% when tumor cells were spiked in PBS and 38% when spiked in blood), whereas ACC-MESO-4 cells were not captured with the CTC-chip coated with the anti-EpCAM antibody. These results indicate that the novel 'CTC-chip' can be useful in sensitive EpCAM-independent detection of CTCs, which may provide new insights into personalized medicine.

  1. T cells kill bacteria captured by transinfection from dendritic cells and confer protection in mice.

    Science.gov (United States)

    Cruz-Adalia, Aránzazu; Ramirez-Santiago, Guillermo; Calabia-Linares, Carmen; Torres-Torresano, Mónica; Feo, Lidia; Galán-Díez, Marta; Fernández-Ruiz, Elena; Pereiro, Eva; Guttmann, Peter; Chiappi, Michele; Schneider, Gerd; Carrascosa, José López; Chichón, Francisco Javier; Martínez Del Hoyo, Gloria; Sánchez-Madrid, Francisco; Veiga, Esteban

    2014-05-14

    Dendritic cells (DCs) phagocytose, process, and present bacterial antigens to T lymphocytes to trigger adaptive immunity. In vivo, bacteria can also be found inside T lymphocytes. However, T cells are refractory to direct bacterial infection, leaving the mechanisms by which bacteria invade T cells unclear. We show that T cells take up bacteria from infected DCs by the process of transinfection, which requires direct contact between the two cells and is enhanced by antigen recognition. Prior to transfer, bacteria localize to the immunological synapse, an intimate DC/T cell contact structure that activates T cells. Strikingly, T cells efficiently eliminate the transinfecting bacteria within the first hours after infection. Transinfected T cells produced high levels of proinflammatory cytokines and were able to protect mice from bacterial challenge following adoptive transfer. Thus, T lymphocytes can capture and kill bacteria in a manner reminiscent of innate immunity.

  2. A Structured Population Model of Cell Differentiation

    CERN Document Server

    Doumic, Marie; Perthame, Benoit; Zubelli, Jorge P

    2010-01-01

    We introduce and analyze several aspects of a new model for cell differentiation. It assumes that differentiation of progenitor cells is a continuous process. From the mathematical point of view, it is based on partial differential equations of transport type. Specifically, it consists of a structured population equation with a nonlinear feedback loop. This models the signaling process due to cytokines, which regulate the differentiation and proliferation process. We compare the continuous model to its discrete counterpart, a multi-compartmental model of a discrete collection of cell subpopulations recently proposed by Marciniak-Czochra et al. in 2009 to investigate the dynamics of the hematopoietic system. We obtain uniform bounds for the solutions, characterize steady state solutions, and analyze their linearized stability. We show how persistence or extinction might occur according to values of parameters that characterize the stem cells self-renewal. We also perform numerical simulations and discuss the q...

  3. Differential estimates of southern flying squirrel (Glaucomys volans) population structure based on capture method

    Science.gov (United States)

    Kevin S. Laves; Susan C. Loeb

    2005-01-01

    It is commonly assumed that population estimates derived from trapping small mammals are accurate and unbiased or that estimates derived from different capture methods are comparable. We captured southern flying squirrels (Glaucmrtys volam) using two methods to study their effect on red-cockaded woodpecker (Picoides bumah) reproductive success. Southern flying...

  4. Differential geometry meets the cell.

    Science.gov (United States)

    Marshall, Wallace F

    2013-07-18

    A new study by Terasaki et al. highlights the role of physical forces in biological form by showing that connections between stacked endoplasmic reticulum cisternae have a shape well known in classical differential geometry, the helicoid, and that this shape is a predictable consequence of membrane physics.

  5. Nucleotide excision repair in differentiated cells

    Energy Technology Data Exchange (ETDEWEB)

    Wees, Caroline van der [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Department of Cardiology, Leiden University Medical Center, Leiden (Netherlands); Jansen, Jacob [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Vrieling, Harry [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Laarse, Arnoud van der [Department of Cardiology, Leiden University Medical Center, Leiden (Netherlands); Zeeland, Albert van [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands); Mullenders, Leon [Department of Toxicogenetics, Leiden University Medical Center, Leiden (Netherlands)]. E-mail: l.mullenders@lumc.nl

    2007-01-03

    Nucleotide excision repair (NER) is the principal pathway for the removal of a wide range of DNA helix-distorting lesions and operates via two NER subpathways, i.e. global genome repair (GGR) and transcription-coupled repair (TCR). Although detailed information is available on expression and efficiency of NER in established mammalian cell lines, little is known about the expression of NER pathways in (terminally) differentiated cells. The majority of studies in differentiated cells have focused on repair of UV-induced cyclobutane pyrimidine dimers (CPD) and 6-4-photoproducts (6-4PP) because of the high frequency of photolesions at low level of toxicity and availability of sensitive technologies to determine photolesions in defined regions of the genome. The picture that emerges from these studies is blurred and rather complex. Fibroblasts and terminally differentiated myocytes of the rat heart display equally efficient GGR of 6-4PP but poor repair of CPD due to the absence of p48 expression. This repair phenotype is clearly different from human terminal differentiated neurons. Furthermore, both cell types were found to carry out TCR of CPD, thus mimicking the repair phenotype of established rodent cell lines. In contrast, in intact rat spermatogenic cells repair was very inefficient at the genome overall level and in transcriptionally active genes indicating that GGR and TCR are non-functional. Also, non-differentiated mouse embryonic stem (ES) cells exhibit low levels of NER after UV irradiation. However, the mechanisms that lead to low NER activity are clearly different: in differentiated spermatogenic cells differences in chromatin compaction and sequestering of NER proteins may underlie the lack of NER activity in pre-meiotic cells, whereas in non-differentiated ES cells NER is impaired by a strong apoptotic response.

  6. Osteogenic Differentiation of Dental Follicle Stem Cells

    Directory of Open Access Journals (Sweden)

    Giorgio Mori, Andrea Ballini, Claudia Carbone, Angela Oranger, Giacomina Brunetti, Adriana Di Benedetto, Biagio Rapone, Stefania Cantore, Mariasevera Di Comite, Silvia Colucci, Maria Grano, Felice R. Grassi

    2012-01-01

    Full Text Available Background: Stem cells are defined as clonogenic cells capable of self-renewal and multi-lineage differentiation. A population of these cells has been identified in human Dental Follicle (DF.Dental Follicle Stem Cells (DFSCs were found in pediatric unerupted wisdom teeth and have been shown to differentiate, under particular conditions, into various cell types of the mesenchymal tissues.Aim: The aim of this study was to investigate if cells isolated from DF show stem features, differentiate toward osteoblastic phenotype and express osteoblastic markers.Methods: We studied the immunophenotype of DFSCs by flow cytometric analysis, the osteoblastic markers of differentiated DFSCs were assayed by histochemical methods and real-time PCR.Results: We demonstrated that DFSCs expressed a heterogeneous assortment of makers associated with stemness. Moreover DFSCs differentiated into osteoblast-like cells, producing mineralized matrix nodules and expressed the typical osteoblastic markers, Alkaline Phosphatase (ALP and Collagen I (Coll I.Conclusion: This study suggests that DFSCs may provide a cell source for tissue engineering of bone.

  7. Mesothelial cell differentiation into osteoblast- and adipocyte-like cells.

    Science.gov (United States)

    Lansley, Sally M; Searles, Richelle G; Hoi, Aina; Thomas, Carla; Moneta, Helena; Herrick, Sarah E; Thompson, Philip J; Newman, Mark; Sterrett, Gregory F; Prêle, Cecilia M; Mutsaers, Steven E

    2011-10-01

    Serosal pathologies including malignant mesothelioma (MM) can show features of osseous and/or cartilaginous differentiation although the mechanism for its formation is unknown. Mesothelial cells have the capacity to differentiate into cells with myofibroblast, smooth muscle and endothelial cell characteristics. Whether they can differentiate into other cell types is unclear. This study tests the hypothesis that mesothelial cells can differentiate into cell lineages of the embryonic mesoderm including osteoblasts and adipocytes. To examine this, a functional assay of bone formation and an adipogenic assay were performed in vitro with primary rat and human mesothelial cells maintained in osteogenic or adipogenic medium (AM) for 0-26 days. Mesothelial cells expressed increasing levels of alkaline phosphatase, an early marker of the osteoblast phenotype, and formed mineralized bone-like nodules. Mesothelial cells also accumulated lipid indicative of a mature adipocyte phenotype when cultured in AM. All cells expressed several key osteoblast and adipocyte markers, including osteoblast-specific runt-related transcription factor 2, and demonstrated changes in mRNA expression consistent with epithelial-to-mesenchymal transition. In conclusion, these studies confirm that mesothelial cells have the capacity to differentiate into osteoblast- and adipocyte-like cells, providing definitive evidence of their multipotential nature. These data strongly support mesothelial cell differentiation as the potential source of different tissue types in MM tumours and other serosal pathologies, and add support for the use of mesothelial cells in regenerative therapies.

  8. Study of differential proteins in lung adenocarcinoma using laser capture microdissection combined with liquid chip-mass spectrometry technology

    Institute of Scientific and Technical Information of China (English)

    BU Li-na; LIN Xiu-li; LIU Yan-feng; LIN Yu-rong; RONG Biao-xue; YANG Shuan-ying; LI Feng-tao; SHANG Wen-li; ZHANG Wei; HUO Shu-fen; NAN Yan-dong; TIAN Ying-xuan; DU Jie

    2010-01-01

    Background In recent years the proportion of lung adenocarcinoma (adCA) which occurs in lung cancer patients has increased. Using laser capture microdissection (LCM) combined with liquid chip-mass spectrometry technology, we aimed to screen lung cancer biomarkers by studying the proteins in the tissues of adCA.Methods We used LCM and magnetic bead based weak cation exchange (MB-WCX) to separate and purify the homogeneous adCA cells and normal calls from six cases of fresh adCA and matched normal lung tissues. The proteins were analyzed and identified by matrix assisted laser desorption/ionization time-of-fight mass spectrometry (MALDI-OF-MS). We screened for the best pattern using a radial basic function neural network algorithm.Results About 2.895x106 and 1.584x106 cells were satisfactorily obtained by LCM from six cases of fresh lung adCA and matched normal lung tissues, respectively. The homogeneities of cell population were estimated to be over 95% as determined by microscopic visualization. Comparing the differentially expressed proteins between the lung adCA and the matched normal lung group, 221 and 239 protein peaks, respectively, were found in the mass-to-charge ration (M/Z)between 800 Da and 10 000 Da. According to t test, the expression of two protein peaks at 7521.5 M/Z and 5079.3 M/Z had the largest difference between tissues. They were more weakly expressed in the lung adCA compared to the matched normal group. The two protein peaks could accurately separate the lung adCA from the matched normal lung group by the sample distribution chart. A discriminatory pattern which can separate the lung adCA from the matched normal lung tissue consisting of three proteins at 3358.1 M/Z, 5079.3 M/Z and 7521.5 M/Z was established by a radial basic function neural network algorithm with a sensitivity of 100% and a specificity of 100%.Conclusions Differential proteins in lung adCA were screened using LCM combined with liquid chip-mass spectrometry technology, and a

  9. Differential Estimates of Southern Flying Squirrel (Glaucomys volans) Population Structure Based on Capture Method.

    Energy Technology Data Exchange (ETDEWEB)

    Laves, Kevin S.; Loeb, Susan C.

    2006-01-01

    ABSTRACT.—It is commonly assumed that population estimates derived from trapping small mammals are accurate and unbiased or that estimates derived from different capture methods are comparable. We captured southern flying squirrels (Glaucomys volans) using two methods to study their effect on red-cockaded woodpecker (Picoides borealis) reproductive success. Southern flying squirrels were captured at and removed from 30 red-cockaded woodpecker cluster sites during March to July 1994 and 1995 using Sherman traps placed in a grid encompassing a red-cockaded woodpecker nest tree and by hand from red-cockaded woodpecker cavities. Totals of 195 (1994) and 190 (1995) red-cockaded woodpecker cavities were examined at least three times each year. Trappability of southern flying squirrels in Sherman traps was significantly greater in 1995 (1.18%; 22,384 trap nights) than in 1994 (0.42%; 20,384 trap nights), and capture rate of southern flying squirrels in cavities was significantly greater in 1994 (22.7%; 502 cavity inspections) than in 1995 (10.8%; 555 cavity inspections). However, more southern flying squirrels were captured per cavity inspection than per Sherman trap night in both years. Male southern flying squirrels were more likely to be captured from cavities than in Sherman traps in 1994, but not in 1995. Both male and female juveniles were more likely to be captured in cavities than in traps in both years. In 1994 males in reproductive condition were more likely to be captured in cavities than in traps and in 1995 we captured significantly more reproductive females in cavities than in traps. Our data suggest that population estimates based solely on one trapping method may not represent true population size or structure of southern flying squirrels.

  10. Nanotopographical Control of Stem Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Laura E. McNamara

    2010-01-01

    Full Text Available Stem cells have the capacity to differentiate into various lineages, and the ability to reliably direct stem cell fate determination would have tremendous potential for basic research and clinical therapy. Nanotopography provides a useful tool for guiding differentiation, as the features are more durable than surface chemistry and can be modified in size and shape to suit the desired application. In this paper, nanotopography is examined as a means to guide differentiation, and its application is described in the context of different subsets of stem cells, with a particular focus on skeletal (mesenchymal stem cells. To address the mechanistic basis underlying the topographical effects on stem cells, the likely contributions of indirect (biochemical signal-mediated and direct (force-mediated mechanotransduction are discussed. Data from proteomic research is also outlined in relation to topography-mediated fate determination, as this approach provides insight into the global molecular changes at the level of the functional effectors.

  11. Biophysical regulation of stem cell differentiation.

    Science.gov (United States)

    Govey, Peter M; Loiselle, Alayna E; Donahue, Henry J

    2013-06-01

    Bone adaptation to its mechanical environment, from embryonic through adult life, is thought to be the product of increased osteoblastic differentiation from mesenchymal stem cells. In parallel with tissue-scale loading, these heterogeneous populations of multipotent stem cells are subject to a variety of biophysical cues within their native microenvironments. Bone marrow-derived mesenchymal stem cells-the most broadly studied source of osteoblastic progenitors-undergo osteoblastic differentiation in vitro in response to biophysical signals, including hydrostatic pressure, fluid flow and accompanying shear stress, substrate strain and stiffness, substrate topography, and electromagnetic fields. Furthermore, stem cells may be subject to indirect regulation by mechano-sensing osteocytes positioned to more readily detect these same loading-induced signals within the bone matrix. Such paracrine and juxtacrine regulation of differentiation by osteocytes occurs in vitro. Further studies are needed to confirm both direct and indirect mechanisms of biophysical regulation within the in vivo stem cell niche.

  12. Translatome analyses capture of opposing tissue-specific brassinosteroid signals orchestrating root meristem differentiation.

    Science.gov (United States)

    Vragović, Kristina; Sela, Ayala; Friedlander-Shani, Lilach; Fridman, Yulia; Hacham, Yael; Holland, Neta; Bartom, Elizabeth; Mockler, Todd C; Savaldi-Goldstein, Sigal

    2015-01-20

    The mechanisms ensuring balanced growth remain a critical question in developmental biology. In plants, this balance relies on spatiotemporal integration of hormonal signaling pathways, but the understanding of the precise contribution of each hormone is just beginning to take form. Brassinosteroid (BR) hormone is shown here to have opposing effects on root meristem size, depending on its site of action. BR is demonstrated to both delay and promote onset of stem cell daughter differentiation, when acting in the outer tissue of the root meristem, the epidermis, and the innermost tissue, the stele, respectively. To understand the molecular basis of this phenomenon, a comprehensive spatiotemporal translatome mapping of Arabidopsis roots was performed. Analyses of wild type and mutants featuring different distributions of BR revealed autonomous, tissue-specific gene responses to BR, implying its contrasting tissue-dependent impact on growth. BR-induced genes were primarily detected in epidermal cells of the basal meristem zone and were enriched by auxin-related genes. In contrast, repressed BR genes prevailed in the stele of the apical meristem zone. Furthermore, auxin was found to mediate the growth-promoting impact of BR signaling originating in the epidermis, whereas BR signaling in the stele buffered this effect. We propose that context-specific BR activity and responses are oppositely interpreted at the organ level, ensuring coherent growth.

  13. Immobilized surfactant-nanotube complexes support selectin-mediated capture of viable circulating tumor cells in the absence of capture antibodies.

    Science.gov (United States)

    Mitchell, Michael J; Castellanos, Carlos A; King, Michael R

    2015-10-01

    The metastatic spread of tumor cells from the primary site to anatomically distant organs leads to a poor patient prognosis. Increasing evidence has linked adhesive interactions between circulating tumor cells (CTCs) and endothelial cells to metastatic dissemination. Microscale biomimetic flow devices hold promise as a diagnostic tool to isolate CTCs and develop metastatic therapies, utilizing E-selectin (ES) to trigger the initial rolling adhesion of tumor cells under flow. To trigger firm adhesion and capture under flow, such devices also typically require antibodies against biomarkers thought to be expressed on CTCs. This approach is challenged by the fact that CTCs are now known to exhibit heterogeneous expression of conventional biomarkers. Here, we describe surfactant-nanotube complexes to enhance ES-mediated capture and isolation of tumor cells without the use of capture antibodies. While the majority of tumor cells exhibited weaker rolling adhesion on halloysite nanotubes (HNT) coated with ES, HNT functionalization with the sodium dodecanoate (NaL) surfactant induced a switch to firm cellular adhesion under flow. Conversely, surfactant-nanotube complexes significantly reduced the number of primary human leukocytes captured via ES-mediated adhesion under flow. The switch in tumor cell adhesion was exploited to capture and isolate tumor cells in the absence of EpCAM antibodies, commonly utilized as the gold standard for CTC isolation. Additionally, HNT-NaL complexes were shown to capture tumor cells with low to negligible EpCAM expression, that are not efficiently captured using conventional approaches.

  14. Differential effects of lenalidomide during plasma cell differentiation

    Science.gov (United States)

    Jourdan, Michel; Cren, Maïlys; Schafer, Peter; Robert, Nicolas; Duperray, Christophe; Vincent, Laure; Ceballos, Patrice; Cartron, Guillaume; Rossi, Jean-François; Moreaux, Jérôme; Chopra, Rajesh; Klein, Bernard

    2016-01-01

    Thalidomide, lenalidomide and pomalidomide have greatly improved the outcome of patients with multiple myeloma. However, their effects on plasma cells, the healthy counterpart of myeloma cells, are unknown. Here, we investigated lenalidomide effects on normal human plasma cell generation using an in vitro model. Lenalidomide inhibited the generation of pre-plasmablasts and early plasma cells, while it moderately affected plasmablast production. It also reduced the expression level of Ikaros, Aiolos, and IRF4 transcription factors, in plasmablasts and early plasma cells. This suggests that their differential sensitivity to lenalidomide is not due to a difference in Ikaros or Aiolos degradation. Lenalidomide also inhibited long-lived plasma cell generation, but did not impair their long-term survival once generated. This last observation is in agreement with the finding that lenalidomide treatment for 3-18 months did not affect the bone marrow healthy plasma cell count in allografted patients with multiple myeloma. Our findings should prompt to investigate whether lenalidomide resistance in patients with multiple myeloma could be associated with the emergence of malignant plasmablasts or long-lived plasma cells that are less sensitive to lenalidomide. PMID:27057635

  15. Differential effects of lenalidomide during plasma cell differentiation.

    Science.gov (United States)

    Jourdan, Michel; Cren, Maïlys; Schafer, Peter; Robert, Nicolas; Duperray, Christophe; Vincent, Laure; Ceballos, Patrice; Cartron, Guillaume; Rossi, Jean-François; Moreaux, Jérôme; Chopra, Rajesh; Klein, Bernard

    2016-05-10

    Thalidomide, lenalidomide and pomalidomide have greatly improved the outcome of patients with multiple myeloma. However, their effects on plasma cells, the healthy counterpart of myeloma cells, are unknown. Here, we investigated lenalidomide effects on normal human plasma cell generation using an in vitro model. Lenalidomide inhibited the generation of pre-plasmablasts and early plasma cells, while it moderately affected plasmablast production. It also reduced the expression level of Ikaros, Aiolos, and IRF4 transcription factors, in plasmablasts and early plasma cells. This suggests that their differential sensitivity to lenalidomide is not due to a difference in Ikaros or Aiolos degradation. Lenalidomide also inhibited long-lived plasma cell generation, but did not impair their long-term survival once generated. This last observation is in agreement with the finding that lenalidomide treatment for 3-18 months did not affect the bone marrow healthy plasma cell count in allografted patients with multiple myeloma. Our findings should prompt to investigate whether lenalidomide resistance in patients with multiple myeloma could be associated with the emergence of malignant plasmablasts or long-lived plasma cells that are less sensitive to lenalidomide.

  16. Differentiation of germ cells and gametes from stem cells.

    Science.gov (United States)

    Marques-Mari, A I; Lacham-Kaplan, O; Medrano, J V; Pellicer, A; Simón, C

    2009-01-01

    Advances in stem cell research have opened new perspectives for regenerative and reproductive medicine. Stem cells (SC) can differentiate under appropriate in vitro and in vivo conditions into different cell types. Several groups have reported their ability to differentiate SCs into germline cells, and some of them have been successful in obtaining male and female gamete-like cells by using different methodologies. This review summarizes the current knowledge in this field and emphasizes significant embryological, genetic and epigenetic aspects of germ cells and gametes in vitro differentiation in humans and other species, highlighting major obstacles that need to be overcome for successful gametogenesis in culture: studies reporting development of germ cell-like cells from murine and human embryonic (ESC) and somatic SCs are critically reviewed. Published studies indicate that germ cells can be consistently differentiated from mouse and human ESC. However, further differentiation of germ cells through gametogenesis still has important genetic and epigenetic obstacles to be efficient. Differentiation of germ cells from SCs has the potential of becoming a future source of gametes for research use, although further investigation is needed to understand and develop the appropriate niches and culture conditions. Additionally, if genetic and epigenetic methodological limitations could be solved, therapeutic opportunities could be also considered.

  17. Engineering cholesterol-based fibers for antibody immobilization and cell capture

    Science.gov (United States)

    Cohn, Celine

    In 2015, the United States is expected to have nearly 600,000 deaths attributed to cancer. Of these 600,000 deaths, 90% will be a direct result of cancer metastasis, the spread of cancer throughout the body. During cancer metastasis, circulating tumor cells (CTCs) are shed from primary tumors and migrate through bodily fluids, establishing secondary cancer sites. As cancer metastasis is incredibly lethal, there is a growing emphasis on developing "liquid biopsies" that can screen peripheral blood, search for and identify CTCs. One popular method for capturing CTCs is the use of a detection platform with antibodies specifically suited to recognize and capture cancer cells. These antibodies are immobilized onto the platform and can then bind and capture cells of interest. However, current means to immobilize antibodies often leave them with drastically reduced function. The antibodies are left poorly suited for cell capture, resulting in low cell capture efficiencies. This body of work investigates the use of lipid-based fibers to immobilize proteins in a way that retains protein function, ultimately leading to increased cell capture efficiencies. The resulting increased efficiencies are thought to arise from the retained three-dimensional structure of the protein as well as having a complete coating of the material surface with antibodies that are capable of interacting with their antigens. It is possible to electrospin cholesterol-based fibers that are similar in design to the natural cell membrane, providing proteins a more natural setting during immobilization. Such fibers have been produced from cholesterol-based cholesteryl succinyl silane (CSS). These fibers have previously illustrated a keen aptitude for retaining protein function and increasing cell capture. Herein the work focuses on three key concepts. First, a model is developed to understand the immobilization mechanism used by electrospun CSS fibers. The antibody immobilization and cell capturing

  18. Directed hepatic differentiation from embryonic stem cells

    OpenAIRE

    Chen, Xuesong; Zeng, Fanyi

    2011-01-01

    The liver is the largest internal organ in mammals, and is important for the maintenance of normal physiological functions of other tissues and organs. Hepatitis, cirrhosis, liver cancer and other chronic liver diseases are serious threats to human health, and these problems are compounded by a scarcity of liver donors for transplantation therapies. Directed differentiation of embryonic stem cells to liver cells is a promising strategy for obtaining hepatocytes that can be used for cell trans...

  19. The Antigen Presenting Cells Instruct Plasma Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Wei eXu

    2014-01-01

    Full Text Available The professional antigen presenting cells (APCs, including many subsets of dendritic cells and macrophages, not only mediate prompt but nonspecific response against microbes, but also bridge the antigen-specific adaptive immune response through antigen presentation. In the latter, typically activated B cells acquire cognate signals from T helper cells in the germinal center of lymphoid follicles to differentiate into plasma cells, which generate protective antibodies. Recent advances have revealed that many APC subsets provide not only signal 1 (the antigen, but also signal 2 to directly instruct the differentiation process of plasma cells in a T cell-independent manner. Herein, the different signals provided by these APC subsets to direct B cell proliferation, survival, class switching and terminal differentiation are discussed. We furthermore propose that the next generation of vaccines for boosting antibody response could be designed by targeting APCs.

  20. Bioprinting and Differentiation of Stem Cells.

    Science.gov (United States)

    Irvine, Scott A; Venkatraman, Subbu S

    2016-09-08

    The 3D bioprinting of stem cells directly into scaffolds offers great potential for the development of regenerative therapies; in particular for the fabrication of organ and tissue substitutes. For this to be achieved; the lineage fate of bioprinted stem cell must be controllable. Bioprinting can be neutral; allowing culture conditions to trigger differentiation or alternatively; the technique can be designed to be stimulatory. Such factors as the particular bioprinting technique; bioink polymers; polymer cross-linking mechanism; bioink additives; and mechanical properties are considered. In addition; it is discussed that the stimulation of stem cell differentiation by bioprinting may lead to the remodeling and modification of the scaffold over time matching the concept of 4D bioprinting. The ability to tune bioprinting properties as an approach to fabricate stem cell bearing scaffolds and to also harness the benefits of the cells multipotency is of considerable relevance to the field of biomaterials and bioengineering.

  1. Bioprinting and Differentiation of Stem Cells

    Directory of Open Access Journals (Sweden)

    Scott A. Irvine

    2016-09-01

    Full Text Available The 3D bioprinting of stem cells directly into scaffolds offers great potential for the development of regenerative therapies; in particular for the fabrication of organ and tissue substitutes. For this to be achieved; the lineage fate of bioprinted stem cell must be controllable. Bioprinting can be neutral; allowing culture conditions to trigger differentiation or alternatively; the technique can be designed to be stimulatory. Such factors as the particular bioprinting technique; bioink polymers; polymer cross-linking mechanism; bioink additives; and mechanical properties are considered. In addition; it is discussed that the stimulation of stem cell differentiation by bioprinting may lead to the remodeling and modification of the scaffold over time matching the concept of 4D bioprinting. The ability to tune bioprinting properties as an approach to fabricate stem cell bearing scaffolds and to also harness the benefits of the cells multipotency is of considerable relevance to the field of biomaterials and bioengineering.

  2. Dazl Promotes Germ Cell Differentiation from Embryonic Stem Cells

    Institute of Scientific and Technical Information of China (English)

    Zhuo Yu; Ping Ji; Jinping Cao; Shu Zhu; Yao Li; Lin Zheng; Xuejin Chen; Lixin Feng

    2009-01-01

    It has been demonstrated that through the formation of embryoid bodies (Ebs) germ cells can be derived from embryonic stem (ES) cells. Here, we describe a transgene expression approach to derive germ cells directly from ES cells in vitro without EB formation. Through the ectopic expression of Deleted in Azoospermia-Like (Dazl), a germ cell-specific RNA-binding protein,both motile tailed-sperm and oocytes were induced from mouse ES (mES) cells in culture. Furthermore, transient overexpression of Dazl led to suppression of Nanog but induced germ cell nuclear antigen in mES cells. Dazl knockdown resulted in reduction in the expression of germ cell markers including Stella, MVH and Prdm1. Our study indicates that Dazl is a master gene controlling germ cell differentiation and that ectopic expression of Dazl promotes the dynamic differentiation of mouse ES cells into gametes in vitro.

  3. Sonic Hedgehog regulates thymic epithelial cell differentiation.

    Science.gov (United States)

    Saldaña, José Ignacio; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Ross, Susan; Furmanski, Anna L; Ono, Masahiro; Holländer, Georg; Crompton, Tessa

    2016-04-01

    Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus.

  4. Signaling involved in stem cell reprogramming and differentiation

    Institute of Scientific and Technical Information of China (English)

    Shihori; Tanabe

    2015-01-01

    Stem cell differentiation is regulated by multiple signaling events. Recent technical advances have reve-aled that differentiated cells can be reprogrammed into stem cells. The signals involved in stem cell pro-gramming are of major interest in stem cell research. The signaling mechanisms involved in regulating stem cell reprogramming and differentiation are the subject of intense study in the field of life sciences. In this review,the molecular interactions and signaling pathways related to stem cell differentiation are discussed.

  5. Stem cell regulation: Implications when differentiated cells regulate symmetric stem cell division.

    Science.gov (United States)

    Høyem, Marte Rørvik; Måløy, Frode; Jakobsen, Per; Brandsdal, Bjørn Olav

    2015-09-07

    We use a mathematical model to show that if symmetric stem cell division is regulated by differentiated cells, then changes in the population dynamics of the differentiated cells can lead to changes in the population dynamics of the stem cells. More precisely, the relative fitness of the stem cells can be affected by modifying the death rate of the differentiated cells. This result is interesting because stem cells are less sensitive than differentiated cells to environmental factors, such as medical therapy. Our result implies that stem cells can be manipulated indirectly by medical treatments that target the differentiated cells. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  6. Transcriptome changes during intestinal cell differentiation

    DEFF Research Database (Denmark)

    Tadjali, Mehrdad; Seidelin, Jakob B; Olsen, Jørgen

    2002-01-01

    The expression of 18149 genes have been analysed during the differentiation of the human intestinal cell line Caco-2. cDNA probes from undifferentiated and differentiated Caco-2 cells were separately hybridised to EST DNAs spotted in an array on a nylon membrane. A remarkable change...... by a general down-regulation of genes in the low abundance class. Similar results were found using mouse small intestinal crypt and villus cells, suggesting that the phenomenon also occurs in the intestine in vivo. The expression data were subsequently used in a search for markers for subsets of epithelial...... cells by performing reverse transcriptase-polymerase chain reaction on RNA extracted from laser dissected intestinal crypt and villi. In a screen of eight transcripts one - SART3 - was identified as a marker for human colonic crypts....

  7. Bioprinting and Differentiation of Stem Cells

    OpenAIRE

    2016-01-01

    The 3D bioprinting of stem cells directly into scaffolds offers great potential for the development of regenerative therapies; in particular for the fabrication of organ and tissue substitutes. For this to be achieved; the lineage fate of bioprinted stem cell must be controllable. Bioprinting can be neutral; allowing culture conditions to trigger differentiation or alternatively; the technique can be designed to be stimulatory. Such factors as the particular bioprinting technique; bioink poly...

  8. Differentiated human stem cells resemble fetal, not adult, β cells.

    Science.gov (United States)

    Hrvatin, Sinisa; O'Donnell, Charles W; Deng, Francis; Millman, Jeffrey R; Pagliuca, Felicia Walton; DiIorio, Philip; Rezania, Alireza; Gifford, David K; Melton, Douglas A

    2014-02-25

    Human pluripotent stem cells (hPSCs) have the potential to generate any human cell type, and one widely recognized goal is to make pancreatic β cells. To this end, comparisons between differentiated cell types produced in vitro and their in vivo counterparts are essential to validate hPSC-derived cells. Genome-wide transcriptional analysis of sorted insulin-expressing (INS(+)) cells derived from three independent hPSC lines, human fetal pancreata, and adult human islets points to two major conclusions: (i) Different hPSC lines produce highly similar INS(+) cells and (ii) hPSC-derived INS(+) (hPSC-INS(+)) cells more closely resemble human fetal β cells than adult β cells. This study provides a direct comparison of transcriptional programs between pure hPSC-INS(+) cells and true β cells and provides a catalog of genes whose manipulation may convert hPSC-INS(+) cells into functional β cells.

  9. Boron neutron capture therapy induces cell cycle arrest and DNA fragmentation in murine melanoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Faiao-Flores, F. [Biochemical and Biophysical Laboratory, Butantan Institute, 1500 Vital Brasil Avenue, Sao Paulo (Brazil)] [Faculty of Medicine, University of Sao Paulo, 455 Doutor Arnaldo Avenue, Sao Paulo (Brazil); Coelho, P.R.P. [Institute for Nuclear and Energy Research, 2242 Lineu Prestes Avenue, Sao Paulo (Brazil); Arruda-Neto, J. [Physics Institute, University of Sao Paulo, 187 Matao Street, Sao Paulo (Brazil)] [FESP, Sao Paulo Engineering School, 5520 Nove de Julho Avenue, Sao Paulo (Brazil); Maria, Durvanei A., E-mail: durvaneiaugusto@yahoo.br [Biochemical and Biophysical Laboratory, Butantan Institute, 1500 Vital Brasil Avenue, Sao Paulo (Brazil)

    2011-12-15

    The melanoma is a highly lethal skin tumor, with a high incidence. Boron Neutron Capture Therapy (BNCT) is a radiotherapy which combines Boron with thermal neutrons, constituting a binary system. B16F10 melanoma and L929 fibroblasts were treated with Boronophenylalanine and irradiated with thermal neutron flux. The electric potential of mitochondrial membrane, cyclin D1 and caspase-3 markers were analyzed. BNCT induced a cell death increase and cyclin D1 amount decreased only in B16F10 melanoma. Besides, there was not caspase-3 phosphorylation.

  10. Cytometric characterization of circulating tumor cells captured by microfiltration and their correlation to the CellSearch(®) CTC test.

    Science.gov (United States)

    Adams, Daniel L; Stefansson, Steingrimur; Haudenschild, Christian; Martin, Stuart S; Charpentier, Monica; Chumsri, Saranya; Cristofanilli, Massimo; Tang, Cha-Mei; Alpaugh, R Katherine

    2015-02-01

    Recent studies reporting hundreds, to thousands, of circulating tumor cells (CTCs) in the blood of cancer patients have raised questions regarding the prevalence of CTCs, as enumerated by the CellSearch(®) CTC Test. Although CellSearch has been shown to consistently detect clinically relevant CTCs; the ability to only capture EpCAM positive cells has led to speculation that it captures limited subsets of CTCs. In contrast, alternative approaches to CTC isolation are often cited as capturing large numbers of CTCs from patient blood. Not surprisingly the number of cells isolated by alternative approaches show poor correlations when compared to CellSearch, even when accounting for EpCAM presence or absence. In an effort to address this discrepancy, we ran an exploratory method comparison study to characterize and compare the CTC subgroups captured from duplicate blood samples from 30 breast and prostate cancer patients using a microfiltration system (CellSieve™) and CellSearch. We then categorized the CellSieve Cytokeratin(CK)+/CD45-/DAPI+ cells into five morphologically distinct subpopulations for correlative analysis. Like other filtration techniques, CellSieve isolated greater numbers of CK+/CD45- cells than CellSearch. Furthermore, analysis showed low correlation between the total CK+/CD45- cells captured by these two assays, regardless of EpCAM presence. However, subgrouping of CK+/CD45-/DAPI+ cells based on distinct cytokeratin staining patterns and nuclear morphologies elucidated a subpopulation correlative to CellSearch. Using method comparison analyses, we identified a specific CTC morphology which is highly correlative between two distinct capture methods. These data suggests that although various morphologic CTCs with similar phenotypic expressions are present in the blood of cancer patients, the clinically relevant cells isolated by CellSearch can potentially be identified using non-EpCAM dependent isolation. © 2014 The Authors. Published by Wiley

  11. A minimal physical model captures the shapes of crawling cells

    Science.gov (United States)

    Tjhung, E.; Tiribocchi, A.; Marenduzzo, D.; Cates, M. E.

    2015-01-01

    Cell motility in higher organisms (eukaryotes) is crucial to biological functions ranging from wound healing to immune response, and also implicated in diseases such as cancer. For cells crawling on hard surfaces, significant insights into motility have been gained from experiments replicating such motion in vitro. Such experiments show that crawling uses a combination of actin treadmilling (polymerization), which pushes the front of a cell forward, and myosin-induced stress (contractility), which retracts the rear. Here we present a simplified physical model of a crawling cell, consisting of a droplet of active polar fluid with contractility throughout, but treadmilling connected to a thin layer near the supporting wall. The model shows a variety of shapes and/or motility regimes, some closely resembling cases seen experimentally. Our work strongly supports the view that cellular motility exploits autonomous physical mechanisms whose operation does not need continuous regulatory effort.

  12. Incidentally detected clear cell renal cell carcinoma with rhabdoid differentiation

    Directory of Open Access Journals (Sweden)

    Venkatesh Krishnamoorthy

    2016-01-01

    Full Text Available Renal cell carcinoma with rhabdoid differentiation (RCC-R has an aggressive biologic behavior and poor prognosis. A recent consensus statement of the International Society of Urological Pathology (ISUP proposed a nucleolar grading system (ISUP grade for RCC to replace Fuhrman system and recommended reporting the presence of rhabdoid differentiation and considering tumors with rhabdoid differentiation to be ISUP Grade 4. We report a case of incidentally detected clear cell RCC-R in a 52-year-old man. This is one of the earliest cases of RCC-R (pT1b detected and first such case from Indian subcontinent.

  13. Incidentally detected clear cell renal cell carcinoma with rhabdoid differentiation.

    Science.gov (United States)

    Krishnamoorthy, Venkatesh; Gowda, Kiran Krishne; Rao, Raman Narayana

    2016-01-01

    Renal cell carcinoma with rhabdoid differentiation (RCC-R) has an aggressive biologic behavior and poor prognosis. A recent consensus statement of the International Society of Urological Pathology (ISUP) proposed a nucleolar grading system (ISUP grade) for RCC to replace Fuhrman system and recommended reporting the presence of rhabdoid differentiation and considering tumors with rhabdoid differentiation to be ISUP Grade 4. We report a case of incidentally detected clear cell RCC-R in a 52-year-old man. This is one of the earliest cases of RCC-R (pT1b) detected and first such case from Indian subcontinent.

  14. Rethinking differentiation: stem cells, regeneration, and plasticity.

    Science.gov (United States)

    Sánchez Alvarado, Alejandro; Yamanaka, Shinya

    2014-03-27

    Cell differentiation is an essential process for the development, growth, reproduction, and longevity of all multicellular organisms, and its regulation has been the focus of intense investigation for the past four decades. The study of natural and induced stem cells has ushered an age of re-examination of what it means to be a stem or a differentiated cell. Past and recent discoveries in plants and animals, as well as novel experimental manipulations, are beginning to erode many of these established concepts and are forcing a re-evaluation of the experimental systems and paradigms presently being used to explore these and other biological process. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Importance of symplasmic communication in cell differentiation.

    Science.gov (United States)

    Marzec, Marek; Kurczynska, Ewa

    2014-01-01

    Symplasmic communication via plasmodesmata (PD) is part of the system of information exchange between plant cells. Molecules that pass through the PD include ions, some hormones, minerals, amino acids, and sugars but also proteins, transcription factors, and different classes of RNA, and as such PD can participate in the coordination of plant growth and development. This review summarizes the current literature on this subject and the role of PD in signal exchange, the importance of symplasmic communication and symplasmic domains in plant cell differentiation, and highlights the future prospective in the exploration of PD functions in plants. Moreover, this review also describes the potential use of barley root epidermis and non-zygotic embryogenesis in study of symplasmic communication during cell differentiation.

  16. Replication of prions in differentiated muscle cells.

    Science.gov (United States)

    Herbst, Allen; Aiken, Judd M; McKenzie, Debbie

    2014-01-01

    We have demonstrated that prions accumulate to high levels in non-proliferative C2C12 myotubes. C2C12 cells replicate as myoblasts but can be differentiated into myotubes. Earlier studies indicated that C2C12 myoblasts are not competent for prion replication. (1) We confirmed that observation and demonstrated, for the first time, that while replicative myoblasts do not accumulate PrP(Sc), differentiated post-mitotic myotube cultures replicate prions robustly. Here we extend our observations and describe the implication and utility of this system for replicating prions.

  17. Dielectrophoretic capture of low abundance cell population using thick electrodes

    Science.gov (United States)

    Marchalot, Julien; Chateaux, Jean-François; Faivre, Magalie; Mertani, Hichem C.; Ferrigno, Rosaria; Deman, Anne-Laure

    2015-01-01

    Enrichment of rare cell populations such as Circulating Tumor Cells (CTCs) is a critical step before performing analysis. This paper presents a polymeric microfluidic device with integrated thick Carbon-PolyDimethylSiloxane composite (C-PDMS) electrodes designed to carry out dielectrophoretic (DEP) trapping of low abundance biological cells. Such conductive composite material presents advantages over metallic structures. Indeed, as it combines properties of both the matrix and doping particles, C-PDMS allows the easy and fast integration of conductive microstructures using a soft-lithography approach while preserving O2 plasma bonding properties of PDMS substrate and avoiding a cumbersome alignment procedure. Here, we first performed numerical simulations to demonstrate the advantage of such thick C-PDMS electrodes over a coplanar electrode configuration. It is well established that dielectrophoretic force (FDEP) decreases quickly as the distance from the electrode surface increases resulting in coplanar configuration to a low trapping efficiency at high flow rate. Here, we showed quantitatively that by using electrodes as thick as a microchannel height, it is possible to extend the DEP force influence in the whole volume of the channel compared to coplanar electrode configuration and maintaining high trapping efficiency while increasing the throughput. This model was then used to numerically optimize a thick C-PDMS electrode configuration in terms of trapping efficiency. Then, optimized microfluidic configurations were fabricated and tested at various flow rates for the trapping of MDA-MB-231 breast cancer cell line. We reached trapping efficiencies of 97% at 20 μl/h and 78.7% at 80 μl/h, for 100 μm thick electrodes. Finally, we applied our device to the separation and localized trapping of CTCs (MDA-MB-231) from a red blood cells sample (concentration ratio of 1:10). PMID:26392836

  18. Differential white cell count by centrifugal microfluidics.

    Energy Technology Data Exchange (ETDEWEB)

    Sommer, Gregory Jon; Tentori, Augusto M.; Schaff, Ulrich Y.

    2010-07-01

    We present a method for counting white blood cells that is uniquely compatible with centrifugation based microfluidics. Blood is deposited on top of one or more layers of density media within a microfluidic disk. Spinning the disk causes the cell populations within whole blood to settle through the media, reaching an equilibrium based on the density of each cell type. Separation and fluorescence measurement of cell types stained with a DNA dye is demonstrated using this technique. The integrated signal from bands of fluorescent microspheres is shown to be proportional to their initial concentration in suspension. Among the current generation of medical diagnostics are devices based on the principle of centrifuging a CD sized disk functionalized with microfluidics. These portable 'lab on a disk' devices are capable of conducting multiple assays directly from a blood sample, embodied by platforms developed by Gyros, Samsung, and Abaxis. [1,2] However, no centrifugal platform to date includes a differential white blood cell count, which is an important metric complimentary to diagnostic assays. Measuring the differential white blood cell count (the relative fraction of granulocytes, lymphocytes, and monocytes) is a standard medical diagnostic technique useful for identifying sepsis, leukemia, AIDS, radiation exposure, and a host of other conditions that affect the immune system. Several methods exist for measuring the relative white blood cell count including flow cytometry, electrical impedance, and visual identification from a stained drop of blood under a microscope. However, none of these methods is easily incorporated into a centrifugal microfluidic diagnostic platform.

  19. Smart interface materials integrated with microfluidics for on-demand local capture and release of cells.

    Science.gov (United States)

    Gurkan, Umut Atakan; Tasoglu, Savas; Akkaynak, Derya; Avci, Oguzhan; Unluisler, Sebnem; Canikyan, Serli; Maccallum, Noah; Demirci, Utkan

    2012-09-01

    Stimuli responsive, smart interface materials are integrated with microfluidic technologies creating new functions for a broad range of biological and clinical applications by controlling the material and cell interactions. Local capture and on-demand local release of cells are demonstrated with spatial and temporal control in a microfluidic system.

  20. Fabrication of endothelial progenitor cell capture surface via DNA aptamer modifying dopamine/polyethyleneimine copolymer film

    Science.gov (United States)

    Li, Xin; Deng, Jinchuan; Yuan, Shuheng; Wang, Juan; Luo, Rifang; Chen, Si; Wang, Jin; Huang, Nan

    2016-11-01

    Endothelial progenitor cells (EPCs) are mainly located in bone marrow and circulate, and play a crucial role in repairmen of injury endothelium. One of the most promising strategies of stents designs were considered to make in-situ endothelialization in vivo via EPC-capture biomolecules on a vascular graft to capture EPCs directly from circulatory blood. In this work, an EPC specific aptamer with a 34 bases single strand DNA sequence was conjugated onto the stent surface via dopamine/polyethyleneimine copolymer film as a platform and linker. The assembled density of DNA aptamer could be regulated by controlling dopamine percentage in this copolymer film. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) and fluorescence test confirmed the successful immobilization of DNA aptamer. To confirm its biofunctionality and cytocompatibility, the capturing cells ability of the aptamer modified surface and the effects on the growth behavior of human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) were investigated. The aptamer functionalized sample revealed a good EPC-capture ability, and had a cellular friendly feature for both EPC and EC growth, while not stimulated the hyperplasia of SMCs. And, the co-culture experiment of three types of cells confirmed the specificity capturing of EPCs to aptamer modified surface, rather than ECs and SMCs. These data suggested that this aptamer functionalized surface may have a large potentiality for the application of vascular grafts with targeted endothelialization.

  1. Innovative Microsystems: Novel Nanostructures to Capture Circulating Breast Cancer Cells

    Science.gov (United States)

    2009-05-01

    cancer, e.g., serum prostate specific antigen (PSA), digital rectal exami- nation, mammography, ultrasonography and needle biopsy .4–6 Since therapeutic...events under fine -controlled conditions. The fabrication starts with thermal growth and patterning of a 3000Å-thick oxide on a silicon wafer, 500μm in...maintained at 37°C in 5% CO2 in humid environment. Prior to experiments, the growth medium was aspirated and the cells were incubated with 4mM

  2. Antitumor potential induction and free radicals production in melanoma cells by Boron Neutron Capture Therapy

    Energy Technology Data Exchange (ETDEWEB)

    Faiao-Flores, F. [Biochemical and Biophysical Laboratory, Butantan Institute, 1500 Vital Brasil Avenue, Sao Paulo (Brazil)] [Faculty of Medicine, University of Sao Paulo, 455 Doutor Arnaldo Avenue, Sao Paulo (Brazil); Coelho, P.R.P.; Muniz, R.O.R.; Souza, G.S. [Institute for Nuclear and Energy Research, 2242 Lineu Prestes Avenue, Sao Paulo (Brazil); Arruda-Neto, J. [Physics Institute, University of Sao Paulo, 187 Matao Street, Sao Paulo (Brazil)] [FESP, Sao Paulo Engineering School, 5520 Nove de Julho Avenue, Sao Paulo (Brazil); Maria, Durvanei A., E-mail: durvaneiaugusto@yahoo.com.br [Biochemical and Biophysical Laboratory, Butantan Institute, 1500 Vital Brasil Avenue, Sao Paulo (Brazil)

    2011-12-15

    Antiproliferative and oxidative damage effects occurring in Boron Neutron Capture Therapy (BNCT) in normal fibroblasts and melanoma cell lines were analyzed. Melanoma cells and normal fibroblasts were treated with different concentrations of Boronophenylalanine and irradiated with thermal neutron flux. The cellular viability and the oxidative stress were determined. BNCT induced free radicals production and proliferative potential inhibition in melanoma cells. Therefore, this therapeutic technique could be considered efficient to inhibit growth of melanoma with minimal effects on normal tissues. - Highlights: Black-Right-Pointing-Pointer Boron Neutron Capture Therapy (BNCT) induces melanoma cell death. Black-Right-Pointing-Pointer BNCT stimulates free radicals production and proliferative inhibition in melanoma cells. Black-Right-Pointing-Pointer It produces tumor membrane degeneration and destruction with apoptotic bodies formation. Black-Right-Pointing-Pointer This therapy damages tumor cells selectively, with minimum effects on normal adjacent tissue.

  3. Molecular mechanisms of male germ cell differentiation.

    Science.gov (United States)

    Hecht, N B

    1998-07-01

    During spermatogenesis, diploid stem cells differentiate, undergo meiosis, and transform into haploid spermatozoa. As this precisely timed series of events proceeds, chromosomal ploidy is reduced and the nucleosomes of the chromatin are replaced by a transcriptionally quiescent protamine-containing nucleus. The premature termination of transcription during the haploid phase of spermatogenesis necessitates an especially prominent role for posttranscriptional regulation in the temporal and spatial expression of many testis-specific proteins and isozymes. In this review article, discussion will focus on novel mechanisms regulating gene expression in mammalian male germ cells from genome to protein.

  4. In Situ Electrochemical ELISA for Specific Identification of Captured Cancer Cells.

    Science.gov (United States)

    Safaei, Tina Saberi; Mohamadi, Reza M; Sargent, Edward H; Kelley, Shana O

    2015-07-08

    Circulating tumor cells (CTCs) are cancer cells disseminated from a tumor into the bloodstream. Their presence in patient blood samples has been associated with metastatic disease. Here, we report a simple system that enables the isolation and detection of these rare cancer cells. By developing a sensitive electrochemical ELISA method integrated within a microfluidic cell capture system, were we able to reliably detect very low levels of cancer cells in whole blood. Our results indicate that the new system provides the clinically relevant specificity and sensitivity needed for a convenient, point-of-need assay for cancer cell counting.

  5. [Human pluripotent stem cell and neural differentiation].

    Science.gov (United States)

    Wataya, Takafumi; Muguruma, Keiko; Sasai, Yoshiki

    2008-10-01

    Recovery of lost brain function is an important issue in medical studies because neurons of the central nervous system (CNS) have poor potential for regeneration. Since few CNS diseases can be treated completely by medicines, regenerative therapy by using stem cells should be studied as a new type of therapeutic intervention. The efficacy of cell replacement therapy in Parkinson's disease has been well investigated. Several studies on fetal tissue transplantation have revealed that quantity and purity of transplanted cells are necessary for recovery of symptoms. SFEB (Serum-free floating culture of embryoid body-like aggregates) method is capable of inducing multi-potential CNS progenitors that can be steered to differentiate into region-specific tissues. On the basis of the existing knowledge of embryology, we have succeeded in the generating of various types of neurons such as telencephalic, cerebeller (Purkinje and granule cells), retinal (photoreceptor cells) and hypothalamic neurons. Application of this culture method to human ES (hES) cells is necessary for clinical purpose: however, poor survival of hES cells in SFEB culture might limit the possibility of using these cells for future medical applications. We found that a selective Rho-associated kinase (ROCK) inhibitor, Y-27632, markedly diminished the dissociation-induced apoptosis of hES cells and enabled the cells to form aggregates in SFEB culture. For both mouse and human ES cells, SFEB culture is a favorable method that can generate large amounts of region-specific neurons. However, stem cell-based therapy continues to face several obstacles. It is important that researchers in the basic sciences and clinical medicine should discuss these problems together to overcome both scientific and ethical issues related to stem cells.

  6. Using Tissue Culture To Investigate Plant Cell Differentiation and Dedifferentiation.

    Science.gov (United States)

    Bozzone, Donna M.

    1997-01-01

    Describes an experimental project that uses plant tissue culture techniques to examine cell differentiation in the carrot. Allows students to gain experience in some important techniques and to explore fundamental questions about cell differentiation. (DDR)

  7. Using Tissue Culture To Investigate Plant Cell Differentiation and Dedifferentiation.

    Science.gov (United States)

    Bozzone, Donna M.

    1997-01-01

    Describes an experimental project that uses plant tissue culture techniques to examine cell differentiation in the carrot. Allows students to gain experience in some important techniques and to explore fundamental questions about cell differentiation. (DDR)

  8. Surfactant functionalization induces robust, differential adhesion of tumor cells and blood cells to charged nanotube-coated biomaterials under flow.

    Science.gov (United States)

    Mitchell, Michael J; Castellanos, Carlos A; King, Michael R

    2015-07-01

    The metastatic spread of cancer cells from the primary tumor to distant sites leads to a poor prognosis in cancers originating from multiple organs. Increasing evidence has linked selectin-based adhesion between circulating tumor cells (CTCs) and endothelial cells of the microvasculature to metastatic dissemination, in a manner similar to leukocyte adhesion during inflammation. Functionalized biomaterial surfaces hold promise as a diagnostic tool to separate CTCs and potentially treat metastasis, utilizing antibody and selectin-mediated interactions for cell capture under flow. However, capture at high purity levels is challenged by the fact that CTCs and leukocytes both possess selectin ligands. Here, a straightforward technique to functionalize and alter the charge of naturally occurring halloysite nanotubes using surfactants is reported to induce robust, differential adhesion of tumor cells and blood cells to nanotube-coated surfaces under flow. Negatively charged sodium dodecanoate-functionalized nanotubes simultaneously enhanced tumor cell capture while negating leukocyte adhesion, both in the presence and absence of adhesion proteins, and can be utilized to isolate circulating tumor cells regardless of biomarker expression. Conversely, diminishing nanotube charge via functionalization with decyltrimethylammonium bromide both abolished tumor cell capture while promoting leukocyte adhesion.

  9. Detection of cells captured with antigens on shear horizontal surface-acoustic-wave sensors.

    Science.gov (United States)

    Hao, Hsu-Chao; Chang, Hwan-You; Wang, Tsung-Pao; Yao, Da-Jeng

    2013-02-01

    Techniques to separate cells are widely applied in immunology. The technique to separate a specific antigen on a microfluidic platform involves the use of a shear horizontal surface-acoustic-wave (SH-SAW) sensor. With specific antibodies conjugated onto the surface of the SH-SAW sensors, this technique can serve to identify specific cells in bodily fluids. Jurkat cells, used as a target in this work, provide a model of cells in small abundance (1:1000) for isolation and purification with the ultimate goal of targeting even more dilute cells. T cells were separated from a mixed-cell medium on a chip (Jurkat cells/K562 cells, 1/1000). A novel microchamber was developed to capture cells during the purification, which required a large biosample. Cell detection was demonstrated through the performance of genetic identification on the chip.

  10. Regulatory T Cells in Skin Facilitate Epithelial Stem Cell Differentiation.

    Science.gov (United States)

    Ali, Niwa; Zirak, Bahar; Rodriguez, Robert Sanchez; Pauli, Mariela L; Truong, Hong-An; Lai, Kevin; Ahn, Richard; Corbin, Kaitlin; Lowe, Margaret M; Scharschmidt, Tiffany C; Taravati, Keyon; Tan, Madeleine R; Ricardo-Gonzalez, Roberto R; Nosbaum, Audrey; Bertolini, Marta; Liao, Wilson; Nestle, Frank O; Paus, Ralf; Cotsarelis, George; Abbas, Abul K; Rosenblum, Michael D

    2017-06-01

    The maintenance of tissue homeostasis is critically dependent on the function of tissue-resident immune cells and the differentiation capacity of tissue-resident stem cells (SCs). How immune cells influence the function of SCs is largely unknown. Regulatory T cells (Tregs) in skin preferentially localize to hair follicles (HFs), which house a major subset of skin SCs (HFSCs). Here, we mechanistically dissect the role of Tregs in HF and HFSC biology. Lineage-specific cell depletion revealed that Tregs promote HF regeneration by augmenting HFSC proliferation and differentiation. Transcriptional and phenotypic profiling of Tregs and HFSCs revealed that skin-resident Tregs preferentially express high levels of the Notch ligand family member, Jagged 1 (Jag1). Expression of Jag1 on Tregs facilitated HFSC function and efficient HF regeneration. Taken together, our work demonstrates that Tregs in skin play a major role in HF biology by promoting the function of HFSCs. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Hematopoietic cell differentiation from embryonic and induced pluripotent stem cells

    Science.gov (United States)

    2013-01-01

    Pluripotent stem cells, both embryonic stem cells and induced pluripotent stem cells, are undifferentiated cells that can self-renew and potentially differentiate into all hematopoietic lineages, such as hematopoietic stem cells (HSCs), hematopoietic progenitor cells and mature hematopoietic cells in the presence of a suitable culture system. Establishment of pluripotent stem cells provides a comprehensive model to study early hematopoietic development and has emerged as a powerful research tool to explore regenerative medicine. Nowadays, HSC transplantation and hematopoietic cell transfusion have successfully cured some patients, especially in malignant hematological diseases. Owing to a shortage of donors and a limited number of the cells, hematopoietic cell induction from pluripotent stem cells has been regarded as an alternative source of HSCs and mature hematopoietic cells for intended therapeutic purposes. Pluripotent stem cells are therefore extensively utilized to facilitate better understanding in hematopoietic development by recapitulating embryonic development in vivo, in which efficient strategies can be easily designed and deployed for the generation of hematopoietic lineages in vitro. We hereby review the current progress of hematopoietic cell induction from embryonic stem/induced pluripotent stem cells. PMID:23796405

  12. Fabrication of endothelial progenitor cell capture surface via DNA aptamer modifying dopamine/polyethyleneimine copolymer film

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xin; Deng, Jinchuan; Yuan, Shuheng; Wang, Juan; Luo, Rifang; Chen, Si [Key Lab. of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China); Wang, Jin, E-mail: jinxxwang@263.net [Key Lab. of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); Huang, Nan [Key Lab. of Advanced Technology for Materials of Education Ministry, Southwest Jiaotong University, Chengdu 610031 (China); School of Material Science and Engineering, Southwest Jiaotong University, Chengdu 610031 (China)

    2016-11-15

    Highlights: • The dopamine/PEI film with controlled amine density was successfully prepared. • The DNA aptamer was assembled onto the film via electrostatic incorporation. • The A@DPfilmscanspecificallyandeffectivelycaptureEPCs. • The A@DP film can support the survival of ECs, control the hyperplasia of SMCs. • The dynamic/co-culture models are useful for studying cells competitive adhesion. - Abstract: Endothelial progenitor cells (EPCs) are mainly located in bone marrow and circulate, and play a crucial role in repairmen of injury endothelium. One of the most promising strategies of stents designs were considered to make in-situ endothelialization in vivo via EPC-capture biomolecules on a vascular graft to capture EPCs directly from circulatory blood. In this work, an EPC specific aptamer with a 34 bases single strand DNA sequence was conjugated onto the stent surface via dopamine/polyethyleneimine copolymer film as a platform and linker. The assembled density of DNA aptamer could be regulated by controlling dopamine percentage in this copolymer film. X-ray photoelectron spectroscopy (XPS), water contact angle (WCA) and fluorescence test confirmed the successful immobilization of DNA aptamer. To confirm its biofunctionality and cytocompatibility, the capturing cells ability of the aptamer modified surface and the effects on the growth behavior of human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs) were investigated. The aptamer functionalized sample revealed a good EPC-capture ability, and had a cellular friendly feature for both EPC and EC growth, while not stimulated the hyperplasia of SMCs. And, the co-culture experiment of three types of cells confirmed the specificity capturing of EPCs to aptamer modified surface, rather than ECs and SMCs. These data suggested that this aptamer functionalized surface may have a large potentiality for the application of vascular grafts with targeted endothelialization.

  13. Laser capture microdissection of bacterial cells targeted by fluorescence in situ hybridization

    DEFF Research Database (Denmark)

    Schou, Kirstine Klitgaard; Mølbak, Lars; Jensen, Tim Kåre

    2005-01-01

    . By this method, a potentially pathogenic strain of the genus Brachyspira from formalin-fixed human colonic biopsies were visualized by fluorescence in situ hybridization (FISH) with a 16S rRNA-targeting oligonucleotide probe, followed by laser capture microdissection (LCM) of the targeted cells. Direct 16S r...

  14. Tunable nanostructured coating for the capture and selective release of viable circulating tumor cells.

    Science.gov (United States)

    Reátegui, Eduardo; Aceto, Nicola; Lim, Eugene J; Sullivan, James P; Jensen, Anne E; Zeinali, Mahnaz; Martel, Joseph M; Aranyosi, Alexander J; Li, Wei; Castleberry, Steven; Bardia, Aditya; Sequist, Lecia V; Haber, Daniel A; Maheswaran, Shyamala; Hammond, Paula T; Toner, Mehmet; Stott, Shannon L

    2015-03-04

    A layer-by-layer gelatin nanocoating is presented for use as a tunable, dual response biomaterial for the capture and release of circulating tumor cells (CTCs) from cancer patient blood. The entire nanocoating can be dissolved from the surface of microfluidic devices through biologically compatible temperature shifts. Alternatively, individual CTCs can be released through locally applied mechanical stress.

  15. Developing a microfluidic-based system to quantify cell capture efficiency

    Institute of Scientific and Technical Information of China (English)

    YANG Fan; GAO YuXin; ZHANG Yan; CHEN Juan; LONG Mian

    2009-01-01

    Micro-fabrication technology has substantial potential for identifying molecular markers expressed on the surfaces of tissue cells and viruses. It has been found in several conceptual prototypes that cells with such markers are able to be captured by their antibodies immobilized on microchannel substrates and unbound cells are flushed out by a driven flow. The feasibility and reliability of such a microfluidic-based assay, however, remains to be further tested. In the current work, we developed a microflu-idic-based system consisting of a microfluidic chip, an image grabbing unit, data acquisition and analysis software, as well as a supporting base. Specific binding of CD59-expressed or BSA-coupled human red blood cells (RBCs) to anti-CD59 or anti-BSA antibody-immobilized chip surfaces was quan-tiffed by capture efficiency and by the fraction of bound cells. Impacts of respective flow rate, cell concentration, antibody concentration and site density were tested systematically. The measured data indicated that the assay was robust. The robustness was further confirmed by capture efficiencies measured from an independent EUSA-based cell binding assay. These results demonstrated that the system developed provided a new plallorm to effectively quantify cellular surface markers effectively, which promoted the potential applications in both biological studies and clinical diagnoses.

  16. Developing a microfluidic-based system to quantify cell capture efficiency

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Micro-fabrication technology has substantial potential for identifying molecular markers expressed on the surfaces of tissue cells and viruses. It has been found in several conceptual prototypes that cells with such markers are able to be captured by their antibodies immobilized on microchannel substrates and unbound cells are flushed out by a driven flow. The feasibility and reliability of such a microflu- idic-based assay, however, remains to be further tested. In the current work, we developed a microflu- idic-based system consisting of a microfluidic chip, an image grabbing unit, data acquisition and analysis software, as well as a supporting base. Specific binding of CD59-expressed or BSA-coupled human red blood cells (RBCs) to anti-CD59 or anti-BSA antibody-immobilized chip surfaces was quan- tified by capture efficiency and by the fraction of bound cells. Impacts of respective flow rate, cell concentration, antibody concentration and site density were tested systematically. The measured data indicated that the assay was robust. The robustness was further confirmed by capture efficiencies measured from an independent ELISA-based cell binding assay. These results demonstrated that the system developed provided a new platform to effectively quantify cellular surface markers effectively, which promoted the potential applications in both biological studies and clinical diagnoses.

  17. Emergence of Rules in Cell Society Differentiation, Hierarchy, and Stability

    CERN Document Server

    Furusawa, C; Furusawa, Chikara; Kaneko, Kunihiko

    1998-01-01

    A dynamic model for cell differentiation is studied, where cells with internal chemical reaction dynamics interact with each other and replicate. It leads to spontaneous differentiation of cells and determination, as is discussed in the isologous diversification. Following features of the differentiation are obtained: (1)Hierarchical differentiation from a ``stem'' cell to other cell types, with the emergence of the interaction-dependent rules for differentiation; (2)Global stability of an ensemble of cells consisting of several cell types, that is sustained by the emergent, autonomous control on the rate of differentiation; (3)Existence of several cell colonies with different cell-type distributions. The results provide a novel viewpoint on the origin of complex cell society, while relevance to some biological problems, especially to the hemopoietic system, is also discussed.

  18. Differentiation of Bone Marrow Mesenchymal Cells to Neural Cells

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To explore the possibility and condition of differentiation of bone marrow mesenchymal cells (BMSCs) to neural cells in vitro, BMSCs from whole bone marrow of rats were cultured. The BMSCs of passage 3 were identified with immunocytochemical staining of CD44 ( + ), CD71 ( + )and CD45(-). There were type Ⅰ and type Ⅱ cells in BMSCs. Type Ⅰ BMSCs were spindleshaped and strong positive in immunocytochemical staining of CD44 and CD71, whereas flat and big type Ⅱ BMSCs were lightly stained. The BMSCs of same passage were induced to differentiate into neural cells by β-mercaptoethanol (BME). After induction by BME, the type Ⅰ BMSCs withdrew to form neuron-like round soma and axon-like and dendrite-like processes, and were stained positively for neurofilament (NF). The type Ⅱ BMSCs did not change in the BME medium and were negatively or slightly stained of NF.

  19. Self-propelled carbon nanotube based microrockets for rapid capture and isolation of circulating tumor cells

    Science.gov (United States)

    Banerjee, Shashwat S.; Jalota-Badhwar, Archana; Zope, Khushbu R.; Todkar, Kiran J.; Mascarenhas, Russel R.; Chate, Govind P.; Khutale, Ganesh V.; Bharde, Atul; Calderon, Marcelo; Khandare, Jayant J.

    2015-05-01

    Here, we report a non-invasive strategy for isolating cancer cells by autonomously propelled carbon nanotube (CNT) microrockets. H2O2-driven oxygen (O2) bubble-propelled microrockets were synthesized using CNT and Fe3O4 nanoparticles in the inner surface and covalently conjugating transferrin on the outer surface. Results show that self-propellant microrockets can specifically capture cancer cells.Here, we report a non-invasive strategy for isolating cancer cells by autonomously propelled carbon nanotube (CNT) microrockets. H2O2-driven oxygen (O2) bubble-propelled microrockets were synthesized using CNT and Fe3O4 nanoparticles in the inner surface and covalently conjugating transferrin on the outer surface. Results show that self-propellant microrockets can specifically capture cancer cells. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr01797a

  20. Effect of leukemia inhibitory factor on embryonic stem cell differentiation: implications for supporting neuronal differentiation

    Institute of Scientific and Technical Information of China (English)

    Zhao HE; Jing-jing LI; Chang-hong ZHEN; Lin-ying FENG; Xiao-yan DING

    2006-01-01

    Aim: Leukemia inhibitory factor (LIF), a pleiotropic cytokine, has been used extensively in the maintenance of mouse embryonic stem cell pluripotency. In this current work, we examined the effect of the LIF signaling pathway in embryonic stem (ES) cell differentiation to a neural fate. Methods: In the presence of LIF (1000 U/mL), the production of neuronal cells derived from embryoid bodies (EB)was tested under various culture conditions. Inhibition of the LIF pathway was examined with specific inhibitors. The effects of cell apoptosis and proliferation on neural differentiation were examined. ES cell differentiation into three-gem layers was compared. Results: Under various culture conditions, neuronal differentiation was increased in the presence of LIF. Blocking the LIF-activated STAT3signaling pathway with specific inhibitors abolished the neuronal differentiation of ES cells, whereas inhibition of the LIF-activated MEK signaling pathway impaired the differentiation of ES cells toward a glial fate. LIF suppressed cell apoptosis and promoted cell proliferation during ES cell differentiation. LIF inhibited the differentiation of ES cells to both mesoderm and extraembryonic endoderm fates, but enhanced the determination of neural progenitors. Conclusion:These results suggest that LIF plays a positive role during the differentiation of ES cells into neuronal cells.

  1. Probing stem cell differentiation using atomic force microscopy

    Science.gov (United States)

    Liang, Xiaobin; Shi, Xuetao; Ostrovidov, Serge; Wu, Hongkai; Nakajima, Ken

    2016-03-01

    A real-time method using atomic force microscopy (AFM) was developed to probe stem cell differentiation by measuring the mechanical properties of cells and the extracellular matrix (ECM). The mechanical properties of stem cells and their ECMs can be used to clearly distinguish specific stem cell-differentiated lineages. It is clear that AFM is a facile and useful tool for monitoring the differentiation of stem cells in a non-invasive manner.

  2. Laser capture microdissection in Ectocarpus siliculosus: the pathway to cell-specific transcriptomics in brown algae

    OpenAIRE

    Denis eSaint-Marcoux; Bernard eBilloud; Jane Alison Langdale; Bénédicte eCharrier

    2015-01-01

    Laser capture microdissection (LCM) facilitates the isolation of individual cells from tissue sections, and when combined with RNA amplification techniques, it is an extremely powerful tool for examining genome-wide expression profiles in specific cell-types. LCM has been widely used to address various biological questions in both animal and plant systems, however, no attempt has been made so far to transfer LCM technology to macroalgae. Macroalgae are a collection of widespread eukaryotes li...

  3. Self-propelled carbon nanotube based microrockets for rapid capture and isolation of circulating tumor cells.

    Science.gov (United States)

    Banerjee, Shashwat S; Jalota-Badhwar, Archana; Zope, Khushbu R; Todkar, Kiran J; Mascarenhas, Russel R; Chate, Govind P; Khutale, Ganesh V; Bharde, Atul; Calderon, Marcelo; Khandare, Jayant J

    2015-05-21

    Here, we report a non-invasive strategy for isolating cancer cells by autonomously propelled carbon nanotube (CNT) microrockets. H2O2-driven oxygen (O2) bubble-propelled microrockets were synthesized using CNT and Fe3O4 nanoparticles in the inner surface and covalently conjugating transferrin on the outer surface. Results show that self-propellant microrockets can specifically capture cancer cells.

  4. Combined cell surface carbonic anhydrase 9 and CD147 antigens enable high-efficiency capture of circulating tumor cells in clear cell renal cell carcinoma patients.

    Science.gov (United States)

    Liu, Shijie; Tian, Zuhong; Zhang, Lei; Hou, Shuang; Hu, Sijun; Wu, Junshen; Jing, Yuming; Sun, Huimin; Yu, Fei; Zhao, Libo; Wang, Ruoxiang; Tseng, Hsian-Rong; Zhau, Haiyen E; Chung, Leland W K; Wu, Kaichun; Wang, Hao; Wu, Jason Boyang; Nie, Yongzhan; Shao, Chen

    2016-09-13

    Circulating tumor cells (CTCs) have emerged as promising tools for noninvasive cancer detection and prognosis. Most conventional approaches for capturing CTCs use an EpCAM-based enrichment strategy, which does not work well in cancers that show low or no expression of EpCAM, such as renal cell carcinoma (RCC). In this study, we developed a new set of cell surface markers including CA9 and CD147 as alternative CTC-capture antigens specifically designed for RCC patients. We showed that the expression of both CA9 and CD147 was prevalent in a RCC patient cohort (n=70) by immunohistochemical analysis, with both molecules in combination covering 97.1% of cases. The NanoVelcro platform combined with CA9-/CD147-capture antibodies demonstrated significantly higher efficiency for capturing both CTC-mimicking renal cancer cells and RCC CTCs in peripheral blood, compared to the conventional EpCAM-based method. Using immunofluorescence cytological validation at the single-cell level, we were able to identify bona fide CTCs in RCC patient blood following the well-accepted criteria in our CTC-capture system. We further demonstrated a significant association of CTC numbers as well as the CTC expression status of Vimentin, a mesenchymal marker, with disease progression, including pathologic features and clinical staging. These results provide new insights into developing novel, effective targets/approaches for capturing CTCs, making CTCs a valuable tool for improved cancer detection, prognosis and treatment in RCC.

  5. Capture and On-chip analysis of Melanoma Cells Using Tunable Surface Shear forces

    Science.gov (United States)

    Tsao, Simon Chang-Hao; Vaidyanathan, Ramanathan; Dey, Shuvashis; Carrascosa, Laura G.; Christophi, Christopher; Cebon, Jonathan; Shiddiky, Muhammad J. A.; Behren, Andreas; Trau, Matt

    2016-01-01

    With new systemic therapies becoming available for metastatic melanoma such as BRAF and PD-1 inhibitors, there is an increasing demand for methods to assist with treatment selection and response monitoring. Quantification and characterisation of circulating melanoma cells (CMCs) has been regarded as an excellent non-invasive candidate but a sensitive and efficient tool to do these is lacking. Herein we demonstrate a microfluidic approach for melanoma cell capture and subsequent on-chip evaluation of BRAF mutation status. Our approach utilizes a recently discovered alternating current electrohydrodynamic (AC-EHD)-induced surface shear forces, referred to as nanoshearing. A key feature of nanoshearing is the ability to agitate fluid to encourage contact with surface-bound antibody for the cell capture whilst removing nonspecific cells from the surface. By adjusting the AC-EHD force to match the binding affinity of antibodies against the melanoma-associated chondroitin sulphate proteoglycan (MCSP), a commonly expressed melanoma antigen, this platform achieved an average recovery of 84.7% from biological samples. Subsequent staining with anti-BRAFV600E specific antibody enabled on-chip evaluation of BRAFV600E mutation status in melanoma cells. We believe that the ability of nanoshearing-based capture to enumerate melanoma cells and subsequent on-chip characterisation has the potential as a rapid screening tool while making treatment decisions.

  6. Engineered decomposable multifunctional nanobioprobes for capture and release of rare cancer cells.

    Science.gov (United States)

    Xie, Min; Lu, Ning-Ning; Cheng, Shi-Bo; Wang, Xue-Ying; Wang, Ming; Guo, Shan; Wen, Cong-Ying; Hu, Jiao; Pang, Dai-Wen; Huang, Wei-Hua

    2014-05-06

    Early detection and isolation of circulating tumor cells (CTCs) can provide helpful information for diagnosis, and functional readouts of CTCs can give deep insight into tumor biology. In this work, we presented a new strategy for simple isolation and release of CTCs using engineered nanobioprobes. The nanobioprobes were constructed by Ca(2+)-assisted layer-by-layer assembly of alginate onto the surface of fluorescent-magnetic nanospheres, followed by immobilization of biotin-labeled anti-EpCAM. As-prepared anti-EpCAM-functionalized nanobioprobes were characterized with integrated features of anti-EpCAM-directed specific recognition, fluorescent magnetic-driven cell capture, and EDTA-assisted cell release, which can specifically recognize 10(2) SK-BR-3 cells spiked in 1 mL of lysed blood or human whole blood samples with 89% and 86% capture efficiency, respectively. Our proof-of-concept experiments demonstrated that 65% of captured SK-BR-3 cells were released after EDTA treatment, and nearly 70% of released SK-BR-3 cells kept their viability, which may facilitate molecular profiling and functional readouts of CTCs.

  7. A novel solar cell fabricated with spiral photo-electrode for capturing sunlight 3-dimensionally

    Institute of Scientific and Technical Information of China (English)

    LIU Yong; SHEN Hui; DENG Youjun

    2006-01-01

    A novel solar cell fabricated with spiral photo-electrode for capturing sunlight 3-dimensionally (3D-cell) is proposed in this paper. We studied its performance both in solar simulator and in nature sunlight. Spiral photo-electrode of 3D-cell can receive sunlight from all directions and therefore can track the sun passively. And it is much insensitive to solar azimuth angle and shade. In addition, it increases the area to obtain scattered sunlight and reflected light. Compared with the dye-sensitized solar cells using sandwich structure, it would be more advantageous in the sealing technique.

  8. Detection and capture of breast cancer cells with photoacoustic flow cytometry

    Science.gov (United States)

    Bhattacharyya, Kiran; Goldschmidt, Benjamin S.; Viator, John A.

    2016-08-01

    According to the Centers for Disease Control and Prevention, breast cancer is the most common cancer and the second leading cause of cancer related deaths among women. Metastasis-the presence of secondary tumors caused by the spread of cancer cells via the circulatory or lymphatic systems-significantly worsens the prognosis of any breast cancer patient. A technique is developed to detect circulating breast cancer cells in human blood using a photoacoustic flow cytometry method. A Q-switched laser is used to interrogate thousands of blood cells with one pulse as they flow through the beam path. Cells that are optically absorbing, either naturally or artificially, emit an ultrasound wave as a result of the photoacoustic (PA) effect. Breast cancer cells are targeted with chromophores through immunochemistry in order to enhance optical absorption. After which, the PA cytometry device is calibrated to demonstrate the ability to detect single cells. Cultured breast cancer cells are added to whole blood to reach a biologically relevant concentration of about 25 to 45 breast cancer cells per 1 mL of blood. An in vitro PA flow cytometer is used to detect and isolate these cells followed by capture with the use of a micromanipulator. This method can not only be used to determine the disease state of the patient and the response to therapy but also it can be used for genetic testing and in vitro drug trials since the circulating cell can be captured and studied.

  9. Detection, isolation, and capture of circulating breast cancer cells with photoacoustic flow cytometry

    Science.gov (United States)

    Bhattacharyya, Kiran; Njoroge, Martin; Goldschmidt, Benjamin S.; Gaffigan, Brian; Rood, Kyle; Viator, John A.

    2013-03-01

    According to the CDC, breast cancer is the most common cancer and the second leading cause of cancer related deaths among women. Metastasis, or the presence of secondary tumors caused by the spread of cancer cells via the circulatory or lymphatic systems, significantly worsens the prognosis of any breast cancer patient. In this study, a technique is developed to detect circulating breast cancer cells in human blood using a photoacoustic flow cytometry method. A Q-switched laser with a 5 ns pulse at 532 nm is used to interrogate thousands of cells with one pulse as they flow through the beam path. Cells which are pigmented, either naturally or artificially, emit an ultrasound wave as a result of the photoacoustic (PA) effect. Breast cancer cells are targeted with chromophores through immunochemistry in order to provide pigment. After which, the device is calibrated to demonstrate a single-cell detection limit. Cultured breast cancer cells are added to whole blood to reach a biologically relevant concentration of about 25-45 breast cancer cells per 1 mL of blood. An in vitro photoacoustic flow cytometer is used to detect and isolate these cells followed by capture with the use of a micromanipulator. This method can not only be used to determine the disease state of the patient and the response to therapy, it can also be used for genetic testing and in vitro drug trials since the circulating cell can be captured and studied.

  10. Comparison among three anion exchange chromatographic supports to capture erythropoietin from cell culture supernatant

    Institute of Scientific and Technical Information of China (English)

    Lourdes HERNNDEZ; Diobel STEWART; Lourdes ZUMALACRREGUI; Daniel AMARO

    2015-01-01

    Affinity and ion exchange conventional chromatography have been used to capture erythropoietin ( EPO)from mammalian cell culture supernatant. Currently,chromatographic adsorbent perfusion is available, however a limited number of applications have been found in the literature. In this work,three anion exchange chromatographic supports( gel,membrane and monolithic)were evaluated in the capture step of the recombi-nant erythropoietin purification process. The influences of load and flow rate on each support performance were analyzed. Also the purity of the EPO molecules was determined. A productivity analysis,as a decision tool for larger scale implementation,was done. As a conclusion,the evaluated supports are technically suitable to cap-ture EPO with adequate recovery and good purity. However,the monolithic column admits high operating velocity,showing the highest adsorption capacity and productivity.

  11. Directed differentiation of pluripotent stem cells to kidney cells.

    Science.gov (United States)

    Lam, Albert Q; Freedman, Benjamin S; Bonventre, Joseph V

    2014-07-01

    Regenerative medicine affords a promising therapeutic strategy for the treatment of patients with chronic kidney disease. Nephron progenitor cell populations exist only during embryonic kidney development. Understanding the mechanisms by which these populations arise and differentiate is integral to the challenge of generating new nephrons for therapeutic purposes. Pluripotent stem cells (PSCs), comprising embryonic stem cells, and induced pluripotent stem cells (iPSCs) derived from adults, have the potential to generate functional kidney cells and tissue. Studies in mouse and human PSCs have identified specific approaches to the addition of growth factors, including Wnt and fibroblast growth factor, that can induce PSC differentiation into cells with phenotypic characteristics of nephron progenitor populations with the capacity to form kidney-like structures. Although significant progress has been made, further studies are necessary to confirm the production of functional kidney cells and to promote their three-dimensional organization into bona fide kidney tissue. Human PSCs have been generated from patients with kidney diseases, including polycystic kidney disease, Alport syndrome, and Wilms tumor, and may be used to better understand phenotypic consequences of naturally occurring genetic mutations and to conduct "clinical trials in a dish". The capability to generate human kidney cells from PSCs has significant translational applications, including the bioengineering of functional kidney tissue, use in drug development to test compounds for efficacy and toxicity, and in vitro disease modeling. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Gene expression profiles of Beta-cell enriched tissue obtained by laser capture microdissection from subjects with type 2 diabetes.

    Directory of Open Access Journals (Sweden)

    Lorella Marselli

    Full Text Available BACKGROUND: Changes in gene expression in pancreatic beta-cells from type 2 diabetes (T2D should provide insights into their abnormal insulin secretion and turnover. METHODOLOGY/PRINCIPAL FINDINGS: Frozen sections were obtained from cadaver pancreases of 10 control and 10 T2D human subjects. Beta-cell enriched samples were obtained by laser capture microdissection (LCM. RNA was extracted, amplified and subjected to microarray analysis. Further analysis was performed with DNA-Chip Analyzer (dChip and Gene Set Enrichment Analysis (GSEA software. There were changes in expression of genes linked to glucotoxicity. Evidence of oxidative stress was provided by upregulation of several metallothionein genes. There were few changes in the major genes associated with cell cycle, apoptosis or endoplasmic reticulum stress. There was differential expression of genes associated with pancreatic regeneration, most notably upregulation of members of the regenerating islet gene (REG family and metalloproteinase 7 (MMP7. Some of the genes found in GWAS studies to be related to T2D were also found to be differentially expressed. IGF2BP2, TSPAN8, and HNF1B (TCF2 were upregulated while JAZF1 and SLC30A8 were downregulated. CONCLUSIONS/SIGNIFICANCE: This study made possible by LCM has identified many novel changes in gene expression that enhance understanding of the pathogenesis of T2D.

  13. Capture of circulating tumor cells using photoacoustic flowmetry and two phase flow.

    Science.gov (United States)

    O'Brien, Christine M; Rood, Kyle D; Bhattacharyya, Kiran; DeSouza, Thiago; Sengupta, Shramik; Gupta, Sagar K; Mosley, Jeffrey D; Goldschmidt, Benjamin S; Sharma, Nikhilesh; Viator, John A

    2012-06-01

    Melanoma is the deadliest form of skin cancer, yet current diagnostic methods are unable to detect early onset of metastatic disease. Patients must wait until macroscopic secondary tumors form before malignancy can be diagnosed and treatment prescribed. Detection of cells that have broken off the original tumor and travel through the blood or lymph system can provide data for diagnosing and monitoring metastatic disease. By irradiating enriched blood samples spiked with cultured melanoma cells with nanosecond duration laser light, we induced photoacoustic responses in the pigmented cells. Thus, we can detect and enumerate melanoma cells in blood samples to demonstrate a paradigm for a photoacoustic flow cytometer. Furthermore, we capture the melanoma cells using microfluidic two phase flow, a technique that separates a continuous flow into alternating microslugs of air and blood cell suspension. Each slug of blood cells is tested for the presence of melanoma. Slugs that are positive for melanoma, indicated by photoacoustic waves, are separated from the cytometer for further purification and isolation of the melanoma cell. In this paper, we evaluate the two phase photoacoustic flow cytometer for its ability to detect and capture metastatic melanoma cells in blood.

  14. Advanced Materials for the Recognition and Capture of Whole Cells and Microorganisms.

    Science.gov (United States)

    Bole, Amanda L; Manesiotis, Panagiotis

    2016-07-01

    Selective cell recognition and capture has recently attracted significant interest due to its potential importance for clinical, diagnostic, environmental, and security applications. Current methods for cell isolation from complex samples are largely dependent on cell size and density, with limited application scope as many of the target cells do not exhibit appreciable differences in this respect. The most recent and forthcoming developments in the area of selective recognition and capture of whole cells, based on natural receptors, as well as synthetic materials utilising physical and chemical properties of the target cell or microorganism, are highlighted. Particular focus is given to the development of cell complementary surfaces using the cells themselves as templating agents, by means of molecular imprinting, and their combination with sensing platforms for rapid cell detection in complex media. The benefits and challenges of each approach are discussed and a perspective of the future of this research area is given. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Dystroglycan depletion inhibits the functions of differentiated HL-60 cells.

    Science.gov (United States)

    Martínez-Zárate, Alma Delia; Martínez-Vieyra, Ivette; Alonso-Rangel, Lea; Cisneros, Bulmaro; Winder, Steve J; Cerecedo, Doris

    2014-06-01

    Dystroglycan has recently been characterized in blood tissue cells, as part of the dystrophin glycoprotein complex but to date nothing is known of its role in the differentiation process of neutrophils. We have investigated the role of dystroglycan in the human promyelocytic leukemic cell line HL-60 differentiated to neutrophils. Depletion of dystroglycan by RNAi resulted in altered morphology and reduced properties of differentiated HL-60 cells, including chemotaxis, respiratory burst, phagocytic activities and expression of markers of differentiation. These findings strongly implicate dystroglycan as a key membrane adhesion protein involved in the differentiation process in HL-60 cells.

  16. A differential model of the complex cell.

    Science.gov (United States)

    Hansard, Miles; Horaud, Radu

    2011-09-01

    The receptive fields of simple cells in the visual cortex can be understood as linear filters. These filters can be modeled by Gabor functions or gaussian derivatives. Gabor functions can also be combined in an energy model of the complex cell response. This letter proposes an alternative model of the complex cell, based on gaussian derivatives. It is most important to account for the insensitivity of the complex response to small shifts of the image. The new model uses a linear combination of the first few derivative filters, at a single position, to approximate the first derivative filter, at a series of adjacent positions. The maximum response, over all positions, gives a signal that is insensitive to small shifts of the image. This model, unlike previous approaches, is based on the scale space theory of visual processing. In particular, the complex cell is built from filters that respond to the 2D differential structure of the image. The computational aspects of the new model are studied in one and two dimensions, using the steerability of the gaussian derivatives. The response of the model to basic images, such as edges and gratings, is derived formally. The response to natural images is also evaluated, using statistical measures of shift insensitivity. The neural implementation and predictions of the model are discussed.

  17. A microfluidic impedance flow cytometer for identification of differentiation state of stem cells.

    Science.gov (United States)

    Song, Hongjun; Wang, Yi; Rosano, Jenna M; Prabhakarpandian, Balabhaskar; Garson, Charles; Pant, Kapil; Lai, Eva

    2013-06-21

    This paper presents a microfluidic electrical impedance flow cytometer (FC) for identifying the differentiation state of single stem cells. This device is comprised of a novel dual micropore design, which not only enhances the processing throughput, but also allows the associated electrodes to be used as a reference for one another. A signal processing algorithm, based on the support vector machine (SVM) theory, and a data classification method were developed to automate the identification of sample types and cell differentiation state based on measured impedance values. The device itself was fabricated using a combination of standard and soft lithography techniques to generate a PDMS-gold electrode construct. Experimental testing with non-biological particles and mouse embryonic carcinoma cells (P19, undifferentiated and differentiated) was carried out using a range of excitation frequencies. The effects of the frequency and the interrogation parameters on sample identification performance were investigated. It was found that the real and imaginary part of the detected impedance signal were adequate for distinguishing the undifferentiated P19 cells from non-biological polystyrene beads at all tested frequencies. A higher frequency and an opacity index were required to resolve the undifferentiated and differentiated P19 cells by capturing capacitive changes in electrophysiological properties arising from differentiation. The experimental results demonstrated salient accuracy of the device and algorithm, and established its feasibility for non-invasive, label-free identification of the differentiation state of the stem cells.

  18. Laser cross-linking protein captures for living cells on a biochip

    Science.gov (United States)

    Lin, Chih-Lang; Pan, Ming-Jeng; Chen, Hai-Wen; Lin, Che-Kuan; Lin, Chuen-Fu; Baldeck, Patrice L.

    2014-03-01

    In this study, bio-sensing pads are proposed to capture living cells, which are fabricated on cover glasses by cross-linking proteins/antibodies using laser induced photochemistry. The biological functions of the cross-linked protein/antibody were verified by capturing Staphylococcus aureus (S. aureus), Leptospira, and red blood cells (RBCs), separately, with associated protein/antibody sensing pads. The experimental results show that S. aureus were bound on GFP-AcmA' pad after minutes of incubation and phosphate buffered saline (PBS) rinsing. No binding was observed with reference pad made of neutral bovine serum albumin (BSA). Second, A-type RBCs were chosen as the model cell to demonstrate the blood typing feasibility of the anti-A pad in microchannel. The A-type RBCs were captured only by the anti-A pad, but not the reference pad made of BSA. The same experimental model was carried out on the Leptospira, which stuck on the blood serum pad after PBS rinsing, but not BSA pad. This study provides a potential platform for simple and direct detection of living full cells without culture that could be used in point-of-care settings.

  19. Carboxybetaine methacrylate-modified nylon surface for circulating tumor cell capture.

    Science.gov (United States)

    Wang, Huiyu; Yue, Guofeng; Dong, Chaoqun; Wu, Fenglei; Wei, Jia; Yang, Yang; Zou, Zhengyun; Wang, Lifeng; Qian, Xiaoping; Zhang, Tao; Liu, Baorui

    2014-03-26

    Conventional in vitro circulating tumor cell (CTC) detection methods are always limited by blood sample volume because of the requirement of a large amount of blood. The aim of this study was to overcome the limitation by designing and making an in vivo CTC capture device. In this study, we designed and prepared a kind of proper material to serve the purpose of intervention. A method employing 3-aminopropyltriethoxysilane (γ-APS) as the coupling reagent to graft carboxybetaine methacrylate (CBMA) and to immobilize an anti-epithelial cell adhesion molecular (EpCAM) antibody on Nylon was developed. The results of X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy proved the successful graft of γ-APS and CBMA to Nylon. Furthermore, the predicted improvement in the biocompatibilities of our modified Nylon was confirmed by water contact angle measurement, bovine serum albumin adhesion, platelet adhesion, plasma recalcification time determination, and cytotoxicity tests. The tumor cells adhesion experiment revealed that Nylon with the antibody immobilized on it had an affinity for EpCAM positive tumor cells higher than that of pristine Nylon. Additionally, the capture ability of the CTCs was demonstrated in a nude mouse tumor model using the interventional device made of the modified Nylon wire. The positive results suggest that CBMA-grafted and anti-EpCAM antibody-immobilized Nylon is a promising new material for in vivo CTC capture devices.

  20. Oligodendrocyte differentiation and implantation : new insights for remyelinating cell therapy

    NARCIS (Netherlands)

    Sher, Falak; Balasubramaniyan, Veerakumar; Boddeke, Erik; Copray, Sjef

    2008-01-01

    Purpose of review Recent research on oligodendrocyte development has yielded new insights on the involvement of morphogens and differentiation factors in oligodendrogenesis. This knowledge has improved strategies to control neural stem cell-derived oligodendrocyte differentiation and functional matu

  1. Neutron capture nuclei-containing carbon nanoparticles for destruction of cancer cells.

    Science.gov (United States)

    Hwang, Kuo Chu; Lai, Po Dong; Chiang, Chi-Shiun; Wang, Pei-Jen; Yuan, Chiun-Jye

    2010-11-01

    HeLa cells were incubated with neutron capture nuclei (boron-10 and gadolinium)-containing carbon nanoparticles, followed by irradiation of slow thermal neutron beam. Under a neutron flux of 6 x 10(11) n/cm(2) (or 10 min irradiation at a neutron flux of 1 x 10(9) n/cm(2) s), the percentages of acute cell death at 8 h after irradiation are 52, 55, and 28% for HeLa cells fed with BCo@CNPs, GdCo@CNPs, and Co@CNPs, respectively. The proliferation capability of the survived HeLa cells was also found to be significantly suppressed. At 48 h after neutron irradiation, the cell viability further decreases to 35 +/- 5% as compared to the control set receiving the same amount of neutron irradiation dose but in the absence of carbon nanoparticles. This work demonstrates "proof-of-concept" examples of neutron capture therapy using (10)B-, (157)Gd-, and (59)Co-containing carbon nanoparticles for effective destruction of cancer cells. It will also be reported the preparation and surface functionalization of boron or gadolinium doped core-shell cobalt/carbon nanoparticles (BCo@CNPs, GdCo@CNPs and Co@CNPs) using a modified DC pulsed arc discharge method, and their characterization by various spectroscopic measurements, including TEM, XRD, SQUID, FT-IR, etc. Tumor cell targeting ability was introduced by surface modification of these carbon nanoparticles with folate moieties.

  2. Genome-wide screen for differential DNA methylation associated with neural cell differentiation in mouse.

    Directory of Open Access Journals (Sweden)

    Rene Cortese

    Full Text Available Cellular differentiation involves widespread epigenetic reprogramming, including modulation of DNA methylation patterns. Using Differential Methylation Hybridization (DMH in combination with a custom DMH array containing 51,243 features covering more than 16,000 murine genes, we carried out a genome-wide screen for cell- and tissue-specific differentially methylated regions (tDMRs in undifferentiated embryonic stem cells (ESCs, in in-vitro induced neural stem cells (NSCs and 8 differentiated embryonic and adult tissues. Unsupervised clustering of the generated data showed distinct cell- and tissue-specific DNA methylation profiles, revealing 202 significant tDMRs (p1.96 enrichment for genes involved in neural differentiation, including, for example, Jag1 and Tcf4. Our results provide robust evidence for the relevance of DNA methylation in early neural development and identify novel marker candidates for neural cell differentiation.

  3. Identification of differentially expressed genes of primary spermatocyte against round spermatid isolated from human testis using the laser capture microdissection technique

    Institute of Scientific and Technical Information of China (English)

    Gang LIANG; Xiao Dong ZHANG; Lu Jing WANG; Yu Shen SHA; Jian Chao ZHANG; Shi Ying MIAO; Shu Dong ZONG; Lin Fang WANG; S.S. KOIDE

    2004-01-01

    The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed during differentiation and development. In the present study, over 10,000 primary spermatocytes and round spermatid cells were successfully isolated by LCM. Using the cDNAs from primary spermatocytes and round spermatids, SSH cDNAs library of primary spermatocyte-specific was constructed. The average insert size of the cDNA isolated from 75randomly picked white clones was 500 bp, ranging from 250 bp to 1.7 kb. Using the dot-blot method, a total of 421clones were examined, resulting in the identification of 390 positive clones emitting strong signals. Partial sequence of cDNAs prepared from each clone was determined with an overall success rate of 84.4%. Genes encoding cytochrome c oxidase Ⅱ and the rescue factor-humanin were most frequently expressed in primary spermatocytes, suggesting their roles involved in meiosis.

  4. Laser capture microdissection and genetic analysis of carbon-labeled Kupffer cells

    Institute of Scientific and Technical Information of China (English)

    Stephan Gehring; Edmond Sabo; Maryann E San Martin; Elizabeth M Dickson; Chao-Wen Cheng; Stephen H Gregory

    2009-01-01

    AIM: To develop a method of labeling and microdissecting mouse Kupffer cells within an extraordinarily short period of time using laser capture microdissection (LCM). METHODS: Tissues are complex structures comprised of a heterogeneous population of interconnected cells. LCM offers a method of isolating a single cell type from specific regions of a tissue section. LCM is an essential approach used in conjunction with molecular analysis to study the functional interaction of cells in their native tissue environment. The process of labeling and acquiring cells by LCM prior to mRNA isolation can be elaborate, thereby subjecting the RNA to considerable degradation. Kupffer cell labeling is achieved by injecting India ink intravenously, thus circumventing the need for in vitro staining. The significance of this novel approach was validated using a cholestatic liver injury model. RESULTS: mRNA extracted from the microdissected cell population displayed marked increases in colonystimulating factor-1 receptor and Kupffer cell receptor message expression, which demonstrated Kupffer cell enrichment. Gene expression by Kupffer cells derived from bile-duct-ligated, versus sham-operated, mice was compared. Microarray analysis revealed a significant (2.5-fold, q value < 10) change in 493 genes. Based on this fold-change and a standardized PubMed search, 10 genes were identified that were relevant to the ability of Kupffer cells to suppress liver injury.CONCLUSION: The methodology outlined herein provides an approach to isolating high quality RNA from Kupffer cells, without altering the tissue integrity.

  5. Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection

    Directory of Open Access Journals (Sweden)

    Helbling Jean-Christophe

    2010-12-01

    Full Text Available Abstract Background Laser-capture microdissection (LCM that enables the isolation of specific cell populations from complex tissues under morphological control is increasingly used for subsequent gene expression studies in cell biology by methods such as real-time quantitative PCR (qPCR, microarrays and most recently by RNA-sequencing. Challenges are i to select precisely and efficiently cells of interest and ii to maintain RNA integrity. The mammary gland which is a complex and heterogeneous tissue, consists of multiple cell types, changing in relative proportion during its development and thus hampering gene expression profiling comparison on whole tissue between physiological stages. During lactation, mammary epithelial cells (MEC are predominant. However several other cell types, including myoepithelial (MMC and immune cells are present, making it difficult to precisely determine the specificity of gene expression to the cell type of origin. In this work, an optimized reliable procedure for producing RNA from alveolar epithelial cells isolated from frozen histological sections of lactating goat, sheep and cow mammary glands using an infrared-laser based Arcturus Veritas LCM (Applied Biosystems® system has been developed. The following steps of the microdissection workflow: cryosectioning, staining, dehydration and harvesting of microdissected cells have been carefully considered and designed to ensure cell capture efficiency without compromising RNA integrity. Results The best results were obtained when staining 8 μm-thick sections with Cresyl violet® (Ambion, Applied Biosystems® and capturing microdissected cells during less than 2 hours before RNA extraction. In addition, particular attention was paid to animal preparation before biopsies or slaughtering (milking and freezing of tissue blocks which were embedded in a cryoprotective compound before being immersed in isopentane. The amount of RNA thus obtained from ca.150 to 250 acini

  6. Directed Differentiation of Human Embryonic Stem Cells into Neural Progenitors.

    Science.gov (United States)

    Banda, Erin; Grabel, Laura

    2016-01-01

    A variety of protocols have been used to produce neural progenitors from human embryonic stem cells. We have focused on a monolayer culture approach that generates neural rosettes. To initiate differentiation, cells are plated in a serum-free nutrient-poor medium in the presence of a BMP inhibitor. Depending on the cell line used, additional growth factor inhibitors may be required to promote neural differentiation. Long-term culture and addition of the Notch inhibitor DAPT can promote terminal neuronal differentiation. Extent of differentiation is monitored using immunocytochemistry for cell type-specific markers.

  7. Distinct T helper cell dependence of memory B-cell proliferation versus plasma cell differentiation.

    Science.gov (United States)

    Zabel, Franziska; Fettelschoss, Antonia; Vogel, Monique; Johansen, Pål; Kündig, Thomas M; Bachmann, Martin F

    2017-03-01

    Several memory B-cell subclasses with distinct functions have been described, of which the most effective is the class-switched (CS) memory B-cell population. We have previously shown, using virus-like particles (VLPs), that the proliferative potential of these CS memory B cells is limited and they fail to re-enter germinal centres (GCs). However, VLP-specific memory B cells quickly differentiated into secondary plasma cells (PCs) with the virtue of elevated antibody production compared with primary PCs. Whereas the induction of VLP(+) memory B cells was strongly dependent on T helper cells, we were wondering whether re-stimulation of VLP(+) memory B cells and their differentiation into secondary PCs would also require T helper cells. Global absence of T helper cells led to strongly impaired memory B cell proliferation and PC differentiation. In contrast, lack of interleukin-21 receptor-dependent follicular T helper cells or CD40 ligand signalling strongly affected proliferation of memory B cells, but differentiation into mature secondary PCs exhibiting increased antibody production was essentially normal. This contrasts with primary B-cell responses, where a strong dependence on CD40 ligand but limited importance of interleukin-21 receptor was seen. Hence, T helper cell dependence differs between primary and secondary B-cell responses as well as between memory B-cell proliferation and PC differentiation.

  8. SETD7 Regulates the Differentiation of Human Embryonic Stem Cells.

    Science.gov (United States)

    Castaño, Julio; Morera, Cristina; Sesé, Borja; Boue, Stephanie; Bonet-Costa, Carles; Martí, Merce; Roque, Alicia; Jordan, Albert; Barrero, Maria J

    2016-01-01

    The successful use of specialized cells in regenerative medicine requires an optimization in the differentiation protocols that are currently used. Understanding the molecular events that take place during the differentiation of human pluripotent cells is essential for the improvement of these protocols and the generation of high quality differentiated cells. In an effort to understand the molecular mechanisms that govern differentiation we identify the methyltransferase SETD7 as highly induced during the differentiation of human embryonic stem cells and differentially expressed between induced pluripotent cells and somatic cells. Knock-down of SETD7 causes differentiation defects in human embryonic stem cell including delay in both the silencing of pluripotency-related genes and the induction of differentiation genes. We show that SETD7 methylates linker histone H1 in vitro causing conformational changes in H1. These effects correlate with a decrease in the recruitment of H1 to the pluripotency genes OCT4 and NANOG during differentiation in the SETD7 knock down that might affect the proper silencing of these genes during differentiation.

  9. Differentiation of pluripotent stem cells for regenerative medicine.

    Science.gov (United States)

    Li, Ke; Kong, Yan; Zhang, Mingliang; Xie, Fei; Liu, Peng; Xu, Shaohua

    2016-02-26

    A long-standing goal in regenerative medicine is to obtain scalable functional cells on demand to replenish cells lost in various conditions, including relevant diseases, injuries, and aging. As an unlimited cell source, pluripotent stem cells (PSCs) are invaluable for regenerative medicine, because they have the potential to give rise to any cell type in an organism. For therapeutic purposes, it is important to develop specific approach to directing PSC differentiation towards desired cell types efficiently. Through directed differentiation, PSCs could give rise to scalable, clinically relevant cells for in vivo transplantation, as well as for studying diseases in vitro and discovering drugs to treat them. Over the past few years, significant progress has been made in directing differentiation of PSCs into a variety of cell types. In this review, we discuss recent progress in directed differentiation of PSCs, clinical translation of PSC-based cell replacement therapies, and remaining challenges.

  10. Primitive human hematopoietic cells give rise to differentially specified daughter cells upon their initial cell division.

    NARCIS (Netherlands)

    Giebel, B.; Zhang, T.; Beckmann, J.; Spanholtz, J.; Wernet, P.; Ho, A.; Punzel, M.

    2006-01-01

    It is often predicted that stem cells divide asymmetrically, creating a daughter cell that maintains the stem-cell capacity, and 1 daughter cell committed to differentiation. While asymmetric stem-cell divisions have been proven to occur in model organisms (eg, in Drosophila), it remains illusive

  11. Primitive human hematopoietic cells give rise to differentially specified daughter cells upon their initial cell division.

    NARCIS (Netherlands)

    Giebel, B.; Zhang, T.; Beckmann, J.; Spanholtz, J.; Wernet, P.; Ho, A.; Punzel, M.

    2006-01-01

    It is often predicted that stem cells divide asymmetrically, creating a daughter cell that maintains the stem-cell capacity, and 1 daughter cell committed to differentiation. While asymmetric stem-cell divisions have been proven to occur in model organisms (eg, in Drosophila), it remains illusive wh

  12. Non-genetic heterogeneity, criticality and cell differentiation

    Science.gov (United States)

    Pal, Mainak; Ghosh, Sayantari; Bose, Indrani

    2015-02-01

    The different cell types in a living organism acquire their identity through the process of cell differentiation in which multipotent progenitor cells differentiate into distinct cell types. Experimental evidence and analysis of large-scale microarray data establish the key role played by a two-gene motif in cell differentiation in a number of cell systems. The two genes express transcription factors which repress each other's expression and autoactivate their own production. A number of theoretical models have recently been proposed based on the two-gene motif to provide a physical understanding of how cell differentiation occurs. In this paper, we study a simple model of cell differentiation which assumes no cooperativity in the regulation of gene expression by the transcription factors. The latter repress each other's activity directly through DNA binding and indirectly through the formation of heterodimers. We specifically investigate how deterministic processes combined with stochasticity contribute in bringing about cell differentiation. The deterministic dynamics of our model give rise to a supercritical pitchfork bifurcation from an undifferentiated stable steady state to two differentiated stable steady states. The stochastic dynamics of our model are studied using the approaches based on the Langevin equations and the linear noise approximation. The simulation results provide a new physical understanding of recent experimental observations. We further propose experimental measurements of quantities like the variance and the lag-1 autocorrelation function in protein fluctuations as the early signatures of an approaching bifurcation point in the cell differentiation process.

  13. An Innovative Configuration for CO2 Capture by High Temperature Fuel Cells

    Directory of Open Access Journals (Sweden)

    Federico Rossi

    2014-09-01

    Full Text Available Many technological solutions have been proposed for CO2 capture in the last few years. Most of them are characterized by high costs in terms of energy consumption and, consequently, higher fossil fuel use and higher economic costs. High temperature fuel cells are technological solutions currently developed for energy production with low environmental impact. In CIRIAF—University of Perugia labs, cylindrical geometry, small-sized molten carbonate fuel cell (MCFC prototypes were built and tested with good energy production and lifetime performances. In the present work, an innovative application for MCFCs is proposed, and an innovative configuration for CO2 capture/separation is investigated. The plant scheme is based on a reformer and a cylindrical MCFC. MCFCs are the most suitable solutions, because CO2 is used in their operating cycle. An analysis in terms of energy consumption/kgCO2 captured is made by coupling the proposed configuration with a gas turbine plant. The proposed configuration is characterized by a theoretical energy consumption of about 500 kJ/kgCO2, which is quite lower than actual sequestration technologies. An experimental campaign will be scheduled to verify the theoretical findings.

  14. Energy Capture from Thermolytic Solutions in Microbial Reverse-Electrodialysis Cells

    KAUST Repository

    Cusick, R. D.

    2012-03-01

    Reverse electrodialysis allows for the capture of energy from salinity gradients between salt and fresh waters, but potential applications are currently limited to coastal areas and the need for a large number of membrane pairs. Using salt solutions that could be continuously regenerated with waste heat (≥40°C) and conventional technologies would allow much wider applications of salinity-gradient power production. We used reverse electrodialysis ion-exchange membrane stacks in microbial reverse- electrodialysis cells to efficiently capture salinity-gradient energy from ammonium bicarbonate salt solutions. The maximum power density using acetate reached 5.6 watts per square meter of cathode surface area, which was five times that produced without the dialysis stack, and 3.0 ± 0.05 watts per square meter with domestic wastewater. Maximum energy recovery with acetate reached 30 ± 0.5%.

  15. Cell differentiation under the influence of rh-BMP-2.

    Science.gov (United States)

    Wilke, A; Traub, F; Kienapfel, H; Griss, P

    2001-06-29

    Bioactive bone growth factors will likely play an important role in the regeneration of bone. BMP-2 is known to promote osteoblastic cell differentiation and osteogenesis. Whether the BMPs act on human osteoblastic cells by increasing immature cell growth and/or differentiation is unknown. The goal of this study was to analyse possible effects of rhBMP-2 on cell differentiation using a human bone marrow cell culture. rhBMP-2 was added to the culture medium once. Fourteen days after addition of rhBMP-2 the cells were incubated with monoclonal antibodies. The cells were counted and analysed in a fluorescence-activating cell sorter (FACS). Compared to the controls there was an increasing effect on granulocytes, B cells and stem cells. The T-cells and monocytes show no increase or decrease after rhBMP-2 treatment.

  16. Five layers of receptor signalling in γδ T cell differentiation and activation

    Directory of Open Access Journals (Sweden)

    Sérgio T. Ribeiro

    2015-01-01

    Full Text Available The contributions of gamma-delta T cells to immunity to infection or tumours critically depend on their activation and differentiation into effectors capable of secreting cytokines and killing infected or transformed cells. These processes are molecularly controlled by surface receptors that capture key extracellular cues and convey downstream intracellular signals that regulate gamma-delta T cell physiology. The understanding of how environmental signals are integrated by gamma-delta T cells is critical for their manipulation in clinical settings. Here we discuss how different classes of surface receptors impact on human and murine gamma-delta T cell differentiation, activation and expansion. In particular, we review the role of five receptor types: the T cell receptor (TCR, costimulatory receptors, cytokine receptors, NK receptors and inhibitory receptors. Some of the key players are the costimulatory receptors CD27 and CD28, which differentially impact on pro-inflammatory subsets of gamma-delta T cells; the cytokine receptors IL-2R, IL-7R and IL-15R, which drive functional differentiation and expansion of gamma-delta T cells; the NK receptor NKG2D and its contribution to gamma-delta T cell cytotoxicity; and the inhibitory receptors PD-1 and BTLA that control gamma-delta T cell homeostasis. We discuss these and other receptors in the context of a five-step model of receptor signalling in gamma-delta T cell differentiation and activation, and discuss its implications for the manipulation of gamma-delta T cells in immunotherapy.

  17. Distinct iPS Cells Show Different Cardiac Differentiation Efficiency.

    Science.gov (United States)

    Ohno, Yohei; Yuasa, Shinsuke; Egashira, Toru; Seki, Tomohisa; Hashimoto, Hisayuki; Tohyama, Shugo; Saito, Yuki; Kunitomi, Akira; Shimoji, Kenichiro; Onizuka, Takeshi; Kageyama, Toshimi; Yae, Kojiro; Tanaka, Tomofumi; Kaneda, Ruri; Hattori, Fumiyuki; Murata, Mitsushige; Kimura, Kensuke; Fukuda, Keiichi

    2013-01-01

    Patient-specific induced pluripotent stem (iPS) cells can be generated by introducing transcription factors that are highly expressed in embryonic stem (ES) cells into somatic cells. This opens up new possibilities for cell transplantation-based regenerative medicine by overcoming the ethical issues and immunological problems associated with ES cells. Despite the development of various methods for the generation of iPS cells that have resulted in increased efficiency, safety, and general versatility, it remains unknown which types of iPS cells are suitable for clinical use. Therefore, the aims of the present study were to assess (1) the differentiation potential, time course, and efficiency of different types of iPS cell lines to differentiate into cardiomyocytes in vitro and (2) the properties of the iPS cell-derived cardiomyocytes. We found that high-quality iPS cells exhibited better cardiomyocyte differentiation in terms of the time course and efficiency of differentiation than low-quality iPS cells, which hardly ever differentiated into cardiomyocytes. Because of the different properties of the various iPS cell lines such as cardiac differentiation efficiency and potential safety hazards, newly established iPS cell lines must be characterized prior to their use in cardiac regenerative medicine.

  18. Distinct iPS Cells Show Different Cardiac Differentiation Efficiency

    Directory of Open Access Journals (Sweden)

    Yohei Ohno

    2013-01-01

    Full Text Available Patient-specific induced pluripotent stem (iPS cells can be generated by introducing transcription factors that are highly expressed in embryonic stem (ES cells into somatic cells. This opens up new possibilities for cell transplantation-based regenerative medicine by overcoming the ethical issues and immunological problems associated with ES cells. Despite the development of various methods for the generation of iPS cells that have resulted in increased efficiency, safety, and general versatility, it remains unknown which types of iPS cells are suitable for clinical use. Therefore, the aims of the present study were to assess (1 the differentiation potential, time course, and efficiency of different types of iPS cell lines to differentiate into cardiomyocytes in vitro and (2 the properties of the iPS cell-derived cardiomyocytes. We found that high-quality iPS cells exhibited better cardiomyocyte differentiation in terms of the time course and efficiency of differentiation than low-quality iPS cells, which hardly ever differentiated into cardiomyocytes. Because of the different properties of the various iPS cell lines such as cardiac differentiation efficiency and potential safety hazards, newly established iPS cell lines must be characterized prior to their use in cardiac regenerative medicine.

  19. The transcriptional landscape of alpha beta T cell differentiation

    NARCIS (Netherlands)

    Mingueneau, Michael; Kreslavsky, Taras; Gray, Daniel; Heng, Tracy; Cruse, Richard; Ericson, Jeffrey; Bendall, Sean; Spitzer, Matt; Nolan, Garry; Kobayashi, Koichi; von Boehmer, Harald; Mathis, Diane; Benoist, Christophe; Best, Adam J.; Knell, Jamie; Goldrath, Ananda; Jojic, Vladimir; Koller, Daphne; Shay, Tal; Regev, Aviv; Cohen, Nadia; Brennan, Patrick; Brenner, Michael; Kim, Francis; Rao, Tata Nageswara; Wagers, Amy; Heng, Tracy; Ericson, Jeffrey; Rothamel, Katherine; Ortiz-Lopez, Adriana; Mathis, Diane; Bezman, Natalie A.; Sun, Joseph C.; Min-Oo, Gundula; Kim, Charlie C.; Lanier, Lewis L.; Miller, Jennifer; Brown, Brian; Merad, Miriam; Gautier, Emmanuel L.; Jakubzick, Claudia; Randolph, Gwendalyn J.; Monach, Paul; Blair, David A.; Dustin, Michael L.; Shinton, Susan A.; Hardy, Richard R.; Laidlaw, David; Collins, Jim; Gazit, Roi; Rossi, Derrick J.; Malhotra, Nidhi; Sylvia, Katelyn; Kang, Joonsoo; Kreslavsky, Taras; Fletcher, Anne; Elpek, Kutlu; Bellemare-Pelletier, Angelique; Malhotra, Deepali; Turley, Shannon

    2013-01-01

    The differentiation of abT cells from thymic precursors is a complex process essential for adaptive immunity. Here we exploited the breadth of expression data sets from the Immunological Genome Project to analyze how the differentiation of thymic precursors gives rise to mature T cell transcriptomes

  20. The transcriptional landscape of alpha beta T cell differentiation

    NARCIS (Netherlands)

    Mingueneau, Michael; Kreslavsky, Taras; Gray, Daniel; Heng, Tracy; Cruse, Richard; Ericson, Jeffrey; Bendall, Sean; Spitzer, Matt; Nolan, Garry; Kobayashi, Koichi; von Boehmer, Harald; Mathis, Diane; Benoist, Christophe; Best, Adam J.; Knell, Jamie; Goldrath, Ananda; Jojic, Vladimir; Koller, Daphne; Shay, Tal; Regev, Aviv; Cohen, Nadia; Brennan, Patrick; Brenner, Michael; Kim, Francis; Rao, Tata Nageswara; Wagers, Amy; Heng, Tracy; Ericson, Jeffrey; Rothamel, Katherine; Ortiz-Lopez, Adriana; Mathis, Diane; Bezman, Natalie A.; Sun, Joseph C.; Min-Oo, Gundula; Kim, Charlie C.; Lanier, Lewis L.; Miller, Jennifer; Brown, Brian; Merad, Miriam; Gautier, Emmanuel L.; Jakubzick, Claudia; Randolph, Gwendalyn J.; Monach, Paul; Blair, David A.; Dustin, Michael L.; Shinton, Susan A.; Hardy, Richard R.; Laidlaw, David; Collins, Jim; Gazit, Roi; Rossi, Derrick J.; Malhotra, Nidhi; Sylvia, Katelyn; Kang, Joonsoo; Kreslavsky, Taras; Fletcher, Anne; Elpek, Kutlu; Bellemare-Pelletier, Angelique; Malhotra, Deepali; Turley, Shannon

    2013-01-01

    The differentiation of abT cells from thymic precursors is a complex process essential for adaptive immunity. Here we exploited the breadth of expression data sets from the Immunological Genome Project to analyze how the differentiation of thymic precursors gives rise to mature T cell transcriptomes

  1. Electrical Property Characterization of Neural Stem Cells in Differentiation.

    Directory of Open Access Journals (Sweden)

    Yang Zhao

    Full Text Available Electrical property characterization of stem cells could be utilized as a potential label-free biophysical approach to evaluate the differentiation process. However, there has been a lack of technology or tools that can quantify the intrinsic cellular electrical markers (e.g., specific membrane capacitance (Cspecific membrane and cytoplasm conductivity (σcytoplasm for a large amount of stem cells or differentiated cells. In this paper, a microfluidic platform enabling the high-throughput quantification of Cspecific membrane and σcytoplasm from hundreds of single neural stem cells undergoing differentiation was developed to explore the feasibility to characterize the neural stem cell differentiation process without biochemical staining. Experimental quantification using biochemical markers (e.g., Nestin, Tubulin and GFAP of neural stem cells confirmed the initiation of the differentiation process featured with gradual loss in cellular stemness and increased cell markers for neurons and glial cells. The recorded electrical properties of neural stem cells undergoing differentiation showed distinctive and unique patterns: 1 in the suspension culture before inducing differentiation, a large distribution and difference in σcytoplasm among individual neural stem cells was noticed, which indicated heterogeneity that may result from the nature of suspension culture of neurospheres; and 2 during the differentiation in adhering monolayer culture, significant changes and a large difference in Cspecific membrane were located indicating different expressions of membrane proteins during the differentiation process, and a small distribution difference in σcytoplasm was less significant that indicated the relatively consistent properties of cytoplasm during the culture. In summary, significant differences in Cspecific membrane and σcytoplasm were observed during the neural stem cell differentiation process, which may potentially be used as label

  2. Two pore channel 2 differentially modulates neural differentiation of mouse embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Zhe-Hao Zhang

    Full Text Available Nicotinic acid adenine dinucleotide phosphate (NAADP is an endogenous Ca(2+ mobilizing nucleotide presented in various species. NAADP mobilizes Ca(2+ from acidic organelles through two pore channel 2 (TPC2 in many cell types and it has been previously shown that NAADP can potently induce neuronal differentiation in PC12 cells. Here we examined the role of TPC2 signaling in the neural differentiation of mouse embryonic stem (ES cells. We found that the expression of TPC2 was markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebounded during the late stages of neurogenesis. Correspondingly, TPC2 knockdown accelerated mouse ES cell differentiation into neural progenitors but inhibited these neural progenitors from committing to neurons. Overexpression of TPC2, on the other hand, inhibited mouse ES cell from entering the early neural lineage. Interestingly, TPC2 knockdown had no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Taken together, our data indicate that TPC2 signaling plays a temporal and differential role in modulating the neural lineage entry of mouse ES cells, in that TPC2 signaling inhibits ES cell entry to early neural progenitors, but is required for late neuronal differentiation.

  3. Derivation and characterization of Chinese human embryonic stem cell line with high potential to differentiate into pancreatic and hepatic cells

    Institute of Scientific and Technical Information of China (English)

    SHI Cheng; SHEN Huan; JIANG Wei; SONG Zhi-hua; WANG Cheng-yan; WEI Li-hui

    2011-01-01

    Background Human embryonic stem cells have prospective uses in regenerative medicine and drug screening. Every human embryonic stem cell line has its own genetic background,which determines its specific ability for differentiation as well as susceptibility to drugs. It is necessary to compile many human embryonic stem cell lines with various backgrounds for future clinical use,especially in China due to its large population. This study contributes to isolating new Chinese human embryonic stem cell lines with clarified directly differentiation ability.Methods Donated embryos that exceeded clinical use in our in vitro fertilization-embryo transfer (IVF-ET) center were collected to establish human embryonic stem cells lines with informed consent. The classic growth factors of basic fibroblast growth factor (bFGF) and recombinant human leukaemia inhibitory factor (hLIF) for culturing embryonic stem cells were used to capture the stem cells from the plated embryos. Mechanical and enzymetic methods were used to propogate the newly established human embryonic stem cells line. The new cell line was checked for pluripotent characteristics with detecting the expression of stemness genes and observing spontaneous differentiation both in vitro and in vivo. Finally similar step-wise protocols from definitive endoderm to target specific cells were used to check the cell line's ability to directly differentiate into pancreatic and hepatic cells.Results We generated a new Chinese human embryonic stem cells line,CH1. This cell line showed the same characteristics as other reported Chinese human embryonic stem cells lines:normal morphology,karyotype and pluripotency in vitro and in vivo. The CH1 cells could be directly differentiated towards pancreatic and hepatic cells with equal efficiency compared to the H1 cell line.Conclusions This newly established Chinese cell line,CH1,which is pluripotent and has high potential to differentiate into pancreatic and hepatic cells,will provide

  4. Laser capture microdissection of bacterial cells targeted by fluorescence in situ hybridization

    DEFF Research Database (Denmark)

    Schou, Kirstine Klitgaard; Mølbak, Lars; Jensen, Tim Kåre

    2005-01-01

    Direct cultivation-independent sequence retrieval of unidentified bacteria from histological tissue sections has been limited by the difficulty of selectively isolating specific bacteria from a complex environment. Here, a new DNA isolation approach is presented for prokaryotic cells....... By this method, a potentially pathogenic strain of the genus Brachyspira from formalin-fixed human colonic biopsies were visualized by fluorescence in situ hybridization (FISH) with a 16S rRNA-targeting oligonucleotide probe, followed by laser capture microdissection (LCM) of the targeted cells. Direct 16S r......RNA gene PCR was performed from the dissected microcolonies, and the subsequent DNA sequence analysis identified the dissected bacterial cells as belonging to the Brachyspira aalborgi cluster 1. The advantage of this technique is the ability to combine the histological recognition of the specific bacteria...

  5. Selective particle and cell capture in a continuous flow using micro-vortex acoustic streaming.

    Science.gov (United States)

    Collins, David J; Khoo, Bee Luan; Ma, Zhichao; Winkler, Andreas; Weser, Robert; Schmidt, Hagen; Han, Jongyoon; Ai, Ye

    2017-05-16

    Acoustic streaming has emerged as a promising technique for refined microscale manipulation, where strong rotational flow can give rise to particle and cell capture. In contrast to hydrodynamically generated vortices, acoustic streaming is rapidly tunable, highly scalable and requires no external pressure source. Though streaming is typically ignored or minimized in most acoustofluidic systems that utilize other acoustofluidic effects, we maximize the effect of acoustic streaming in a continuous flow using a high-frequency (381 MHz), narrow-beam focused surface acoustic wave. This results in rapid fluid streaming, with velocities orders of magnitude greater than that of the lateral flow, to generate fluid vortices that extend the entire width of a 400 μm wide microfluidic channel. We characterize the forces relevant for vortex formation in a combined streaming/lateral flow system, and use these acoustic streaming vortices to selectively capture 2 μm from a mixed suspension with 1 μm particles and human breast adenocarcinoma cells (MDA-231) from red blood cells.

  6. Early specification of dopaminergic phenotype during ES cell differentiation

    Directory of Open Access Journals (Sweden)

    Li Meng

    2007-07-01

    Full Text Available Abstract Background Understanding how lineage choices are made during embryonic stem (ES cell differentiation is critical for harnessing strategies for controlled production of therapeutic somatic cell types for cell transplantation and pharmaceutical drug screens. The in vitro generation of dopaminergic neurons, the type of cells lost in Parkinson's disease patients' brains, requires the inductive molecules sonic hedgehog and FGF8, or an unknown stromal cell derived inducing activity (SDIA. However, the exact identity of the responding cells and the timing of inductive activity that specify a dopaminergic fate in neural stem/progenitors still remain elusive. Results Using ES cells carrying a neuroepithelial cell specific vital reporter (Sox1-GFP and FACS purification of Sox1-GFP neural progenitors, we have investigated the temporal aspect of SDIA mediated dopaminergic neuron specification during ES cell differentiation. Our results establish that SDIA induces a dopaminergic neuron fate in nascent neural stem or progenitor cells at, or prior to, Sox1 expression and does not appear to have further instructive role or neurotrophic activity during late neuronal differentiation of neural precursors. Furthermore, we show that dopaminergic neurons could be produced efficiently in a monolayer differentiation paradigm independent of SDIA activity or exogenous signalling molecules. In this case, the competence for dopaminergic neuron differentiation is also established at the level of Sox1 expression. Conclusion Dopaminergic neurons are specified early during mouse ES cell differentiation. The subtype specification seems to be tightly linked with the acquisition of a pan neuroectoderm fate.

  7. Lentivirus Live Cell Array for Quantitative Assessment of Gene and Pathway Activation during Myogenic Differentiation of Mesenchymal Stem Cells.

    Directory of Open Access Journals (Sweden)

    Janhavi Moharil

    Full Text Available Stem cell differentiation involves multiple cascades of transcriptional regulation that govern the cell fate. To study the real-time dynamics of this complex process, quantitative and high throughput live cell assays are required. Herein, we developed a lentiviral library of promoters and transcription factor binding sites to quantitatively capture the gene expression dynamics over a period of several days during myogenic differentiation of human mesenchymal stem cells (MSCs harvested from two different anatomic locations, bone marrow and hair follicle. Our results enabled us to monitor the sequential activation of signaling pathways and myogenic gene promoters at various stages of differentiation. In conjunction with chemical inhibitors, the lentiviral array (LVA results also revealed the relative contribution of key signaling pathways that regulate the myogenic differentiation. Our study demonstrates the potential of LVA to monitor the dynamics of gene and pathway activation during MSC differentiation as well as serve as a platform for discovery of novel molecules, genes and pathways that promote or inhibit complex biological processes.

  8. NOV/CCN3 impairs muscle cell commitment and differentiation.

    Science.gov (United States)

    Calhabeu, Frederico; Lafont, Jérome; Le Dreau, Gwenvael; Laurent, Maryvonne; Kazazian, Chantal; Schaeffer, Laurent; Martinerie, Cécile; Dubois, Catherine

    2006-06-10

    NOV (nephroblastoma overexpressed) is a member of a family of proteins which encodes secreted matrix-associated proteins. NOV is expressed during development in dermomyotome and limb buds, but its functions are still poorly defined. In order to understand the role of NOV in myogenic differentiation, C2C12 cells overexpressing NOV (C2-NOV) were generated. These cells failed to engage into myogenic differentiation, whereas they retained the ability to differentiate into osteoblasts. In differentiating conditions, C2-NOV cells remained proliferative, failed to express differentiation markers and lost their ability to form myotubes. Inhibition of differentiation by NOV was also observed with human primary muscle cells. Further examination of C2-NOV cells revealed a strong downregulation of the myogenic determination genes MyoD and Myf5 and of IGF-II expression. MyoD forced expression in C2-NOV was sufficient to restore differentiation and IGF-II induction whereas 10(-6) M insulin treatment had no effects. NOV therefore acts upstream of MyoD and does not affect IGF-II induction and signaling. HES1, a target of Notch, previously proposed to mediate NOV action, was not implicated in the inhibition of differentiation. We propose that NOV is a specific cell fate regulator in the myogenic lineage, acting negatively on key myogenic genes thus controlling the transition from progenitor cells to myoblasts.

  9. Derivation, propagation and differentiation of human embryonic stem cells.

    Science.gov (United States)

    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

  10. Vascular Mural Cells Promote Noradrenergic Differentiation of Embryonic Sympathetic Neurons.

    Science.gov (United States)

    Fortuna, Vitor; Pardanaud, Luc; Brunet, Isabelle; Ola, Roxana; Ristori, Emma; Santoro, Massimo M; Nicoli, Stefania; Eichmann, Anne

    2015-06-23

    The sympathetic nervous system controls smooth muscle tone and heart rate in the cardiovascular system. Postganglionic sympathetic neurons (SNs) develop in close proximity to the dorsal aorta (DA) and innervate visceral smooth muscle targets. Here, we use the zebrafish embryo to ask whether the DA is required for SN development. We show that noradrenergic (NA) differentiation of SN precursors temporally coincides with vascular mural cell (VMC) recruitment to the DA and vascular maturation. Blocking vascular maturation inhibits VMC recruitment and blocks NA differentiation of SN precursors. Inhibition of platelet-derived growth factor receptor (PDGFR) signaling prevents VMC differentiation and also blocks NA differentiation of SN precursors. NA differentiation is normal in cloche mutants that are devoid of endothelial cells but have VMCs. Thus, PDGFR-mediated mural cell recruitment mediates neurovascular interactions between the aorta and sympathetic precursors and promotes their noradrenergic differentiation.

  11. Differentiation of neuroepithelia from human embryonic stem cells

    OpenAIRE

    2009-01-01

    We describe the method for in vitro differentiation of neuroepithelial cells from human embryonic stem cells under a chemically defined condition. The protocol is established following the fundamental principle of in vivo neuroectodermal specification. The primitive neuroepithelial cells generated by this protocol can be further induced into neuronal and glia cells with forebrain, mid/hind brain, and spinal cord identities.

  12. Differentiation of embryonic stem cells into corneal epithelium

    Institute of Scientific and Technical Information of China (English)

    WANG Zhichong; LIU Jingbo; GE Jian; HUANG Bing; GAO Qianying; LIU Bingqian; WANG Linghua; YU Ling; FAN Zhigang; LU Xiaoming

    2005-01-01

    Our project was to determine whether embryonic stem (ES) cells could be induced to differentiate into corneal epithelia by superficial corneoscleral limbal stroma. To achieve this goal, ES-GFP cell line D3 was pre-induced by retinoic acid (RA). The pre-induced cells were seeded on deepithelialized superficial corneoscleral slices (SCSS) to form a monolayer, and divided into three groups. Group 1 was cultured and passaged in vitro for direct detection. Group 2 was exposed to air-liquid interfaces for 10 days and implanted into the subcutaneous layer of nude mice for 2 weeks for further induction in vivo. Group 3 was cultured in vitro without any inducing factors for control. There were no teratomas found in nude mice which were implanted with differentiated ES cells after two weeks. The differentiated cells showed an appearance of epithelia both in vitro and in vivo. Expression of CK3, P63 and PCNA was detected by immunohistochemical staining in the differentiated cells in group 1 and 2. Microvillis and zonula occludens were observed on the surface of the differentiated cells under an electron microscope. In the control group, ES cells differentiated freely without any inducing factors. Most cells were shed and formed a neuronal dendrite-like structure, and a minority of cells appeared polymorphic. These results demonstrate that ES cells can differentiate into corneal epithelia on the surface of SCSS under the controlled condition. Differentiated ES cells could be used as epithelial seeding cells for the reconstruction of ocular surface and corneal tissue engineering in the future.

  13. Differentiation of monkey embryonic stem cells into neural lineages.

    Science.gov (United States)

    Kuo, Hung-Chih; Pau, K-Y Francis; Yeoman, Richard R; Mitalipov, Shoukhrat M; Okano, Hideyuki; Wolf, Don P

    2003-05-01

    Embryonic stem (ES) cells are self-renewing, pluripotent, and capable of differentiating into all of the cell types found in the adult body. Therefore, they have the potential to replace degenerated or damaged cells, including those in the central nervous system. For ES cell-based therapy to become a clinical reality, translational research involving nonhuman primates is essential. Here, we report monkey ES cell differentiation into embryoid bodies (EBs), neural progenitor cells (NPCs), and committed neural phenotypes. The ES cells were aggregated in hanging drops to form EBs. The EBs were then plated onto adhesive surfaces in a serum-free medium to form NPCs and expanded in serum-free medium containing fibroblast growth factor (FGF)-2 before neural differentiation was induced. Cells were characterized at each step by immunocytochemistry for the presence of specific markers. The majority of cells in complex/cystic EBs expressed antigens (alpha-fetal protein, cardiac troponin I, and vimentin) representative of all three embryonic germ layers. Greater than 70% of the expanded cell populations expressed antigenic markers (nestin and musashi1) for NPCs. After removal of FGF-2, approximately 70% of the NPCs differentiated into neuronal phenotypes expressing either microtubule-associated protein-2C (MAP2C) or neuronal nuclear antigen (NeuN), and approximately 28% differentiated into glial cell types expressing glial fibrillary acidic protein. Small populations of MAP2C/NeuN-positive cells also expressed tyrosine hydroxylase (approximately 4%) or choline acetyltransferase (approximately 13%). These results suggest that monkey ES cells spontaneously differentiate into cells of all three germ layers, can be induced and maintained as NPCs, and can be further differentiated into committed neural lineages, including putative neurons and glial cells.

  14. Proteomic analysis of cellular response induced by boron neutron capture reaction in human squamous cell carcinoma SAS cells.

    Science.gov (United States)

    Sato, Akira; Itoh, Tasuku; Imamichi, Shoji; Kikuhara, Sota; Fujimori, Hiroaki; Hirai, Takahisa; Saito, Soichiro; Sakurai, Yoshinori; Tanaka, Hiroki; Nakamura, Hiroyuki; Suzuki, Minoru; Murakami, Yasufumi; Baiseitov, Diaz; Berikkhanova, Kulzhan; Zhumadilov, Zhaxybay; Imahori, Yoshio; Itami, Jun; Ono, Koji; Masunaga, Shinichiro; Masutani, Mitsuko

    2015-12-01

    To understand the mechanism of cell death induced by boron neutron capture reaction (BNCR), we performed proteome analyses of human squamous tumor SAS cells after BNCR. Cells were irradiated with thermal neutron beam at KUR after incubation under boronophenylalanine (BPA)(+) and BPA(-) conditions. BNCR mainly induced typical apoptosis in SAS cells 24h post-irradiation. Proteomic analysis in SAS cells suggested that proteins functioning in endoplasmic reticulum, DNA repair, and RNA processing showed dynamic changes at early phase after BNCR and could be involved in the regulation of cellular response to BNCR. We found that the BNCR induces fragments of endoplasmic reticulum-localized lymphoid-restricted protein (LRMP). The fragmentation of LRMP was also observed in the rat tumor graft model 20 hours after BNCT treatment carried out at the National Nuclear Center of the Republic of Kazakhstan. These data suggest that dynamic changes of LRMP could be involved during cellular response to BNCR.

  15. Parallel differentiation of embryonic stem cells into different cell types by a single gene-based differentiation system.

    Science.gov (United States)

    Thoma, Eva C; Maurus, Katja; Wagner, Toni U; Schartl, Manfred

    2012-04-01

    The generation of defined somatic cell types from pluripotent stem cells represents a promising system for many applications for regenerative therapy or developmental studies. Certain key developmental genes have been shown to be able to influence the fate determination of differentiating stem cells suggesting an alternative differentiation strategy to conventional medium-based methods. Here, we present a system allowing controlled, directed differentiation of embryonic stem cells (ESCs) solely by ectopic expression of single genes. We demonstrate that the myogenic master regulator myoD1 is sufficient to induce formation of skeletal muscle. In contrast to previous studies, our data suggest that myoD1-induced differentiation is independent of additional differentiation-inducing or lineage-promoting signals and occurs even under pluripotency-promoting conditions. Moreover, we demonstrate that single gene-induced differentiation enables the controlled formation of two distinct cell types in parallel. By mixing ES cell lines expressing myoD1 or the neural transcription factor ngn2, respectively, we generated a mixed culture of myocytes and neurons. Our findings provide new insights in the role of key developmental genes during cell fate decisions. Furthermore, this study represents an interesting strategy to obtain mixed cultures of different cells from stem cells, suggesting a valuable tool for cellular development and cell-cell interaction studies.

  16. The Effect of Spaceflight on Cartilage Cell Cycle and Differentiation

    Science.gov (United States)

    Doty, Stephen B.; Stiner, Dalina; Telford, William G.

    2000-01-01

    In vivo studies have shown that spaceflight results in loss of bone and muscle. In an effort to understand the mechanisms of these changes, cell cultures of cartilage, bone and muscle have been subjected to spaceflight to study the microgravity effects on differentiated cells. However it now seems possible that the cell differentiation process itself may be the event(s) most affected by spaceflight. For example, osteoblast-like cells have been shown to have reduced cellular activity in microgravity due to an underdifferentiated state (Carmeliet, et al, 1997). And reduced human lymphocyte growth in spaceflight was related to increased apoptosis (Lewis, et al, 1998). Which brings us to the question of whether reduced cellular activity in space is due to an effect on the differentiated cell, an effect on the cell cycle and cell proliferation, or an effect on cell death. This question has not been specifically addressed on previous flights and was the question behind die present study.

  17. Differentiation of stem cells upon deprivation of exogenous FGF2

    DEFF Research Database (Denmark)

    Kjartansdóttir, Kristín Rós; Gabrielsen, Anette; Reda, Ahmed

    2012-01-01

    Establishing a model for in vitro differentiation of human embryonic stem cells (hESCs) towards the germ cell lineage could be used to identify molecular mechanisms behind germ cell differentiation that may help in understanding human infertility. Here, we evaluate whether a lack of exogenous...... fibroblast growth factor 2 (FGF2) is supporting spontaneous differentiation of hESCs cultured on human foreskin fibroblast (hFF) monolayers towards germ cell lineage. Additionally to depriving the hESCs of exogenous FGF2, cells were stimulated with all-trans retinoic acid (ATRA). To get a more comprehensive...... impression on effects of removal of FGF2 and stimulation with ATRA, we combined the results of three cell lines for each experimental setting. When combining gene expression profiles of three cell lines for 96 genes, only 6 genes showed a significant up-regulation in all cell lines, when no FGF2 was added...

  18. HIV-1 capture and transmission by dendritic cells: the role of viral glycolipids and the cellular receptor Siglec-1.

    Directory of Open Access Journals (Sweden)

    Nuria Izquierdo-Useros

    2014-07-01

    Full Text Available Dendritic cells (DCs are essential in order to combat invading viruses and trigger antiviral responses. Paradoxically, in the case of HIV-1, DCs might contribute to viral pathogenesis through trans-infection, a mechanism that promotes viral capture and transmission to target cells, especially after DC maturation. In this review, we highlight recent evidence identifying sialyllactose-containing gangliosides in the viral membrane and the cellular lectin Siglec-1 as critical determinants for HIV-1 capture and storage by mature DCs and for DC-mediated trans-infection of T cells. In contrast, DC-SIGN, long considered to be the main receptor for DC capture of HIV-1, plays a minor role in mature DC-mediated HIV-1 capture and trans-infection.

  19. Cyclosporin A induces cardiac differentiation but inhibits hemato-endothelial differentiation of P19 cells.

    Directory of Open Access Journals (Sweden)

    Seung-Cheol Choi

    Full Text Available Little is known about the mechanisms underlying the effects of Cyclosporin A (CsA on the fate of stem cells, including cardiomyogenic differentiation. Therefore, we investigated the effects and the molecular mechanisms behind the actions of CsA on cell lineage determination of P19 cells. CsA induced cardiomyocyte-specific differentiation of P19 cells, with the highest efficiency at a concentration of 0.32 μM during embryoid body (EB formation via activation of the Wnt signaling pathway molecules, Wnt3a, Wnt5a, and Wnt8a, and the cardiac mesoderm markers, Mixl1, Mesp1, and Mesp2. Interestingly, cotreatment of P19 cells with CsA plus dimethyl sulfoxide (DMSO during EB formation significantly increases cardiac differentiation. In contrast, mRNA expression levels of hematopoietic and endothelial lineage markers, including Flk1 and Er71, were severely reduced in CsA-treated P19 cells. Furthermore, expression of Flk1 protein and the percentage of Flk1+ cells were severely reduced in 0.32 μM CsA-treated P19 cells compared to control cells. CsA significantly modulated mRNA expression levels of the cell cycle molecules, p53 and Cyclins D1, D2, and E2 in P19 cells during EB formation. Moreover, CsA significantly increased cell death and reduced cell number in P19 cells during EB formation. These results demonstrate that CsA induces cardiac differentiation but inhibits hemato-endothelial differentiation via activation of the Wnt signaling pathway, followed by modulation of cell lineage-determining genes in P19 cells during EB formation.

  20. Novel Application of Carbonate Fuel Cell for Capturing Carbon Dioxide from Flue Gas Streams

    Energy Technology Data Exchange (ETDEWEB)

    Jolly, Stephen; Ghezel-Ayagh, Hossein; Willman, Carl; Patel, Dilip; DiNitto, M.; Marina, Olga A.; Pederson, Larry R.; Steen, William A.

    2015-09-30

    To address concerns about climate change resulting from emission of CO2 by coal-fueled power plants, FuelCell Energy, Inc. has developed the Combined Electric Power and Carbon-dioxide Separation (CEPACS) system concept. The CEPACS system utilizes Electrochemical Membrane (ECM) technology derived from the Company’s Direct FuelCell® products. The system separates the CO2 from the flue gas of other plants and produces electric power using a supplementary fuel. FCE is currently evaluating the use of ECM to cost effectively separate CO2 from the flue gas of Pulverized Coal (PC) power plants under a U.S. Department of Energy contract. The overarching objective of the project is to verify that the ECM can achieve at least 90% CO2 capture from the flue gas with no more than 35% increase in the cost of electricity. The project activities include: 1) laboratory scale operational and performance tests of a membrane assembly, 2) performance tests of the membrane to evaluate the effects of impurities present in the coal plant flue gas, in collaboration with Pacific Northwest National Laboratory, 3) techno-economic analysis for an ECM-based CO2 capture system applied to a 550 MW existing PC plant, in partnership with URS Corporation, and 4) bench scale (11.7 m2 area) testing of an ECM-based CO2 separation and purification system.

  1. Hypercholesterolemia Induces Differentiation of Regulatory T Cells in the Liver.

    Science.gov (United States)

    Mailer, Reiner K W; Gisterå, Anton; Polyzos, Konstantinos A; Ketelhuth, Daniel F J; Hansson, Göran K

    2017-05-26

    The liver is the central organ that responds to dietary cholesterol intake and facilitates the release and clearance of lipoprotein particles. Persistent hypercholesterolemia leads to immune responses against lipoprotein particles that drive atherosclerosis. However, the effect of hypercholesterolemia on hepatic T-cell differentiation remains unknown. To investigate hepatic T-cell subsets upon hypercholesterolemia. We observed that hypercholesterolemia elevated the intrahepatic regulatory T (Treg) cell population and increased the expression of transforming growth factor-β1 in the liver. Adoptive transfer experiments revealed that intrahepatically differentiated Treg cells relocated to the inflamed aorta in atherosclerosis-prone low-density lipoprotein receptor deficient (Ldlr(-/-)) mice. Moreover, hypercholesterolemia induced the differentiation of intrahepatic, but not intrasplenic, Th17 cells in wild-type mice, whereas the disrupted liver homeostasis in hypercholesterolemic Ldlr(-/-) mice led to intrahepatic Th1 cell differentiation and CD11b(+)CD11c(+) leukocyte accumulation. Our results elucidate a new mechanism that controls intrahepatic T-cell differentiation during atherosclerosis development and indicates that intrahepatically differentiated T cells contribute to the CD4(+) T-cell pool in the atherosclerotic aorta. © 2017 American Heart Association, Inc.

  2. Power generation enhancement in novel microbial carbon capture cells with immobilized Chlorella vulgaris

    Science.gov (United States)

    Zhou, Minghua; He, Huanhuan; Jin, Tao; Wang, Hongyu

    2012-09-01

    With the increasing concerns for global climate change, a sustainable, efficient and renewable energy production from wastewater is imperative. In this study, a novel microbial carbon capture cell (MCC), is constructed for the first time by the introduction of immobilized microalgae (Chlorella vulgaris) into the cathode chamber of microbial fuel cells (MFCs) to fulfill the zero discharge of carbon dioxide. This process can achieve an 84.8% COD removal, and simultaneously the maximum power density can reach 2485.35 mW m-3 at a current density of 7.9 A m-3 and the Coulombic efficiency is 9.40%, which are 88% and 57.7% greater than that with suspended C. vulgaris, respectively. These enhancements in performance demonstrate the feasibility of an economical and effective approach for the simultaneous wastewater treatment, electricity generation and biodiesel production from microalgae.

  3. Cell cycle-dependent differentiation dynamics balances growth and endocrine differentiation in the pancreas

    DEFF Research Database (Denmark)

    Kim, Yung Hae; Larsen, Hjalte List; Rué, Paul

    2015-01-01

    Organogenesis relies on the spatiotemporal balancing of differentiation and proliferation driven by an expanding pool of progenitor cells. In the mouse pancreas, lineage tracing at the population level has shown that the expanding pancreas progenitors can initially give rise to all endocrine......, ductal, and acinar cells but become bipotent by embryonic day 13.5, giving rise to endocrine cells and ductal cells. However, the dynamics of individual progenitors balancing self-renewal and lineage-specific differentiation has never been described. Using three-dimensional live imaging and in vivo...... differentiation process is consistent with a simple model of cell cycle-dependent stochastic priming of progenitors to endocrine fate. The findings provide insights to define control parameters to optimize the generation of β-cells in vitro....

  4. Boron neutron capture therapy induces apoptosis of glioma cells through Bcl-2/Bax

    Directory of Open Access Journals (Sweden)

    Mao Xinggang

    2010-12-01

    Full Text Available Abstract Background Boron neutron capture therapy (BNCT is an alternative treatment modality for patients with glioma. The aim of this study was to determine whether induction of apoptosis contributes to the main therapeutic efficacy of BNCT and to compare the relative biological effect (RBE of BNCT, γ-ray and reactor neutron irradiation. Methods The neutron beam was obtained from the Xi'an Pulsed Reactor (XAPR and γ-rays were obtained from [60Co] γ source of the Fourth Military Medical University (FMMU in China. Human glioma cells (the U87, U251, and SHG44 cell lines were irradiated by neutron beams at the XAPR or [60Co] γ-rays at the FMMU with different protocols: Group A included control nonirradiated cells; Group B included cells treated with 4 Gy of [60Co] γ-rays; Group C included cells treated with 8 Gy of [60Co] γ-rays; Group D included cells treated with 4 Gy BPA (p-borono-phenylalanine-BNCT; Group E included cells treated with 8 Gy BPA-BNCT; Group F included cells irradiated in the reactor for the same treatment period as used for Group D; Group G included cells irradiated in the reactor for the same treatment period as used for Group E; Group H included cells irradiated with 4 Gy in the reactor; and Group I included cells irradiated with 8 Gy in the reactor. Cell survival was determined using the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium (MTT cytotoxicity assay. The morphology of cells was detected by Hoechst33342 staining and transmission electron microscope (TEM. The apoptosis rate was detected by flow cytometer (FCM. The level of Bcl-2 and Bax protein was measured by western blot analysis. Results Proliferation of U87, U251, and SHG44 cells was much more strongly inhibited by BPA-BNCT than by irradiation with [60Co] γ-rays (P 60Co] γ-rays (P P Conclusions Compared with ��-ray and reactor neutron irradiation, a higher RBE can be achieved upon treatment of glioma cells with BNCT. Glioma cell apoptosis induced by

  5. In vitro germ cell differentiation from cynomolgus monkey embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Kaori Yamauchi

    Full Text Available BACKGROUND: Mouse embryonic stem (ES cells can differentiate into female and male germ cells in vitro. Primate ES cells can also differentiate into immature germ cells in vitro. However, little is known about the differentiation markers and culture conditions for in vitro germ cell differentiation from ES cells in primates. Monkey ES cells are thus considered to be a useful model to study primate gametogenesis in vitro. Therefore, in order to obtain further information on germ cell differentiation from primate ES cells, this study examined the ability of cynomolgus monkey ES cells to differentiate into germ cells in vitro. METHODS AND FINDINGS: To explore the differentiation markers for detecting germ cells differentiated from ES cells, the expression of various germ cell marker genes was examined in tissues and ES cells of the cynomolgus monkey (Macaca fascicularis. VASA is a valuable gene for the detection of germ cells differentiated from ES cells. An increase of VASA expression was observed when differentiation was induced in ES cells via embryoid body (EB formation. In addition, the expression of other germ cell markers, such as NANOS and PIWIL1 genes, was also up-regulated as the EB differentiation progressed. Immunocytochemistry identified the cells expressing stage-specific embryonic antigen (SSEA 1, OCT-4, and VASA proteins in the EBs. These cells were detected in the peripheral region of the EBs as specific cell populations, such as SSEA1-positive, OCT-4-positive cells, OCT-4-positive, VASA-positive cells, and OCT-4-negative, VASA-positive cells. Thereafter, the effect of mouse gonadal cell-conditioned medium and growth factors on germ cell differentiation from monkey ES cells was examined, and this revealed that the addition of BMP4 to differentiating ES cells increased the expression of SCP1, a meiotic marker gene. CONCLUSION: VASA is a valuable gene for the detection of germ cells differentiated from ES cells in monkeys, and the

  6. Boolean Modeling Reveals the Necessity of Transcriptional Regulation for Bistability in PC12 Cell Differentiation.

    Science.gov (United States)

    Offermann, Barbara; Knauer, Steffen; Singh, Amit; Fernández-Cachón, María L; Klose, Martin; Kowar, Silke; Busch, Hauke; Boerries, Melanie

    2016-01-01

    The nerve growth factor NGF has been shown to cause cell fate decisions toward either differentiation or proliferation depending on the relative activity of downstream pERK, pAKT, or pJNK signaling. However, how these protein signals are translated into and fed back from transcriptional activity to complete cellular differentiation over a time span of hours to days is still an open question. Comparing the time-resolved transcriptome response of NGF- or EGF-stimulated PC12 cells over 24 h in combination with protein and phenotype data we inferred a dynamic Boolean model capturing the temporal sequence of protein signaling, transcriptional response and subsequent autocrine feedback. Network topology was optimized by fitting the model to time-resolved transcriptome data under MEK, PI3K, or JNK inhibition. The integrated model confirmed the parallel use of MAPK/ERK, PI3K/AKT, and JNK/JUN for PC12 cell differentiation. Redundancy of cell signaling is demonstrated from the inhibition of the different MAPK pathways. As suggested in silico and confirmed in vitro, differentiation was substantially suppressed under JNK inhibition, yet delayed only under MEK/ERK inhibition. Most importantly, we found that positive transcriptional feedback induces bistability in the cell fate switch. De novo gene expression was necessary to activate autocrine feedback that caused Urokinase-Type Plasminogen Activator (uPA) Receptor signaling to perpetuate the MAPK activity, finally resulting in the expression of late, differentiation related genes. Thus, the cellular decision toward differentiation depends on the establishment of a transcriptome-induced positive feedback between protein signaling and gene expression thereby constituting a robust control between proliferation and differentiation.

  7. Selective uptake of p-boronophenylalanine by osteosarcoma cells for boron neutron capture therapy

    Energy Technology Data Exchange (ETDEWEB)

    Ferrari, C. [Department of Surgery, Experimental Surgery Laboratory, University of Pavia, Piazza Botta, Pavia (Italy)], E-mail: ferraric@unipv.it; Zonta, C.; Cansolino, L.; Clerici, A.M.; Gaspari, A. [Department of Surgery, Experimental Surgery Laboratory, University of Pavia, Piazza Botta, Pavia (Italy); Altieri, S.; Bortolussi, S.; Stella, S. [Department of Nuclear and Theoretical Physics of University, Via Bassi, 6, Pavia (Italy); National Institute of Nuclear Physics (INFN) Section of Pavia, Via Bassi, 6, Pavia (Italy); Bruschi, P. [Department of Nuclear and Theoretical Physics of University, Via Bassi, 6, Pavia (Italy); Dionigi, P.; Zonta, A. [Department of Surgery, Experimental Surgery Laboratory, University of Pavia, Piazza Botta, Pavia (Italy)

    2009-07-15

    Osteosarcoma is the most common non-hematologic primary cancer type that develops in bone. Current osteosarcoma treatments combine multiagent chemotherapy with extensive surgical resection, which in some cases makes necessary the amputation of the entire limb. Nevertheless its infiltrative growth leads to a high incidence of local and distant recurrences that reduce the percentage of cured patients to less than 60%. These poor data required to set up a new therapeutic approach aimed to restrict the surgical removal meanwhile performing a radical treatment. Boron neutron capture therapy (BNCT), a particular radiotherapy based on the nuclear capture and fission reactions by atoms of {sup 10}B, when irradiated with thermal neutrons, could be a valid alternative or integrative option in case of osteosarcoma management, thanks to its peculiarity in selectively destroying neoplastic cells without damaging normal tissues. Aim of the present work is to investigate the feasibility of employing BNCT to treat the limb osteosarcoma. Boronophenylalanine (BPA) is used to carry {sup 10}B inside the neoplastic cells. As a first step the endocellular BPA uptake is tested in vitro on the UMR-106 osteosarcoma cell line. The results show an adequate accumulation capability. For the in vivo experiments, an animal tumor model is developed in Sprague-Dawley rats by means of an intrafemoral injection of UMR-106 cells at the condyle site. The absolute amounts of boron loading and the tumor to normal tissue {sup 10}B ratio are evaluated 2 h after the i.v. administration of BPA. The boron uptake by the neoplastic tissue is almost twice the normal one. However, higher values of boron concentration in tumor are requested before upholding BNCT as a valid therapeutic option in the treatment of osteosarcoma.

  8. Alpha-adrenergic blocker mediated osteoblastic stem cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Yoon Jung [Craniomaxillofacial Reconstructive Sciences Major, College of Dentistry, Seoul National University, Seoul 110-749 (Korea, Republic of); Lee, Jue Yeon [Craniomaxillofacial Reconstructive Sciences Major, College of Dentistry, Seoul National University, Seoul 110-749 (Korea, Republic of); Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Lee, Seung Jin [Department of Industrial Pharmacy, College of Pharmacy, Ewha Womans University, Seoul (Korea, Republic of); Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Chung, Chong-Pyoung [Department of Periodontology, School of Dentistry, Seoul National University, Seoul (Korea, Republic of); Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of); Park, Yoon Jeong, E-mail: parkyj@snu.ac.kr [Craniomaxillofacial Reconstructive Sciences Major, College of Dentistry, Seoul National University, Seoul 110-749 (Korea, Republic of); Research Center, Nano Intelligent Biomedical Engineering Corporation (NIBEC), Seoul (Korea, Republic of)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Doxazocin directly up-regulated bone metabolism at a low dose. Black-Right-Pointing-Pointer Doxazocin induced osteoblastic stem cell differentiation without affecting cell proliferation. Black-Right-Pointing-Pointer This osteogenic stem cell differentiation is mediated by ERK-signal dependent pathway. -- Abstract: Recent researches have indicated a role for antihypertensive drugs including alpha- or beta-blockers in the prevention of bone loss. Some epidemiological studies reported the protective effects of those agents on fracture risk. However, there is limited information on the association with those agents especially at the mechanism of action. In the present study, we investigated the effects of doxazosin, an alpha-blocker that is clinically used for the treatment of benign prostatic hyperplasia (BPH) along with antihypertensive medication, on the osteogenic stem cell differentiation. We found that doxazosin increased osteogenic differentiation of human mesenchymal stem cells, detected by Alizarin red S staining and calcein. Doxazosin not only induced expression of alkaline phosphatase, type I collagen, osteopontin, and osteocalcin, it also resulted in increased phosphorylation of extracellular signal-regulated kinase (ERK1/2), a MAP kinase involved in osteoblastic differentiation. Treatment with U0126, a MAP kinase inhibitor, significantly blocked doxazosin-induced osteoblastic differentiation. Unrelated to activation of osteogenic differentiation by doxazosin, we found that there were no significant changes in adipogenic differentiation or in the expression of adipose-specific genes, including peroxisome proliferator-activated receptor {gamma}, aP2, or LPL. In this report, we suggest that doxazosin has the ability to increase osteogenic cell differentiation via ERK1/2 activation in osteogenic differentiation of adult stem cells, which supports the protective effects of antihypertensive drug on fracture risk and

  9. Efficient differentiation of mouse embryonic stem cells into motor neurons.

    Science.gov (United States)

    Wu, Chia-Yen; Whye, Dosh; Mason, Robert W; Wang, Wenlan

    2012-06-09

    Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb9. Using this robust protocol, we achieved 51 ± 0.8% of differentiation efficiency (n = 3; p motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

  10. Collision rates for rare cell capture in periodic obstacle arrays strongly depend on density of cell suspension.

    Science.gov (United States)

    Cimrák, I

    2016-11-01

    Recently, computational modelling has been successfully used for determination of collision rates for rare cell capture in periodic obstacle arrays. The models were based on particle advection simulations where the cells were advected according to velocity field computed from two dimensional Navier-Stokes equations. This approach may be used under the assumption of very dilute cell suspensions where no mutual cell collisions occur. We use the object-in-fluid framework to demonstrate that even with low cell-to-fluid ratio, the optimal geometry of the obstacle array significantly changes. We show computational simulations for ratios of 3.5, 6.9 and 10.4% determining the optimal geometry of the periodic obstacle arrays. It was already previously demonstrated that cells in periodic obstacle arrays follow trajectories in two modes: the colliding mode and the zig-zag mode. The colliding mode maximizes the cell-obstacle collision frequency. Our simulations reveal that for dilute suspensions and for suspensions with cell-to-fluid ratio 3.5%, there is a range of column shifts for which the cells follow colliding trajectories. However we showed, that for 6.9 and 10.4%, the cells never follow colliding trajectories.

  11. Interplay of Matrix Stiffness and Cell-Cell Contact in Regulating Differentiation of Stem Cells.

    Science.gov (United States)

    Ye, Kai; Cao, Luping; Li, Shiyu; Yu, Lin; Ding, Jiandong

    2016-08-31

    Stem cells are capable of sensing and responding to the mechanical properties of extracellular matrixes (ECMs). It is well-known that, while osteogenesis is promoted on the stiff matrixes, adipogenesis is enhanced on the soft ones. Herein, we report an "abnormal" tendency of matrix-stiffness-directed stem cell differentiation. Well-defined nanoarrays of cell-adhesive arginine-glycine-aspartate (RGD) peptides were modified onto the surfaces of persistently nonfouling poly(ethylene glycol) (PEG) hydrogels to achieve controlled specific cell adhesion and simultaneously eliminate nonspecific protein adsorption. Mesenchymal stem cells were cultivated on the RGD-nanopatterned PEG hydrogels with the same RGD nanospacing but different hydrogel stiffnesses and incubated in the induction medium to examine the effect of matrix stiffness on osteogenic and adipogenic differentiation extents. When stem cells were kept at a low density during the induction period, the differentiation tendency was consistent with the previous reports in the literature; however, both lineage commitments were favored on the stiff matrices at a high cell density. We interpreted such a complicated stiffness effect at a high cell density in two-dimensional culture as the interplay of matrix stiffness and cell-cell contact. As a result, this study strengthens the essence of the stiffness effect and highlights the combinatory effects of ECM cues and cell cues on stem cell differentiation.

  12. Granulosa cell proliferation differentiation and its role in follicular development

    Institute of Scientific and Technical Information of China (English)

    LU Cuiling; YANG Wei; HU Zhaoyuan; LIU Yixun

    2005-01-01

    Granuiosa cells (GCs) are the most important cells in the ovary that undergo serious changes morphologically and physiologically during the processes of follicular proliferation, differentiation, ovulation, lutenization and atresia. Oocyte (OC) directs GC proliferation and differentiation, while GCs influence OC maturation. Many ovarian factors are involved in the regulation of these processes via different molecular mechanisms and signal pathways. P38MAPK can selectively regulate steroidogenesis in GCs controlled by FSH; Transcript factors LRH-1 and DAX-1 play an important role in this process; FSH induces GC prolfferation and differentiation by stimulating PCNA and StAR expression and steroidogenesis. Activated ERK1/2 signal pathway may be involved in the FSH-regulated GC proliferation and differentiation. Therefore, GC is an ideal model for studying cell proliferation, differentiation and interaction,as well as signal transduction. This review briefly summarizes the latest data in the literature, including the results achieved in our laboratory.

  13. Differentiation of Dental Pulp Stem Cells into Neuron-Like Cells in Serum-Free Medium

    Directory of Open Access Journals (Sweden)

    Shahrul Hisham Zainal Ariffin

    2013-01-01

    Full Text Available Dental pulp tissue contains dental pulp stem cells (DPSCs. Dental pulp cells (also known as dental pulp-derived mesenchymal stem cells are capable of differentiating into multilineage cells including neuron-like cells. The aim of this study was to examine the capability of DPSCs to differentiate into neuron-like cells without using any reagents or growth factors. DPSCs were isolated from teeth extracted from 6- to 8-week-old mice and maintained in complete medium. The cells from the fourth passage were induced to differentiate by culturing in medium without serum or growth factors. RT-PCR molecular analysis showed characteristics of Cd146+, Cd166+, and Cd31− in DPSCs, indicating that these cells are mesenchymal stem cells rather than hematopoietic stem cells. After 5 days of neuronal differentiation, the cells showed neuron-like morphological changes and expressed MAP2 protein. The activation of Nestin was observed at low level prior to differentiation and increased after 5 days of culture in differentiation medium, whereas Tub3 was activated only after 5 days of neuronal differentiation. The proliferation of the differentiated cells decreased in comparison to that of the control cells. Dental pulp stem cells are induced to differentiate into neuron-like cells when cultured in serum- and growth factor-free medium.

  14. Pathways in pluripotency and differentiation of embryonic cells

    NARCIS (Netherlands)

    du Puy, L.

    2010-01-01

    Pluripotency - the potential to differentiate into derivatives of the three embryonic germ layers endoderm, ectoderm and mesoderm - is the main characteristic of embryonic stem (ES) cells. ES cells are derived from the inner cell mass (ICM) of a pre-implantation blastocyst and can self-renew

  15. Differentiation ability of rat postnatal dental pulp cells in vitro.

    NARCIS (Netherlands)

    Zhang, W.; Walboomers, X.F.; Wolke, J.G.C.; Bian, Z.; Fan, M.W.; Jansen, J.A.

    2005-01-01

    The current rapid progression in stem cell research has enhanced our knowledge of dental tissue regeneration. In this study, rat dental pulp cells were isolated and their differentiation ability was evaluated. First, dental pulp cells were obtained from maxillary incisors of male Wistar rats. Immuno

  16. Stem cell differentiation: Post-degradation forces kick in

    Science.gov (United States)

    Vincent, Ludovic G.; Engler, Adam J.

    2013-05-01

    Stem cells alter their morphology and differentiate to particular lineages in response to biophysical cues from the surrounding matrix. When the matrix is degradable, however, cell fate is morphology-independent and is directed by the traction forces that the cells actively apply after they have degraded the matrix.

  17. Pathways in pluripotency and differentiation of embryonic cells

    NARCIS (Netherlands)

    du Puy, L.

    2010-01-01

    Pluripotency - the potential to differentiate into derivatives of the three embryonic germ layers endoderm, ectoderm and mesoderm - is the main characteristic of embryonic stem (ES) cells. ES cells are derived from the inner cell mass (ICM) of a pre-implantation blastocyst and can self-renew indefin

  18. Division of Labor in Biofilms : the Ecology of Cell Differentiation

    NARCIS (Netherlands)

    van Gestel, Jordi; Vlamakis, Hera; Kolter, Roberto

    2015-01-01

    The dense aggregation of cells on a surface, as seen in biofilms, inevitably results in both environmental and cellular heterogeneity. For example, nutrient gradients can trigger cells to differentiate into various phenotypic states. Not only do cells adapt physiologically to the local environmental

  19. Functional Implications of Neuroendocrine Differentiated Cells in Prostate Cancer

    NARCIS (Netherlands)

    J. Jongsma (Johan)

    2000-01-01

    textabstractThis thesis focuses on NE differentiation in prostate cancer, especially in prostate cancer models. We studied the effects of androgen depletion on the NE differentiated status of in vivo and in vitro prostatic tumor models. Knowledge concerning the function of NE cells in the normal hum

  20. Functional Implications of Neuroendocrine Differentiated Cells in Prostate Cancer

    NARCIS (Netherlands)

    J. Jongsma (Johan)

    2000-01-01

    textabstractThis thesis focuses on NE differentiation in prostate cancer, especially in prostate cancer models. We studied the effects of androgen depletion on the NE differentiated status of in vivo and in vitro prostatic tumor models. Knowledge concerning the function of NE cells in the normal

  1. NEW DESIGNED HMBA AGENTS AS INDUCERS OF ERYTHROLEUKEMIA CELL DIFFERENTIATION

    Institute of Scientific and Technical Information of China (English)

    王华力; 张世馥; 周建平; 章静波

    2002-01-01

    Objective.Searching for more potent and less toxic HMBA related agents. Methods.Human erythroleukemia cell K562,murine erythroleukemia cell (MEL) and its sub line MEL DS19 were used as target cells to select a cell line which is the most sensitive to HMBA,then analyzed the activity of inducing differentiation of two new designed HMBA derivatives:HMBPA [hexamethylenebi (3 pyridin) amide] and Co HDTA (ethylenediaminetetra acetic acid cobalt) using cell biology,cytochemical and molecular biology techniques. Results.We found that the MEL DS19 cells were most sensitive to HMBA (benzidine positive,B+ ~76% ).Co HDTA can inhibit the growth of MEL DS19,but induces differentiation just in a small population (B+ 2% ~4.5% ).Between 0.02~5μ mol/L,HMBPA induces 3% ~8% cells committed to differentiation with little inhibition of cell proliferation.1μ mol/L HMBPA and 2mmol/L HMBA together,can obviously increase the percentage of differentiated cell (B+ ~72% ),inhibit DNA synthesis and accelerate β globin transcription. Conclusion.The new HMBA derivatives may provide potential cancer differentiation inducers.

  2. Dedifferentiated fat cells differentiate into osteoblasts in titanium fiber mesh.

    Science.gov (United States)

    Kishimoto, Naotaka; Momota, Yoshihiro; Hashimoto, Yoshiya; Ando, Kayoko; Omasa, Takeshi; Kotani, Junichiro

    2013-01-01

    Mature adipocyte-derived dedifferentiated fat (DFAT) cells rapidly differentiate into osteoblasts under three-dimensional culture conditions. However, it has not been demonstrated that DFAT cells can differentiate into osteoblasts in a rigid scaffold consisting of titanium fiber mesh (TFM). We examined the proliferation and osteogenic differentiation ability of DFAT cells using TFM as a scaffold. DFAT cells derived from rabbit subcutaneous fat were seeded into TFM and cultured in osteogenic medium containing dexamethasone, L-ascorbic acid 2-phosphate and β-glycerophosphate for 14 days. In scanning electron microscopy (SEM) analysis, well-spread cells covered the titanium fibers on day 3, and appeared to increase in number from day 3 to 7. Numerous globular accretions were found and almost completely covered the fibers on day 14. Cell proliferation, as measured by DNA content in the TFM, was significantly higher on day 7 compared with that of day 1. Osteocalcin and calcium content in the TFM were significantly higher on day 14 compared to those of days 1, 3, and 7, indicating DFAT cells differentiated into osteoblasts. We theorize that globular accretions observed in SEM analysis may be calcified matrix resulting from osteocalcin secreted by osteoblasts binding calcium contained in fetal bovine serum. In this study, we demonstrated that DFAT cells differentiate into osteoblasts and deposit mineralized matrices in TFM. Therefore, the combination of DFAT cells and TFM may be an attractive option for bone tissue engineering.

  3. Mouse bone marrow stromal cells differentiate to neuron-like cells upon inhibition of BMP signaling.

    Science.gov (United States)

    Saxena, Monika; Prashar, Paritosh; Yadav, Prem Swaroop; Sen, Jonaki

    2016-01-01

    Bone marrow stromal cells (BMSCs) are a source of autologous stem cells that have the potential for undergoing differentiation into multiple cell types including neurons. Although the neuronal differentiation of mesenchymal stem cells has been studied for a long time, the molecular players involved are still not defined. Here we report that the genetic deletion of two members of the bone morphogenetic protein (Bmp) family, Bmp2 and Bmp4 in mouse BMSCs causes their differentiation into cells with neuron-like morphology. Surprisingly these cells expressed certain markers characteristic of both neuronal and glial cells. Based on this observation, we inhibited BMP signaling in mouse BMSCs through a brief exposure to Noggin protein which also led to their differentiation into cells expressing both neuronal and glial markers. Such cells seem to have the potential for further differentiation into subtypes of neuronal and glial cells and thus could be utilized for cell-based therapeutic applications.

  4. The Therapeutic Potential of Differentiated Lung Cells from Embryonic Stem Cells in Lung Diseases.

    Science.gov (United States)

    Mokhber Dezfouli, Mohammad Reza; Chaleshtori, Sirous Sadeghian; Dehghan, Mohammad Mehdi; Tavanaeimanesh, Hamid; Baharvand, Hossein; Tahamtani, Yaser

    2017-01-01

    Lung diseases cause great morbidity and mortality. The choice of effective medical treatment is limited and the number of lung diseases are difficult to treat with current treatments. The embryonic stem cells (ESCs) have the potential to differentiate into cell types of all three germinal layers, including lung epithelial cells. So they can be a potential source for new cell therapies for hereditary or acquired diseases of the airways and lungs. One method for treatment of lung diseases is cell therapy and the use of ESCs that can replace the damaged epithelial and endothelial cells. Progress using ESCs has developed slowly for lung regeneration because differentiation of lung cells from ESCs is more difficult as compared to differentiation of other cells. The review studies the therapeutic effects of differentiated lung cells from embryonic stem cells in lung diseases. There are few studies of differentiation of ESCs into a lineage of respiratory and then investigation of this cell in experimental model of lung diseases.

  5. Electro-osmotic-based catholyte production by Microbial Fuel Cells for carbon capture.

    Science.gov (United States)

    Gajda, Iwona; Greenman, John; Melhuish, Chris; Santoro, Carlo; Li, Baikun; Cristiani, Pierangela; Ieropoulos, Ioannis

    2015-12-01

    In Microbial Fuel Cells (MFCs), the recovery of water can be achieved with the help of both active (electro-osmosis), and passive (osmosis) transport pathways of electrolyte through the semi-permeable selective separator. The electrical current-dependent transport, results in cations and electro-osmotically dragged water molecules reaching the cathode. The present study reports on the production of catholyte on the surface of the cathode, which was achieved as a direct result of electricity generation using MFCs fed with wastewater, and employing Pt-free carbon based cathode electrodes. The highest pH levels (>13) of produced liquid were achieved by the MFCs with the activated carbon cathodes producing the highest power (309 μW). Caustic catholyte formation is presented in the context of beneficial cathode flooding and transport mechanisms, in an attempt to understand the effects of active and passive diffusion. Active transport was dominant under closed circuit conditions and showed a linear correlation with power performance, whereas osmotic (passive) transport was governing the passive flux of liquid in open circuit conditions. Caustic catholyte was mineralised to a mixture of carbonate and bicarbonate salts (trona) thus demonstrating an active carbon capture mechanism as a result of the MFC energy-generating performance. Carbon capture would be valuable for establishing a carbon negative economy and environmental sustainability of the wastewater treatment process.

  6. Simultaneous Wastewater Treatment, Algal Biomass Production and Electricity Generation in Clayware Microbial Carbon Capture Cells.

    Science.gov (United States)

    Jadhav, Dipak A; Jain, Sumat C; Ghangrekar, Makarand M

    2017-05-02

    Performance of microbial carbon capture cells (MCCs), having a low-cost clayware separator, was evaluated in terms of wastewater treatment and electricity generation using algae Chlorella pyrenoidosa in MCC-1 and Anabaena ambigua in MCC-2 and without algae in a cathodic chamber of MCC-3. Higher power production was achieved in MCC-1 (6.4 W/m(3)) compared to MCC-2 (4.29 W/m(3)) and MCC-3 (3.29 W/m(3)). Higher coulombic efficiency (15.23 ± 1.30%) and biomass production (66.4 ± 4.7 mg/(L*day)) in MCC-1 indicated the superiority of Chlorella over Anabaena algae for carbon capture and oxygen production to facilitate the cathodic reduction. Algal biofilm formation on the cathode surface of MCC-1 increased dissolved oxygen in the catholyte and decreased the cathodic charge transfer resistance with increase in reduction current. Electrochemical analyses revealed slow cathodic reactions and increase in internal resistance in MCC-2 (55 Ω) than MCC-1 (30 Ω), due to lower oxygen produced by Anabaena algae. Thus, biomass production in conjunction with wastewater treatment, CO2 sequestration and electricity generation can be achieved using Chlorella algal biocathode in MCC.

  7. COMPUTATION MODELING OF TCDD DISRUPTION OF B CELL TERMINAL DIFFERENTIATION

    Science.gov (United States)

    In this study, we established a computational model describing the molecular circuit underlying B cell terminal differentiation and how TCDD may affect this process by impinging upon various molecular targets.

  8. Differentiation of Stem Cells: Strategies for Modifying Surface Biomaterials

    National Research Council Canada - National Science Library

    Fu, Ru-Huei; Wang, Yu-Chi; Liu, Shih-Ping; Huang, Chin-Mao; Kang, Yun-Han; Tsai, Chang-Hai; Shyu, Woei-Cherng; Lin, Shinn-Zong

    2011-01-01

    ... in vivo, and their ability to generate tissue constructs in vitro. Determining how to efficiently drive stem cell differentiation to a lineage of choice is critical for the success of cellular therapeutics...

  9. Bone marrow cells differentiation into organ cells using stem cell therapy.

    Science.gov (United States)

    Yang, Y-J; Li, X-L; Xue, Y; Zhang, C-X; Wang, Y; Hu, X; Dai, Q

    2016-07-01

    Bone marrow cells (BMC) are progenitors of bone, cartilage, skeletal tissue, the hematopoiesis-supporting stroma and adipocyte cells. BMCs have the potential to differentiate into neural cells, cardiac myocytes, liver hepatocytes, chondrocytes, renal, corneal, blood, and myogenic cells. The bone marrow cell cultures from stromal and mesenchymal cells are called multipotent adult progenitor cells (MAPCs). MAPCs can differentiate into mesenchymal cells, visceral mesoderm, neuroectoderm and endoderm in vitro. It has been shown that the stem cells derived from bone marrow cells (BMCs) can regenerate cardiac myocytes after myocardial infarction (MI). Adult bone marrow mesenchymal stem cells have the ability to regenerate neural cells. Neural stem/progenitor cells (NS/PC) are ideal for treating central nervous system (CNS) diseases, such as Alzheimer's, Parkinson's and Huntington disease. However, there are important ethical issues about the therapeutic use of stem cells. Neurons, cardiac myocytes, hepatocytes, renal cells, blood cells, chondrocytes and adipocytes regeneration from BMCs are very important in disease control. It is known that limbal epithelial stem cells in the cornea can repair the eye sight and remove symptoms of blindness. Stem cell therapy (SCT) is progressing well in animal models, but the use of SCT in human remains to be explored further.

  10. Laser capture microdissection in Ectocarpus siliculosus: the pathway to cell-specific transcriptomics in brown algae

    Directory of Open Access Journals (Sweden)

    Denis eSaint-Marcoux

    2015-02-01

    Full Text Available Laser capture microdissection (LCM facilitates the isolation of individual cells from tissue sections, and when combined with RNA amplification techniques, it is an extremely powerful tool for examining genome-wide expression profiles in specific cell-types. LCM has been widely used to address various biological questions in both animal and plant systems, however, no attempt has been made so far to transfer LCM technology to macroalgae. Macroalgae are a collection of widespread eukaryotes living in fresh and marine water. In line with the collective effort to promote molecular investigations of macroalgal biology, here we demonstrate the feasibility of using LCM and cell-specific transcriptomics to study development of the brown alga, Ectocarpus siliculosus. We describe a workflow comprising cultivation and fixation of algae on glass slides, laser microdissection, and RNA amplification. To illustrate the effectiveness of the procedure, we show qPCR data and metrics obtained from cell-specific transcriptomes generated from both upright and prostrate filaments of Ectocarpus.

  11. Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin

    OpenAIRE

    2015-01-01

    Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34+ hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex...

  12. Extracellular vesicles derived from preosteoblasts influence embryonic stem cell differentiation.

    Science.gov (United States)

    Nair, Rekha; Santos, Lívia; Awasthi, Siddhant; von Erlach, Thomas; Chow, Lesley W; Bertazzo, Sergio; Stevens, Molly M

    2014-07-15

    Embryonic stem cells (ESCs) can differentiate into all cell types of the body and, therefore, hold tremendous promise for cell-based regenerative medicine therapies. One significant challenge that should be addressed before using ESCs in the clinic is to improve methods of efficiently and effectively directing the differentiation of this heterogeneous cell population. The work presented here examines the potential of harnessing naturally derived extracellular vesicles to deliver genetic material from mature cells to undifferentiated ESCs for the purpose of manipulating stem cell fate. Vesicles were isolated from preosteoblast cells and were found to be ∼170 nm in diameter and to express the CD40 surface marker. Multiple interactions were visualized between vesicles and ESCs using confocal microscopy, and no significant difference in cell viability was noted. Incubation with vesicles caused significant changes in ESC gene expression, including persistence of pluripotent gene levels as well as increased neurectoderm differentiation. Genetic cargo of the vesicles as well as the cells from which they were derived were examined using a small microRNA (miRNA) gene array. Interestingly, ∼20% of the examined miRNAs were increased more than twofold in the vesicles compared with preosteoblast cells. Together, these results suggest that extracellular vesicles may be utilized as a novel method of directing stem cell differentiation. Future work examining methods for controlled delivery of vesicles may improve the clinical potential of these physiological liposomes for therapeutic applications.

  13. Multiplicity of Sites for Extrathymic T-cell Differentiation

    OpenAIRE

    Abo, Toru; Watanabe, Hisami; Sekikawa, Hiroho

    1993-01-01

    In addition to an intrathymic pathway of T-cell differentiation, extrathymic pathways of T-cell differentiation have been found to exist at multiple sites in the living bodies of both mice and humans. Such sites include the sinusoids in the liver, intraepithelial sites in the intestine, the splenic red pulp, the thymic medulla, the decidua in the uterus, and the omentum in the peritoneal cavity. Although extrathymic pathways are minimal in youth, they become predominant with aging and under c...

  14. A systems biology approach identifies a regulatory network in parotid acinar cell terminal differentiation.

    Directory of Open Access Journals (Sweden)

    Melissa A Metzler

    Full Text Available The transcription factor networks that drive parotid salivary gland progenitor cells to terminally differentiate, remain largely unknown and are vital to understanding the regeneration process.A systems biology approach was taken to measure mRNA and microRNA expression in vivo across acinar cell terminal differentiation in the rat parotid salivary gland. Laser capture microdissection (LCM was used to specifically isolate acinar cell RNA at times spanning the month-long period of parotid differentiation.Clustering of microarray measurements suggests that expression occurs in four stages. mRNA expression patterns suggest a novel role for Pparg which is transiently increased during mid postnatal differentiation in concert with several target gene mRNAs. 79 microRNAs are significantly differentially expressed across time. Profiles of statistically significant changes of mRNA expression, combined with reciprocal correlations of microRNAs and their target mRNAs, suggest a putative network involving Klf4, a differentiation inhibiting transcription factor, which decreases as several targeting microRNAs increase late in differentiation. The network suggests a molecular switch (involving Prdm1, Sox11, Pax5, miR-200a, and miR-30a progressively decreases repression of Xbp1 gene transcription, in concert with decreased translational repression by miR-214. The transcription factor Xbp1 mRNA is initially low, increases progressively, and may be maintained by a positive feedback loop with Atf6. Transfection studies show that Xbp1 activates the Mist1 promoter [corrected]. In addition, Xbp1 and Mist1 each activate the parotid secretory protein (Psp gene, which encodes an abundant salivary protein, and is a marker of terminal differentiation.This study identifies novel expression patterns of Pparg, Klf4, and Sox11 during parotid acinar cell differentiation, as well as numerous differentially expressed microRNAs. Network analysis identifies a novel stemness arm, a

  15. Differentiation of mammary stem cells in vivo and in vitro.

    Science.gov (United States)

    Barraclough, R; Rudland, P S

    1989-03-01

    The fully differentiated cells of the rat mammary parenchyma, the ductal epithelial, alveolar, and myoepithelial cells, are distinguished by their ultrastructure and by their accumulation of immunocytochemically detectable marker proteins. The different cell types probably develop from primative ductal structures called terminal end buds, which are present in the developing rat mammary glands, and these structures contain relatively undifferentiated cells. Clonal epithelial stem cell lines, obtained from normal rat mammary glands or benign mammary tumors, differentiate under appropriate conditions along a pathway to droplet-cell/doming cultures of primative alveolarlike cells. Under different culture conditions, the epithelial stem cells differentiate along a separate pathway to myoepitheliallike cells. They accumulate some of the specific marker proteins of myoepithelial cells in vivo, including type IV collagen, laminin, and Thy-1 antigen. In addition, these myoepitheliallike cells in culture contain an abundance of a potential calcium-binding protein, p9Ka, which also occurs in myoepithelial cells of histological sections from mammary glands. The accumulation of type IV collagen, laminin, Thy-1, and p9Ka occurs asynchronously along the pathway to the myoepitheliallike cells in vitro. Furthermore, the steady-state levels of these different marker proteins arise by alterations in the controls at the transcriptional, the posttranscriptional processing, and the translational stages of their production. These results suggest a stepwise control of synthesis of myoepithelial cell marker proteins, and in the case of p9Ka and Thy-1 antigen, this altered control may arise through their possession of novel transcriptional promoters.

  16. Pituitary cell differentiation from stem cells and other cells: toward restorative therapy for hypopituitarism?

    Science.gov (United States)

    Willems, Christophe; Vankelecom, Hugo

    2014-01-01

    The pituitary gland, key regulator of our endocrine system, produces multiple hormones that steer essential physiological processes. Hence, deficient pituitary function (hypopituitarism) leads to severe disorders. Hypopituitarism can be caused by defective embryonic development, or by damage through tumor growth/resection and traumatic brain injury. Lifelong hormone replacement is needed but associated with significant side effects. It would be more desirable to restore pituitary tissue and function. Recently, we showed that the adult (mouse) pituitary holds regenerative capacity in which local stem cells are involved. Repair of deficient pituitary may therefore be achieved by activating these resident stem cells. Alternatively, pituitary dysfunction may be mended by cell (replacement) therapy. The hormonal cells to be transplanted could be obtained by (trans-)differentiating various kinds of stem cells or other cells. Here, we summarize the studies on pituitary cell regeneration and on (trans-)differentiation toward hormonal cells, and speculate on restorative therapies for pituitary deficiency.

  17. Lactobacilli Differentially Activate Natural Killer Cells

    DEFF Research Database (Denmark)

    Fink, Lisbeth Nielsen; Christensen, Hanne Risager; Frøkiær, Hanne

    Bacteria translocating across the gastrointestinal mucosa are presumed to gain access to NK cell compartments, as consumption of certain lactic acid bacteria has been shown to increase in vivo NK cytotoxicity. On-going research in our lab aims at describing strain-dependent effects of lactic acid...... bacteria on regulatory functions of NK-cells. Here, we have investigated how human gut flora-derived non-pathogenic lactobacilli affect NK cells in vitro, by measuring proliferation and IFN-gamma production of human peripheral blood NK cells upon bacterial stimulation. CD3-CD56+ NK cells were isolated from...... buffy coats by negative isolation using non-NK lineage specific antibodies and magnetic beads. NK cells were incubated with 10 microg/ml UV-inactivated bacteria for four days. Proliferation was assessed by incorporation of radioactive thymidine into NK cell DNA and cytokine concentrations were...

  18. Sambucus williamsii induced embryonic stem cells differentiated into neurons.

    Science.gov (United States)

    Liu, Shih-Ping; Hsu, Chien-Yu; Fu, Ru-Huei; Huang, Yu-Chuen; Chen, Shih-Yin; Lin, Shinn-Zong; Shyu, Woei-Cherng

    2015-01-01

    The pluripotent stem cells, including embryonic stem cells (ESCs), are capable of self-renewal and differentiation into any cell type, thus making them the focus of many clinical application studies. However, the efficiency of ESCs differentiated into neurons needs to improve. In this study, we tried to increase efficiently to a neural fate in the presence of various transitional Chinese medicines through a three-step differentiation strategy. From extracts of 10 transitional Chinese medicine candidates, we determined that Sambucus williamsii (SW) extract triggers the up-regulation of Nestin and Tuj1 (neuron cells markers) gene expression levels. After determining the different concentrations of SW extract, the number of neurons in the 200 μg/ml SW extract group was higher than the control, 50, 100, and 400 μg/ml SW extract groups. In addition, the number of neurons in the 200 μg/ml SW extract group was higher and higher after each time passage (three times). We also detected the Oct4, Sox2 (stem cells markers), Tuj1, and Nestin genes expression levels by RT-PCR. In the differentiated process, Oct4 and Sox2 genes decreased while the Tuj1 and Nestin genes expression levels increased. In summary, we demonstrated that SW could induce pluripotent stem cells differentiated into neurons. Thus, SW might become a powerful material for neurons-differentiating strategies.

  19. Hepatocyte-like cells from directed differentiation of mouse bone marrow cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Xiao-lei SHI; Yu-dong QIU; Qiang LI; Ting XIE; Zhang-hua ZHU; Lei-lei CHEN; Lei LI; Yi-tao DING

    2005-01-01

    Aim: To design the effective directed differentiation medium to differentiate bone marrow cells into hepatocyte-like cells. Methods: Bone marrow cells were cultured in the directed differentiation media including fibroblast growth factor-4 (FGF-4) and oncostatin M (OSM). Hepatocyte-like cells from directed differentia tion of bone marrow cells were identified through cell morphology, RNA expressions by reverse transcriptase-polymerase chain reaction (RT-PCR), protein expressions by Western blot, and hepatocellular synthesis and metabolism functions by albumin ELISA, Periodic acid-Shiff staining and urea assay. Results:Some epithelial-like cells or polygonal cells appeared and increased in the course of the cell directed differentiation. Hepatocyte nucleur factor-3β (HNF-3β), albumin (ALB), cytokeratin 18 (CK18), transthyretin (TFR), glucose-6-phosphate (G6-Pase), and tyrosine aminotransferase (TAT) mRNA were expressed in the course of the directed differentiation. The directed differentiated cells on d 21 expressed HNF-3β, ALB, and CK18 proteins. The directed differentiated cells produced albumin and synthesized urea in a time-dependent manner. They could also synthesize glycogen. Conclusion: Our differentiation media, including FGF-4 and OSM, are effective to differentiate bone marrow cells into hepatocyte-like cells, which could be used for hepatocyte resources for bioartificial liver or hepatocyte transplantation.

  20. Molten Carbonate Fuel Cell performance analysis varying cathode operating conditions for carbon capture applications

    Science.gov (United States)

    Audasso, Emilio; Barelli, Linda; Bidini, Gianni; Bosio, Barbara; Discepoli, Gabriele

    2017-04-01

    The results of a systematic experimental campaign to verify the impact of real operating conditions on the performance of a complete Molten Carbonate Fuel Cell (MCFC) are presented. In particular, the effects of ageing and composition of water, oxygen and carbon dioxide in the cathodic feeding stream are studied through the analysis of current-voltage curves and Electrochemical Impedance Spectroscopy (EIS). Based on a proposed equivalent electrical circuit model and a fitting procedure, a correlation is found among specific operating parameters and single EIS coefficients. The obtained results suggest a new performance monitoring approach to be applied to MCFC for diagnostic purpose. Particular attention is devoted to operating conditions characteristic of MCFC application as CO2 concentrators, which, by feeding the cathode with exhaust gases, is a promising route for efficient and cheap carbon capture.

  1. Adlayer-mediated antibody immobilization to stainless steel for potential application to endothelial progenitor cell capture.

    Science.gov (United States)

    Benvenuto, Pasquale; Neves, Miguel A D; Blaszykowski, Christophe; Romaschin, Alexander; Chung, Timothy; Kim, Sa Rang; Thompson, Michael

    2015-05-19

    This work describes the straightforward surface modification of 316L stainless steel with BTS, S-(11-trichlorosilylundecanyl)-benzenethiosulfonate, a thiol-reactive trichlorosilane cross-linker molecule designed to form intermediary coatings with subsequent biofunctionalization capability. The strategy is more specifically exemplified with the immobilization of intact antibodies and their Fab' fragments. Both surface derivatization steps are thoroughly characterized by means of X-ray photoelectron spectroscopy. The antigen binding capability of both types of biofunctionalized surfaces is subsequently assessed by fluorescence microscopy. It was determined that BTS adlayers achieve robust immobilization of both intact and fragmented antibodies, while preserving antigen binding activity. Another key finding was the observation that the Fab' fragment immobilization strategy would constitute a preferential option over that involving intact antibodies in the context of in vivo capture of endothelial progenitor cells in stent applications.

  2. Intrinsic differentiation potential of adolescent human tendon tissue: an in-vitro cell differentiation study

    Directory of Open Access Journals (Sweden)

    Weinans Harrie

    2007-02-01

    Full Text Available Abstract Background Tendinosis lesions show an increase of glycosaminoglycan amount, calcifications, and lipid accumulation. Therefore, altered cellular differentiation might play a role in the etiology of tendinosis. This study investigates whether adolescent human tendon tissue contains a population of cells with intrinsic differentiation potential. Methods Cells derived from adolescent non-degenerative hamstring tendons were characterized by immunohistochemistry and FACS-analysis. Cells were cultured for 21 days in osteogenic, adipogenic, and chondrogenic medium and phenotypical evaluation was carried out by immunohistochemical and qPCR analysis. The results were compared with the results of similar experiments on adult bone marrow-derived stromal cells (BMSCs. Results Tendon-derived cells stained D7-FIB (fibroblast-marker positive, but α-SMA (marker for smooth muscle cells and pericytes negative. Tendon-derived cells were 99% negative for CD34 (endothelial cell marker, and 73% positive for CD105 (mesenchymal progenitor-cell marker. In adipogenic medium, intracellular lipid vacuoles were visible and tendon-derived fibroblasts showed upregulation of adipogenic markers FABP4 (fatty-acid binding protein 4 and PPARG (peroxisome proliferative activated receptor γ. In chondrogenic medium, some cells stained positive for collagen 2 and tendon-derived fibroblasts showed upregulation of collagen 2 and collagen 10. In osteogenic medium Von Kossa staining showed calcium deposition although osteogenic markers remained unaltered. Tendon-derived cells and BMCSs behaved largely comparable, although some distinct differences were present between the two cell populations. Conclusion This study suggests that our population of explanted human tendon cells has an intrinsic differentiation potential. These results support the hypothesis that there might be a role for altered tendon-cell differentiation in the pathophysiology of tendinosis.

  3. BMP2 induces chondrogenic differentiation, osteogenic differentiation and endochondral ossification in stem cells.

    Science.gov (United States)

    Zhou, Nian; Li, Qi; Lin, Xin; Hu, Ning; Liao, Jun-Yi; Lin, Liang-Bo; Zhao, Chen; Hu, Zhen-Ming; Liang, Xi; Xu, Wei; Chen, Hong; Huang, Wei

    2016-10-01

    Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-β (TGF-β) super-family, is one of the main chondrogenic growth factors involved in cartilage regeneration. BMP2 is known to induce chondrogenic differentiation in various types of stem cells in vitro. However, BMP2 also induces osteogenic differentiation and endochondral ossification in mesenchymal stem cells (MSCs). Although information regarding BMP2-induced chondrogenic and osteogenic differentiation within the same system might be essential for cartilage tissue engineering, few studies concerning these issues have been conducted. In this study, BMP2 was identified as a regulator of chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. BMP2 was used to regulate chondrogenic and osteogenic differentiation in stem cells within the same culture system in vitro and in vivo. Any changes in the differentiation markers were assessed. BMP2 was found to induce chondrogenesis and osteogenesis in vitro via the expression of Sox9, Runx2 and its downstream markers. According to the results of the subcutaneous stem cell implantation studies, BMP2 not only induced cartilage formation but also promoted endochondral ossification during ectopic bone/cartilage formation. In fetal limb cultures, BMP2 promoted chondrocyte hypertrophy and endochondral ossification. Our data reveal that BMP2 can spontaneously induce chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. Thus, BMP2 can be used in cartilage tissue engineering to regulate cartilage formation but has to be properly regulated for cartilage tissue engineering in order to retain the cartilage phenotype.

  4. Integrating human stem cell expansion and neuronal differentiation in bioreactors

    Directory of Open Access Journals (Sweden)

    Costa Eunice M

    2009-09-01

    Full Text Available Abstract Background Human stem cells are cellular resources with outstanding potential for cell therapy. However, for the fulfillment of this application, major challenges remain to be met. Of paramount importance is the development of robust systems for in vitro stem cell expansion and differentiation. In this work, we successfully developed an efficient scalable bioprocess for the fast production of human neurons. Results The expansion of undifferentiated human embryonal carcinoma stem cells (NTera2/cl.D1 cell line as 3D-aggregates was firstly optimized in spinner vessel. The media exchange operation mode with an inoculum concentration of 4 × 105 cell/mL was the most efficient strategy tested, with a 4.6-fold increase in cell concentration achieved in 5 days. These results were validated in a bioreactor where similar profile and metabolic performance were obtained. Furthermore, characterization of the expanded population by immunofluorescence microscopy and flow cytometry showed that NT2 cells maintained their stem cell characteristics along the bioreactor culture time. Finally, the neuronal differentiation step was integrated in the bioreactor process, by addition of retinoic acid when cells were in the middle of the exponential phase. Neurosphere composition was monitored and neuronal differentiation efficiency evaluated along the culture time. The results show that, for bioreactor cultures, we were able to increase significantly the neuronal differentiation efficiency by 10-fold while reducing drastically, by 30%, the time required for the differentiation process. Conclusion The culture systems developed herein are robust and represent one-step-forward towards the development of integrated bioprocesses, bridging stem cell expansion and differentiation in fully controlled bioreactors.

  5. Hepatic differentiation of embryonic stem cells by murine fetal liver mesenchymal cells.

    Science.gov (United States)

    Ishii, Takamichi; Yasuchika, Kentaro; Ikai, Iwao

    2013-01-01

    Hepatocytes derived from embryonic stem cells (ESCs) are a potential cell source for regenerative medicine. However, it has been technically difficult to differentiate ESCs into mature hepatocytes because the definitive growth factors and molecular mechanisms governing hepatocyte differentiation have not yet been well defined. The CD45(-)CD49f(+/-)Thy1(+)gp38(+) mesenchymal cells that reside in murine fetal livers induce hepatic progenitor cells to differentiate into mature hepatocytes by direct cell-cell contact. Utilizing these cells, we employ a two-step procedure for hepatic maturation of ESCs: first, ESCs are differentiated into endodermal cells or hepatic progenitor cells, and second, ESC-derived endodermal cells are matured into functional hepatocytes by coculture with murine fetal liver mesenchymal cells. The ESC-derived hepatocyte-like cells possess hepatic functions, including ammonia removal activity, albumin secretion ability, glycogen synthesis and storage, and cytochrome P450 enzymatic activity.

  6. Differentiation of rat marrow mesenchymal stem cells into pancreatic islet beta-cells

    Institute of Scientific and Technical Information of China (English)

    Li-Bo Chen; Xiao-Bing Jiang; Lian Yang

    2004-01-01

    AIM: To explore the possibility of marrow mesenchymal stem cells (MSC)in vitro differentiating into functional isletlike cells and to test the diabetes therapeutic potency of Islet-like cells.METHODS: Rat MSCs were isolated from Wistar rats and cultured. Passaged MSCs were induced to differentiate into islet-like cells under following conditions: pre-induction with L-DMEM including 10 mmol/L nicotinamide+1 mmol/L β-mercaptoethanol+200 mL/L fetal calf serum (FSC) for 24 h,followed by induction with serum free H-DMEM solution including 10 mmol/L nicotinamide+ 1 mmol/L,β-mercaptoethanol for 10 h. Differentiated cells were observed under inverse microscopy, insulin and nestin expressed in differentiated cells were detected with immunocytochemistry. Insulin excreted from differentiated cells was tested with radioimmunoassay. Rat diabetic models were made to test in vivo function of differentiated MSCs.RESULTS: Typical islet -like clustered cells were observed.Insulin mRNA and protein expressions were positive in differentiated cells, and nestin could be detected in predifferentiated cells. Insulin excreted from differentiated MSCs (446.93±102.28 IU/L) was much higher than that from pre-differentiated MSCs (2.45±0.81 IU/L (P<0.01).Injected differentiated MSCs cells could down-regulate glucose level in diabetic rats.CONCLUSION: Islet-like functional cells can be differentiated from marrow mesenchymal stem cells, which may be a new procedure for clinical diabetes stem -cell therapy, these cells can control blood glucose level in diabetic rats. MSCs may play an important role in diabetes therapy by islet differentiation and transplantation.

  7. Communication is key: Reducing DEK1 activity reveals a link between cell-cell contacts and epidermal cell differentiation status.

    Science.gov (United States)

    Galletti, Roberta; Ingram, Gwyneth C

    2015-01-01

    Plant epidermis development requires not only the initial acquisition of tissue identity, but also the ability to differentiate specific cell types over time and to maintain these differentiated states throughout the plant life. To set-up and maintain differentiation, plants activate specific transcriptional programs. Interfering with these programs can prevent differentiation and/or force differentiated cells to lose their identity and re-enter a proliferative state. We have recently shown that the Arabidopsis Defective Kernel 1 (DEK1) protein is required both for the differentiation of epidermal cells and for the maintenance of their fully differentiated state. Defects in DEK1 activity lead to a deregulation of the expression of epidermis-specific differentiation-promoting HD-ZIP IV transcription factors. Here we propose a working model in which DEK1, by maintaining cell-cell contacts, and thus communication between neighboring cells, influences HD-ZIP IV gene expression and epidermis differentiation.

  8. Influence of Porcine Intervertebral Disc Matrix on Stem Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Hans-Lothar Fuchsbauer

    2011-08-01

    Full Text Available For back disorders, cell therapy is one approach for a real regeneration of a degenerated nucleus pulposus. Human mesenchymal stem cells (hMSC could be differentiated into nucleus pulposus (NP-like cells and used for cell therapy. Therefore it is necessary to find a suitable biocompatible matrix, which supports differentiation. It could be shown that a differentiation of hMSC in a microbial transglutaminase cross-linked gelatin matrix is possible, but resulted in a more chondrocyte-like cell type. The addition of porcine NP extract to the gelatin matrix caused a differentiation closer to the desired NP cell phenotype. This concludes that a hydrogel containing NP extract without any other supplements could be suitable for differentiation of hMSCs into NP cells. The NP extract itself can be cross-linked by transglutaminase to build a hydrogel free of NP atypical substrates. As shown by side-specific biotinylation, the NP extract contains molecules with free glutamine and lysine residues available for the transglutaminase.

  9. Molecular ties between the cell cycle and differentiation in embryonic stem cells.

    Science.gov (United States)

    Li, Victor C; Kirschner, Marc W

    2014-07-01

    Attainment of the differentiated state during the final stages of somatic cell differentiation is closely tied to cell cycle progression. Much less is known about the role of the cell cycle at very early stages of embryonic development. Here, we show that molecular pathways involving the cell cycle can be engineered to strongly affect embryonic stem cell differentiation at early stages in vitro. Strategies based on perturbing these pathways can shorten the rate and simplify the lineage path of ES differentiation. These results make it likely that pathways involving cell proliferation intersect at various points with pathways that regulate cell lineages in embryos and demonstrate that this knowledge can be used profitably to guide the path and effectiveness of cell differentiation of pluripotent cells.

  10. Cell Shape and Cardiosphere Differentiation: A Revelation by Proteomic Profiling

    Directory of Open Access Journals (Sweden)

    Nanako Kawaguchi

    2013-01-01

    Full Text Available Stem cells (embryonic stem cells, somatic stem cells such as neural stem cells, and cardiac stem cells and cancer cells are known to aggregate and form spheroid structures. This behavior is common in undifferentiated cells and may be necessary for adapting to certain conditions such as low-oxygen levels or to maintain undifferentiated status in microenvironments including stem cell niches. In order to decipher the meaning of this spheroid structure, we established a cardiosphere clone (CSC-21E derived from the rat heart which can switch its morphology between spheroid and nonspheroid. Two forms, floating cardiospheres and dish-attached flat cells, could be switched reversibly by changing the cell culture condition. We performed differential proteome analysis studies and obtained protein profiles distinct between spherical forms and flat cells. From protein profiling analysis, we found upregulation of glycolytic enzymes in spheroids with some stress proteins switched in expression levels between these two forms. Evidence has been accumulating that certain chaperone/stress proteins are upregulated in concert with cellular changes including proliferation and differentiation. We would like to discuss the possible mechanism of how these aggregates affect cell differentiation and/or other cellular functions.

  11. Cartilage stem cells: regulation of differentiation.

    Science.gov (United States)

    Solursh, M

    1989-01-01

    The developing limb bud is a useful source of cartilage stem cells for studies on the regulation of chondrogenesis. In high density cultures these cells can progress through all stages of chondrogenesis to produce mineralized hypertrophic cartilage. If the cells are maintained in a spherical shape, single stem cells can progress through a similar sequence. The actin cytoskeleton is implicated in the regulation of chondrogenesis since conditions that favor its disruption promote chondrogenesis and conditions that favor actin assembly inhibit chondrogenesis. Since a number of extracellular matrix receptors mediate effects of the extracellular matrix on cytoskeletal organization and some of these receptors are developmentally regulated, it is proposed that matrix receptor expression plays a central role in the divergence of connective tissue cells during development.

  12. Differentiation of breast cancer stem cells by knockdown of CD44: promising differentiation therapy

    Directory of Open Access Journals (Sweden)

    Pham Phuc V

    2011-12-01

    Full Text Available Abstract Background Breast cancer stem cells (BCSCs are the source of breast tumors. Compared with other cancer cells, cancer stem cells show high resistance to both chemotherapy and radiotherapy. Targeting of BCSCs is thus a potentially promising and effective strategy for breast cancer treatment. Differentiation therapy represents one type of cancer stem-cell-targeting therapy, aimed at attacking the stemness of cancer stem cells, thus reducing their chemo- and radioresistance. In a previous study, we showed that down-regulation of CD44 sensitized BCSCs to the anti-tumor agent doxorubicin. This study aimed to determine if CD44 knockdown caused BCSCs to differentiate into breast cancer non-stem cells (non-BCSCs. Methods We isolated a breast cancer cell population (CD44+CD24- cells from primary cultures of malignant breast tumors. These cells were sorted into four sub-populations based on their expression of CD44 and CD24 surface markers. CD44 knockdown in the BCSC population was achieved using small hairpin RNA lentivirus particles. The differentiated status of CD44 knock-down BCSCs was evaluated on the basis of changes in CD44+CD24- phenotype, tumorigenesis in NOD/SCID mice, and gene expression in relation to renewal status, metastasis, and cell cycle in comparison with BCSCs and non-BCSCs. Results Knockdown of CD44 caused BCSCs to differentiate into non-BCSCs with lower tumorigenic potential, and altered the cell cycle and expression profiles of some stem cell-related genes, making them more similar to those seen in non-BCSCs. Conclusions Knockdown of CD44 is an effective strategy for attacking the stemness of BCSCs, resulting in a loss of stemness and an increase in susceptibility to chemotherapy or radiation. The results of this study highlight a potential new strategy for breast cancer treatment through the targeting of BCSCs.

  13. Oncogenic NRAS Primes Primary Acute Myeloid Leukemia Cells for Differentiation.

    Directory of Open Access Journals (Sweden)

    Cornelia Brendel

    Full Text Available RAS mutations are frequently found among acute myeloid leukemia patients (AML, generating a constitutively active signaling protein changing cellular proliferation, differentiation and apoptosis. We have previously shown that treatment of AML patients with high-dose cytarabine is preferentially beneficial for those harboring oncogenic RAS. On the basis of a murine AML cell culture model, we ascribed this effect to a RAS-driven, p53-dependent induction of differentiation. Hence, in this study we sought to confirm the correlation between RAS status and differentiation of primary blasts obtained from AML patients. The gene expression signature of AML blasts with oncogenic NRAS indeed corresponded to a more mature profile compared to blasts with wildtype RAS, as demonstrated by gene set enrichment analysis (GSEA and real-time PCR analysis of myeloid ecotropic viral integration site 1 homolog (MEIS1 in a unique cohort of AML patients. In addition, in vitro cell culture experiments with established cell lines and a second set of primary AML cells showed that oncogenic NRAS mutations predisposed cells to cytarabine (AraC driven differentiation. Taken together, our findings show that AML with inv(16 and NRAS mutation have a differentiation gene signature, supporting the notion that NRAS mutation may predispose leukemic cells to AraC induced differentiation. We therefore suggest that promotion of differentiation pathways by specific genetic alterations could explain the superior treatment outcome after therapy in some AML patient subgroups. Whether a differentiation gene expression status may generally predict for a superior treatment outcome in AML needs to be addressed in future studies.

  14. An Evolutionarily Conserved Plant RKD Factor Controls Germ Cell Differentiation.

    Science.gov (United States)

    Koi, Satoshi; Hisanaga, Tetsuya; Sato, Katsutoshi; Shimamura, Masaki; Yamato, Katsuyuki T; Ishizaki, Kimitsune; Kohchi, Takayuki; Nakajima, Keiji

    2016-07-11

    In contrast to animals, in which the germ cell lineage is established during embryogenesis, plant germ cells are generated in reproductive organs via reprogramming of somatic cells. The factors that control germ cell differentiation and reprogramming in plants are poorly understood. Members of the RKD subfamily of plant-specific RWP-RK transcription factors have been implicated in egg cell formation in Arabidopsis based on their expression patterns and ability to cause an egg-like transcriptome upon ectopic expression [1]; however, genetic evidence of their involvement is lacking, due to possible genetic redundancy, haploid lethality, and the technical difficulty of analyzing egg cell differentiation in angiosperms. Here we analyzed the factors that govern germ cell formation in the liverwort Marchantia polymorpha. This recently revived model bryophyte has several characteristics that make it ideal for studies of germ cell formation, such as low levels of genetic redundancy, readily accessible germ cells, and the ability to propagate asexually via gemma formation [2, 3]. Our analyses revealed that MpRKD, a single RWP-RK factor closely related to angiosperm RKDs, is preferentially expressed in developing eggs and sperm precursors in M. polymorpha. Targeted disruption of MpRKD had no effect on the gross morphology of the vegetative and reproductive organs but led to striking defects in egg and sperm cell differentiation, demonstrating that MpRKD is an essential regulator of germ cell differentiation. Together with previous findings [1, 4-6], our results suggest that RKD factors are evolutionarily conserved regulators of germ cell differentiation in land plants. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Differential Effects of Tacrolimus versus Sirolimus on the Proliferation, Activation and Differentiation of Human B Cells.

    Directory of Open Access Journals (Sweden)

    Opas Traitanon

    Full Text Available The direct effect of immunosuppressive drugs calcineurin inhibitor (Tacrolimus, TAC and mTOR inhibitor (Sirolimus, SRL on B cell activation, differentiation and proliferation is not well documented. Purified human B cells from healthy volunteers were stimulated through the B Cell Receptor with Anti-IgM + anti-CD40 + IL21 in the absence / presence of TAC or SRL. A variety of parameters of B cell activity including activation, differentiation, cytokine productions and proliferation were monitored by flow cytometry. SRL at clinically relevant concentrations (6 ng/ml profoundly inhibited CD19(+ B cell proliferation compared to controls whereas TAC at similar concentrations had a minimal effect. CD27(+ memory B cells were affected more by SRL than naïve CD27- B cells. SRL effectively blocked B cell differentiation into plasma cells (CD19(+CD138(+ and Blimp1(+/Pax5(low cells even at low dose (2 ng/ml, and totally eliminated them at 6 ng/ml. SRL decreased absolute B cell counts, but the residual responding cells acquired an activated phenotype (CD25(+/CD69(+ and increased the expression of HLA-DR. SRL-treated stimulated B cells on a per cell basis were able to enhance the proliferation of allogeneic CD4(+CD25(- T cells and induce a shift toward the Th1 phenotype. Thus, SRL and TAC have different effects on B lymphocytes. These data may provide insights into the clinical use of these two agents in recipients of solid organ transplants.

  16. Heterogeneous differentiation patterns of individual CD8+ T cells.

    Science.gov (United States)

    Gerlach, Carmen; Rohr, Jan C; Perié, Leïla; van Rooij, Nienke; van Heijst, Jeroen W J; Velds, Arno; Urbanus, Jos; Naik, Shalin H; Jacobs, Heinz; Beltman, Joost B; de Boer, Rob J; Schumacher, Ton N M

    2013-05-03

    Upon infection, antigen-specific CD8(+) T lymphocyte responses display a highly reproducible pattern of expansion and contraction that is thought to reflect a uniform behavior of individual cells. We tracked the progeny of individual mouse CD8(+) T cells by in vivo lineage tracing and demonstrated that, even for T cells bearing identical T cell receptors, both clonal expansion and differentiation patterns are heterogeneous. As a consequence, individual naïve T lymphocytes contributed differentially to short- and long-term protection, as revealed by participation of their progeny during primary versus recall infections. The discordance in fate of individual naïve T cells argues against asymmetric division as a singular driver of CD8(+) T cell heterogeneity and demonstrates that reproducibility of CD8(+) T cell responses is achieved through population averaging.

  17. Human embryonic stem cells differentiate into functional renal proximal tubular-like cells.

    Science.gov (United States)

    Narayanan, Karthikeyan; Schumacher, Karl M; Tasnim, Farah; Kandasamy, Karthikeyan; Schumacher, Annegret; Ni, Ming; Gao, Shujun; Gopalan, Began; Zink, Daniele; Ying, Jackie Y

    2013-04-01

    Renal cells are used in basic research, disease models, tissue engineering, drug screening, and in vitro toxicology. In order to provide a reliable source of human renal cells, we developed a protocol for the differentiation of human embryonic stem cells into renal epithelial cells. The differentiated stem cells expressed markers characteristic of renal proximal tubular cells and their precursors, whereas markers of other renal cell types were not expressed or expressed at low levels. Marker expression patterns of these differentiated stem cells and in vitro cultivated primary human renal proximal tubular cells were comparable. The differentiated stem cells showed morphological and functional characteristics of renal proximal tubular cells, and generated tubular structures in vitro and in vivo. In addition, the differentiated stem cells contributed in organ cultures for the formation of simple epithelia in the kidney cortex. Bioreactor experiments showed that these cells retained their functional characteristics under conditions as applied in bioartificial kidneys. Thus, our results show that human embryonic stem cells can differentiate into renal proximal tubular-like cells. Our approach would provide a source for human renal proximal tubular cells that are not affected by problems associated with immortalized cell lines or primary cells.

  18. Differentiation of neuron-like cells from mouse parthenogenetic embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    Xingrong Yan; Liwen Li; Fulin Chen; Yanhong Yang; Wei Liu; Wenxin Geng; Huichong Du; Jihong Cui; Xin Xie; Jinlian Hua; Shumin Yu

    2013-01-01

    Parthenogenetic embryonic stem cells have pluripotent differentiation potentials, akin to fertilized embryo-derived embryonic stem cells. The aim of this study was to compare the neuronal differentiation potential of parthenogenetic and fertilized embryo-derived embryonic stem cells. Before differentiation, karyotype analysis was performed, with normal karyotypes detected in both parthenogenetic and fertilized embryo-derived embryonic stem cells. Sex chromosomes were identified as XX. Immunocytochemistry and quantitative real-time PCR detected high expression of the pluripotent gene, Oct4, at both the mRNA and protein levels, indicating pluripotent differentiation potential of the two embryonic stem cell subtypes. Embryonic stem cells were induced with retinoic acid to form embryoid bodies, and then dispersed into single cells. Single cells were differentiated in N2 differentiation medium for 9 days. Immunocytochemistry showed parthenogenetic and fertilized embryo-derived embryonic stem cells both express the neuronal cell markers nestin, βIII-tubulin and myelin basic protein. Quantitative real-time PCR found expression of neurogenesis related genes (Sox-1, Nestin, GABA, Pax6, Zic5 and Pitx1) in both types of embryonic stem cells, and Oct4 expression was significantly decreased. Nestin and Pax6 expression in parthenogenetic embryonic stem cells was significantly higher than that in fertilized embryo-derived embryonic stem cells. Thus, our experimental findings indicate that parthenogenetic embryonic stem cells have stronger neuronal differentiation potential than fertilized embryo-derived embryonic stem cells.

  19. Tinospora cordifolia Induces Differentiation and Senescence Pathways in Neuroblastoma Cells.

    Science.gov (United States)

    Mishra, Rachana; Kaur, Gurcharan

    2015-08-01

    Children diagnosed with neuroblastomas often suffer from severe side as well as late effects of conventional treatments like chemotherapy and radiotherapy. Recent advances in understanding of molecular pathways involved in cellular differentiation and apoptosis have helped in the development of new therapeutic approach based on differentiation-based therapy of malignant tumours. Natural medicines with their holistic therapeutic approach are known to selectively eliminate cancer cells thus provide a better substitute for the conventional treatment modes. The current study was aimed to investigate the anti-cancer potential of aqueous ethanolic extract of Tinospora cordifolia (TCE) using IMR-32 human neuroblastoma cell line as a model system. TCE is highly recommended in Ayurveda for its general body and metal health-promoting properties. TCE treatment was seen to arrest the majority of cells in G0/G1 phase and modulated the expression of DNA clamp sliding protein (PCNA) and cyclin D1. Further, TCE-treated cells showed differentiation as revealed by their morphology and the expression of neuronal cell specific differentiation markers NF200, MAP-2 and NeuN in neuroblastoma cells. The differentiated phenotype was associated with induction of senescence and pro-apoptosis pathways by enhancing expression of senescence marker mortalin and Rel A subunit of nuclear factor kappa beta (NFkB) along with decreased expression of anti-apoptotic marker, Bcl-xl. TCE exhibited anti-metastatic activity and significantly reduced cell migration in the scratched area along with downregulation of neural cell adhesion molecule (NCAM) polysialylation and secretion of matrix metalloproteinases (MMPs). Our data suggest that crude extract or active phytochemicals from this plant may be a potential candidate for differentiation-based therapy of malignant neuroblastoma cells.

  20. Cripto is essential to capture mouse epiblast stem cell and human embryonic stem cell pluripotency.

    Science.gov (United States)

    Fiorenzano, Alessandro; Pascale, Emilia; D'Aniello, Cristina; Acampora, Dario; Bassalert, Cecilia; Russo, Francesco; Andolfi, Gennaro; Biffoni, Mauro; Francescangeli, Federica; Zeuner, Ann; Angelini, Claudia; Chazaud, Claire; Patriarca, Eduardo J; Fico, Annalisa; Minchiotti, Gabriella

    2016-09-02

    Known molecular determinants of developmental plasticity are mainly transcription factors, while the extrinsic regulation of this process has been largely unexplored. Here we identify Cripto as one of the earliest epiblast markers and a key extracellular determinant of the naive and primed pluripotent states. We demonstrate that Cripto sustains mouse embryonic stem cell (ESC) self-renewal by modulating Wnt/β-catenin, whereas it maintains mouse epiblast stem cell (EpiSC) and human ESC pluripotency through Nodal/Smad2. Moreover, we provide unprecedented evidence that Cripto controls the metabolic reprogramming in ESCs to EpiSC transition. Remarkably, Cripto deficiency attenuates ESC lineage restriction in vitro and in vivo, and permits ESC transdifferentiation into trophectoderm lineage, suggesting that Cripto has earlier functions than previously recognized. All together, our studies provide novel insights into the current model of mammalian pluripotency and contribute to the understanding of the extrinsic regulation of the first cell lineage decision in the embryo.

  1. E-cadherin interactions are required for Langerhans cell differentiation

    Science.gov (United States)

    Mayumi, Nobuko; Watanabe, Eri; Norose, Yoshihiko; Watari, Eiji; Kawana, Seiji; Geijtenbeek, Teunis B H; Takahashi, Hidemi

    2013-01-01

    Human skin contains the following two distinct DC subsets: (i) Langerhans cells (LCs), expressing Langerin but not DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), are predominantly localized in the epidermis; and (ii) dermal DCs, expressing DC-SIGN but not Langerin, are observed mainly in the dermis. It is not known whether localization in the epidermis provides cues for LC differentiation. Here, we show that E-cadherin expressed by epidermal keratinocytes (KCs) is crucial for differentiation of LCs. Monocytes differentiated into LC-like cells in presence of IL-4, GM-CSF, and TGF-β1. However, these LC-like cells expressed not only Langerin but also DC-SIGN. Notably, co-culturing of these LC-like cells with KCs expressing E-cadherin or recombinant E-cadherin strongly decreased expression of DC-SIGN and further induced a phenotype similar to purified epidermal LCs. Moreover, pretreatment of LC-like cells with anti-E-cadherin-specific antibody completely abolished their Langerin expression, indicating the requirement of E-cadherin–E-cadherin interactions for the differentiation into Langerin+ cells. These findings suggest that E-cadherin expressed by KCs provide environmental cues that induce differentiation of LCs in the epidermis. PMID:23135957

  2. Hyaluronan scaffold supports osteogenic differentiation of bone marrow concentrate cells.

    Science.gov (United States)

    Cavallo, C; Desando, G; Ferrari, A; Zini, N; Mariani, E; Grigolo, B

    2016-01-01

    Osteochondral lesions are considered a challenge for orthopedic surgeons. Currently, the treatments available are often unsatisfactory and unable to stimulate tissue regeneration. Tissue engineering offers a new therapeutic strategy, taking into account the role exerted by cells, biomaterial and growth factors in restoring tissue damage. In this light, Mesenchymal Stem Cells (MSCs) have been indicated as a fascinating tool for regenerative medicine thanks to their ability to differentiate into bone, cartilage and adipose tissue. However, in vitro-cultivation of MSCs could be associated with some risks such as de-differentiation/reprogramming, infection and contaminations of the cells. To overcome these shortcomings, a new approach is represented by the use of Bone Marrow Concentrate (BMC), that could allow the delivery of cells surrounded by their microenvironment in injured tissue. For this purpose, cells require a tridimensional scaffold that can support their adhesion, proliferation and differentiation. This study is focused on the potentiality of BMC seeded onto a hyaluronan-based scaffold (Hyaff-11) to differentiate into osteogenic lineage. This process depends on the specific interaction between cells derived from bone marrow (surrounded by their niche) and scaffold, that create an environment able to support the regeneration of damaged tissue. The data obtained from the present study demonstrate that BMC grown onto Hyaff-11 are able to differentiate toward osteogenic sense, producing specific osteogenic genes and matrix proteins.

  3. The Preliminary Experimental Study of Induced Differentiation of Embryonic Stem Cells into Corneal Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    Ling Yu; Jian Ge; Zhichong Wang; Bing Huang; Keming Yu; Chongde Long; Xigu Chen

    2001-01-01

    Purpose:To study preliminarily induced differentiation of embryonic stem cells intocorneal epithelial cells in vitro.Methods: Murine embryonic stem cells were co-cultured with Rabbit limbal cornealepithelial cells in Transwell system to induce differentiation. Mophological andimmunohistochemical examination were implemented.Results: The induced cells from embryonic stem cells have an epithelial appearance.The cells formed a network and were confluent into film gradually after beingco-cultured with rabbit limbal corneal epithelial cells for 24 ~ 96 hours. The cells rangedmosaic structure and localized together with clear rim. Most of the cells showedpolygonal appearance. Transmission electron microscope showed lots of microvilli on thesurface of induced cells and tight junctions between them. These epithelial-like cellsexpressed the corneal epithelial cell specific marker cytokeratin3/cytokeratinl2.Conclusion: The potential mechanism of the differentiation of murine embryonic stemcells into corneal epithelial cells induced by limbal corneal epithelial cell-derivedinducing activity is to be further verified.

  4. Differential expression of cell adhesion genes

    DEFF Research Database (Denmark)

    Stein, Wilfred D; Litman, Thomas; Fojo, Tito

    2005-01-01

    It is well known that tumors arising from tissues such as kidney, pancreas, liver and stomach are particularly refractory to treatment. Searching for new anticancer drugs using cells in culture has yielded some effective therapies, but these refractory tumors remain intractable. Studies that comp......It is well known that tumors arising from tissues such as kidney, pancreas, liver and stomach are particularly refractory to treatment. Searching for new anticancer drugs using cells in culture has yielded some effective therapies, but these refractory tumors remain intractable. Studies...... in cell adhesion and the cytoskeleton. If the proteins involved in tethering cells to the extracellular matrix are important in conferring drug resistance, it may be possible to improve chemotherapy by designing drugs that target these proteins....

  5. Proteomic cornerstones of hematopoietic stem cell differentiation

    DEFF Research Database (Denmark)

    Klimmeck, Daniel; Hansson, Jenny; Raffel, Simon

    2012-01-01

    Regenerative tissues such as the skin epidermis, the intestinal mucosa or the hematopoietic system are organized in a hierarchical manner with stem cells building the top of this hierarchy. Somatic stem cells harbor the highest self-renewal activity and generate a series of multipotent progenitors...... related to immune defence mechanisms, centering around the RIG-I and type-1 interferon response systems, which are installed in multipotent progenitors but not evident in myeloid committed cells. This suggests that specific, and so far unrecognized, mechanisms protect these immature cells before...... they mature. In conclusion, this study indicates that the transition of hematopoietic stem/progenitors towards myeloid commitment is accompanied by a profound change in processing of cellular resources, adding novel insights into the molecular mechanisms at the interface between multipotency and lineage...

  6. [Cell therapy using stem cells: trophic factor, differentiation, and cell transplantation].

    Science.gov (United States)

    Hida, Hideki

    2013-02-01

    Our research of stem cell transplantation using mouse embryonic stem (ES) cells and induced pluripotent (iPS) cells was carried out from the aspect of trophic factor, cell differentiation, and better survival of grafted cells. Pleiotrophin, an enhanced trophic factor in the dopamine (DA)-depleted striatum, increased the number of DAergic neurons from ES-derived neural stem cells (ES-NSCs), increased cell survival of cultured DAergic neurons, and affected cell survival of grafted DAergic cells in Parkinson model rats. It was shown that DAergic differentiation from ES-NSCs was mediated by hypoxia inducible factor 1-alpha. Our challenges of the transplantation of ES-NSCs and iPS-derived oligodendrocyte progenitor cells (iPS-OPCs) into periventricular leukomalasia (PVL) model rats are also presented. It was found that grafted ES-NSCs survived better in the corpus callosum without immunosuppressant and most of them differentiated into neurons near the grafted site. It was also revealed that only a few of the grafted iPS-OPCs induced by a stepwise culture method with no use of serum could survive in PVL model rats, indicating that trophic factor (s) and improvement of graft techniques will be needed for better survival of grafted iPS-OPCs.

  7. A novel system for xylem cell differentiation in Arabidopsis thaliana.

    Science.gov (United States)

    Kondo, Yuki; Fujita, Takashi; Sugiyama, Munetaka; Fukuda, Hiroo

    2015-04-01

    During vascular development, procambial and cambial cells give rise to xylem and phloem cells. Because the vascular tissue is deeply embedded, it has been difficult to analyze the processes of vascular development in detail. Here, we establish a novel in vitro experimental system in which vascular development is induced in Arabidopsis thaliana leaf-disk cultures using bikinin, an inhibitor of glycogen synthase kinase 3 proteins. Transcriptome analysis reveals that mesophyll cells in leaf disks synchronously turn into procambial cells and then differentiate into tracheary elements. Leaf-disk cultures from plants expressing the procambial cell markers TDR(pro):GUS and TDR(pro):YFP can be used for spatiotemporal visualization of procambial cell formation. Further analysis with the tdr mutant and TDIF (tracheary element differentiation inhibitory factor) indicates that the key signaling TDIF-TDR-GSK3s regulates xylem differentiation in leaf-disk cultures. This new culture system can be combined with analysis using the rich material resources for Arabidopsis including cell-marker lines and mutants, thus offering a powerful tool for analyzing xylem cell differentiation. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

  8. Phosphorylation dynamics during early differentiation of human embryonic stem cells

    NARCIS (Netherlands)

    van Hoof, D.; Munoz, J.; Braam, S.R.; Pinkse, M.W.H.; Linding, R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.

    2009-01-01

    Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during

  9. Phosphorylation dynamics during early differentiation of human embryonic stem cells

    NARCIS (Netherlands)

    van Hoof, D.; Munoz, J.; Braam, S.R.; Pinkse, M.W.H.; Linding, R.; Heck, A.J.R.; Mummery, C.L.; Krijgsveld, J.

    2009-01-01

    Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during different

  10. Dexamethasone Suppresses Oxysterol-Induced Differentiation of Monocytic Cells

    Directory of Open Access Journals (Sweden)

    Yonghae Son

    2016-01-01

    Full Text Available Oxysterol like 27-hydroxycholesterol (27OHChol has been reported to induce differentiation of monocytic cells into a mature dendritic cell phenotype. We examined whether dexamethasone (Dx affects 27OHChol-induced differentiation using THP-1 cells. Treatment of monocytic cells with Dx resulted in almost complete inhibition of transcription and surface expression of CD80, CD83, and CD88 induced by 27OHChol. Elevated surface levels of MHC class I and II molecules induced by 27OHChol were reduced to basal levels by treatment with Dx. A decreased endocytosis ability caused by 27OHChol was recovered by Dx. We also examined effects of Dx on expression of CD molecules involved in atherosclerosis. Increased levels of surface protein and transcription of CD105, CD137, and CD166 by treatment with 27OHChol were significantly inhibited by cotreatment with Dx. These results indicate that Dx inhibits 27OHChol-induced differentiation of monocytic cells into a mature dendritic cell phenotype and expression of CD molecules whose levels are associated with atherosclerosis. In addition, we examined phosphorylation of AKT induced by 27OHChol and effect of Dx, where cotreatment with Dx inhibited the phosphorylation of AKT. The current study reports that Dx regulates oxysterol-mediated dendritic cell differentiation of monocytic cells.

  11. Endogenous collagen influences differentiation of human multipotent mesenchymal stromal cells

    NARCIS (Netherlands)

    Fernandes, H.; Mentink, A.; Bank, R.; Stoop, R.; Blitterswijk, C. van; Boer, J. de

    2010-01-01

    Human multipotent mesenchymal stromal cells (hMSCs) are multipotent cells that, in the presence of appropriate stimuli, can differentiate into different lineages such as the osteogenic, chondrogenic, and adipogenic lineages. In the presence of ascorbic acid, MSCs secrete an extracellular matrix main

  12. Phosphorylation dynamics during early differentiation of human embryonic stem cells

    DEFF Research Database (Denmark)

    Van Hoof, Dennis; Muñoz, Javier; Braam, Stefan R

    2009-01-01

    Pluripotent stem cells self-renew indefinitely and possess characteristic protein-protein networks that remodel during differentiation. How this occurs is poorly understood. Using quantitative mass spectrometry, we analyzed the (phospho)proteome of human embryonic stem cells (hESCs) during...

  13. Differential Protein Network Analysis of the Immune Cell Lineage

    Directory of Open Access Journals (Sweden)

    Trevor Clancy

    2014-01-01

    Full Text Available Recently, the Immunological Genome Project (ImmGen completed the first phase of the goal to understand the molecular circuitry underlying the immune cell lineage in mice. That milestone resulted in the creation of the most comprehensive collection of gene expression profiles in the immune cell lineage in any model organism of human disease. There is now a requisite to examine this resource using bioinformatics integration with other molecular information, with the aim of gaining deeper insights into the underlying processes that characterize this immune cell lineage. We present here a bioinformatics approach to study differential protein interaction mechanisms across the entire immune cell lineage, achieved using affinity propagation applied to a protein interaction network similarity matrix. We demonstrate that the integration of protein interaction networks with the most comprehensive database of gene expression profiles of the immune cells can be used to generate hypotheses into the underlying mechanisms governing the differentiation and the differential functional activity across the immune cell lineage. This approach may not only serve as a hypothesis engine to derive understanding of differentiation and mechanisms across the immune cell lineage, but also help identify possible immune lineage specific and common lineage mechanism in the cells protein networks.

  14. Pathway-focused PCR array profiling of enriched populations of laser capture microdissected hippocampal cells after traumatic brain injury.

    Directory of Open Access Journals (Sweden)

    Deborah R Boone

    Full Text Available Cognitive deficits in survivors of traumatic brain injury (TBI are associated with irreversible neurodegeneration in brain regions such as the hippocampus. Comparative gene expression analysis of dying and surviving neurons could provide insight into potential therapeutic targets. We used two pathway-specific PCR arrays (RT2 Profiler Apoptosis and Neurotrophins & Receptors PCR arrays to identify and validate TBI-induced gene expression in dying (Fluoro-Jade-positive or surviving (Fluoro-Jade-negative pyramidal neurons obtained by laser capture microdissection (LCM. In the Apoptosis PCR array, dying neurons showed significant increases in expression of genes associated with cell death, inflammation, and endoplasmic reticulum (ER stress compared with adjacent, surviving neurons. Pro-survival genes with pleiotropic functions were also significantly increased in dying neurons compared to surviving neurons, suggesting that even irreversibly injured neurons are able to mount a protective response. In the Neurotrophins & Receptors PCR array, which consists of genes that are normally expected to be expressed in both groups of hippocampal neurons, only a few genes were expressed at significantly different levels between dying and surviving neurons. Immunohistochemical analysis of selected, differentially expressed proteins supported the gene expression data. This is the first demonstration of pathway-focused PCR array profiling of identified populations of dying and surviving neurons in the brain after TBI. Combining precise laser microdissection of identifiable cells with pathway-focused PCR array analysis is a practical, low-cost alternative to microarrays that provided insight into neuroprotective signals that could be therapeutically targeted to ameliorate TBI-induced neurodegeneration.

  15. Cloning mice and ES cells by nuclear transfer from somatic stem cells and fully differentiated cells.

    Science.gov (United States)

    Wang, Zhongde

    2011-01-01

    Cloning animals by nuclear transfer (NT) has been successful in several mammalian species. In addition to cloning live animals (reproductive cloning), this technique has also been used in several species to establish cloned embryonic stem (ntES) cell lines from somatic cells. It is the latter application of this technique that has been heralded as being the potential means to produce isogenic embryonic stem cells from patients for cell therapy (therapeutic cloning). These two types of cloning differ only in the steps after cloned embryos are produced: for reproductive cloning the cloned embryos are transferred to surrogate mothers to allow them to develop to full term and for therapeutic cloning the cloned embryos are used to derive ntES cells. In this chapter, a detailed NT protocol in mouse by using somatic stem cells (neuron and skin stem cells) and fully differentiated somatic cells (cumulus cells and fibroblast cells) as nuclear donors is described.

  16. Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Met, Özcan;

    2011-01-01

    Standard cell culture plastic was surface modified by passive adsorption or covalent attachment of interleukin (IL)-4 and investigated for its ability to induce differentiation of human monocytes into mature dendritic cells, a process dose-dependently regulated by IL-4. Covalent attachment of IL-4...... proceeded via anthraquinone photochemistry to introduce amine functionalities at the surface followed by coupling of IL-4 through a bifunctional amine-reactive linker. X-ray photoelectron spectroscopy showed that undesirable multilayer formation of the photoactive compound could be avoided by reaction...... in water instead of phosphate-buffered saline. Passively adsorbed IL-4 was observed to induce differentiation to dendritic cells, but analysis of cell culture supernatants revealed that leakage of IL-4 into solution could account for the differentiation observed. Covalent attachment resulted in bound IL-4...

  17. Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (Keystone Sym)

    Science.gov (United States)

    Our goal is to establish an in vitro model system to evaluate chemical effects using a single stem cell culture technique that would improve throughput and provide quantitative markers of differentiation and cell number. To this end, we have used an adherent cell differentiation ...

  18. Hypergravity Stimulation Enhances PC12 Neuron-Like Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Giada Graziana Genchi

    2015-01-01

    Full Text Available Altered gravity is a strong physical cue able to elicit different cellular responses, representing a largely uninvestigated opportunity for tissue engineering/regenerative medicine applications. Our recent studies have shown that both proliferation and differentiation of C2C12 skeletal muscle cells can be enhanced by hypergravity treatment; given these results, PC12 neuron-like cells were chosen to test the hypothesis that hypergravity stimulation might also affect the behavior of neuronal cells, in particular promoting an enhanced differentiated phenotype. PC12 cells were thus cultured under differentiating conditions for either 12 h or 72 h before being stimulated with different values of hypergravity (50 g and 150 g. Effects of hypergravity were evaluated at transcriptional level 1 h and 48 h after the stimulation, and at protein level 48 h from hypergravity exposure, to assess its influence on neurite development over increasing differentiation times. PC12 differentiation resulted strongly affected by the hypergravity treatments; in particular, neurite length was significantly enhanced after exposure to high acceleration values. The achieved results suggest that hypergravity might induce a faster and higher neuronal differentiation and encourage further investigations on the potential of hypergravity in the preparation of cellular constructs for regenerative medicine and tissue engineering purposes.

  19. PPARγ agonists promote oligodendrocyte differentiation of neural stem cells by modulating stemness and differentiation genes.

    Directory of Open Access Journals (Sweden)

    Saravanan Kanakasabai

    Full Text Available Neural stem cells (NSCs are a small population of resident cells that can grow, migrate and differentiate into neuro-glial cells in the central nervous system (CNS. Peroxisome proliferator-activated receptor gamma (PPARγ is a nuclear receptor transcription factor that regulates cell growth and differentiation. In this study we analyzed the influence of PPARγ agonists on neural stem cell growth and differentiation in culture. We found that in vitro culture of mouse NSCs in neurobasal medium with B27 in the presence of epidermal growth factor (EGF and basic fibroblast growth factor (bFGF induced their growth and expansion as neurospheres. Addition of all-trans retinoic acid (ATRA and PPARγ agonist ciglitazone or 15-Deoxy-Δ(12,14-Prostaglandin J(2 (15d-PGJ2 resulted in a dose-dependent inhibition of cell viability and proliferation of NSCs in culture. Interestingly, NSCs cultured with PPARγ agonists, but not ATRA, showed significant increase in oligodendrocyte precursor-specific O4 and NG2 reactivity with a reduction in NSC marker nestin, in 3-7 days. In vitro treatment with PPARγ agonists and ATRA also induced modest increase in the expression of neuronal β-III tubulin and astrocyte-specific GFAP in NSCs in 3-7 days. Further analyses showed that PPARγ agonists and ATRA induced significant alterations in the expression of many stemness and differentiation genes associated with neuro-glial differentiation in NSCs. These findings highlight the influence of PPARγ agonists in promoting neuro-glial differentiation of NSCs and its significance in the treatment of neurodegenerative diseases.

  20. Capturing Thoughts, Capturing Minds?

    DEFF Research Database (Denmark)

    Nielsen, Janni

    2004-01-01

    Think Aloud is cost effective, promises access to the user's mind and is the applied usability technique. But 'keep talking' is difficult, besides, the multimodal interface is visual not verbal. Eye-tracking seems to get around the verbalisation problem. It captures the visual focus of attention...

  1. Role of Hox genes in stem cell differentiation

    Institute of Scientific and Technical Information of China (English)

    Anne Seifert; David F Werheid; Silvana M Knapp; Edda Tobiasch

    2015-01-01

    Hox genes are an evolutionary highly conserved genefamily. They determine the anterior-posterior body axisin bilateral organisms and influence the developmentalfate of cells. Embryonic stem cells are usually devoidof any Hox gene expression, but these transcriptionfactors are activated in varying spatial and temporalpatterns defining the development of various bodyregions. In the adult body, Hox genes are among othersresponsible for driving the differentiation of tissuestem cells towards their respective lineages in order torepair and maintain the correct function of tissues andorgans. Due to their involvement in the embryonic andadult body, they have been suggested to be useable forimproving stem cell differentiations in vitro and in vivo .In many studies Hox genes have been found as drivingfactors in stem cell differentiation towards adipogenesis,in lineages involved in bone and joint formation, mainlychondrogenesis and osteogenesis, in cardiovascularlineages including endothelial and smooth muscle celldifferentiations, and in neurogenesis. As life expectancyis rising, the demand for tissue reconstruction continuesto increase. Stem cells have become an increasinglypopular choice for creating therapies in regenerativemedicine due to their self-renewal and differentiationpotential. Especially mesenchymal stem cells are usedmore and more frequently due to their easy handlingand accessibility, combined with a low tumorgenicityand little ethical concerns. This review therefore intendsto summarize to date known correlations betweennatural Hox gene expression patterns in body tissuesand during the differentiation of various stem cellstowards their respective lineages with a major focus onmesenchymal stem cell differentiations. This overviewshall help to understand the complex interactions of Hoxgenes and differentiation processes all over the bodyas well as in vitro for further improvement of stem celltreatments in future regenerative medicine approaches.

  2. Isolation of Circulating Plasma Cells in Multiple Myeloma Using CD138 Antibody-Based Capture in a Microfluidic Device

    Science.gov (United States)

    Qasaimeh, Mohammad A.; Wu, Yichao C.; Bose, Suman; Menachery, Anoop; Talluri, Srikanth; Gonzalez, Gabriel; Fulciniti, Mariateresa; Karp, Jeffrey M.; Prabhala, Rao H.; Karnik, Rohit

    2017-04-01

    The necessity for bone marrow aspiration and the lack of highly sensitive assays to detect residual disease present challenges for effective management of multiple myeloma (MM), a plasma cell cancer. We show that a microfluidic cell capture based on CD138 antigen, which is highly expressed on plasma cells, permits quantitation of rare circulating plasma cells (CPCs) in blood and subsequent fluorescence-based assays. The microfluidic device is based on a herringbone channel design, and exhibits an estimated cell capture efficiency of ~40-70%, permitting detection of <10 CPCs/mL using 1-mL sample volumes, which is difficult using existing techniques. In bone marrow samples, the microfluidic-based plasma cell counts exhibited excellent correlation with flow cytometry analysis. In peripheral blood samples, the device detected a baseline of 2-5 CD138+ cells/mL in healthy donor blood, with significantly higher numbers in blood samples of MM patients in remission (20-24 CD138+ cells/mL), and yet higher numbers in MM patients exhibiting disease (45-184 CD138+ cells/mL). Analysis of CPCs isolated using the device was consistent with serum immunoglobulin assays that are commonly used in MM diagnostics. These results indicate the potential of CD138-based microfluidic CPC capture as a useful ‘liquid biopsy’ that may complement or partially replace bone marrow aspiration.

  3. Curcumin increases rat mesenchymal stem cell osteoblast differentiation but inhibits adipocyte differentiation

    Directory of Open Access Journals (Sweden)

    Qiaoli Gu

    2012-01-01

    Full Text Available Background: Curcumin is a phenolic natural product isolated from the rhizome of Curcuma longa (turmeric and has effects on bone health and fat formation. The bone marrow mesenchymal stem cells (MSCs are multipotent cells capable of differentiating into osteoblasts and adipocytes. Osteoblast differentiation of MSCs can be a result of upregulation of heme oxygenase (HO-1 expression. Curcumin can potently induce HO-1 expression. Objective: The present study describes the effects of curcumin on rat MSC (rMSCs differentiation into osteoblasts and adipocytes. Materials and Methods: Rat bone marrow MSCs were isolated and treated with or without curcumin. Osteoblast differentiation was confirmed and determined by alkaline phosphatase (ALP activity, mineralized nodule formation, the expression of Runx2 (runt-related transcription factor 2 and osteocalcin. Adipocyte differentiation was determined by Oil red O staining and the expression of peroxisome proliferator-activated receptor-γ 2 (PPARγ2 and CCAAT/enhancer-binding protein (C/EBP α. Results: Curcumin increased ALP activity and osteoblast-specific mRNA expression of Runx2 and osteocalcin when rMSCs were cultured in osteogenic medium. In contrast, curcumin decreased adipocyte differentiation and inhibited adipocyte-specific mRNA expression of PPARγ2 and C/EBPα when rMSCs were cultured in adipogenic medium. HO-1 expression was increased during osteogenic differentiation of rMSCs. Conclusions: These findings demonstrate that curcumin can promote osteogenic differentiation of rMSCs and inhibit adipocyte formation. The effect of curcumin on osteogenic differentiation of rMSCs is correlated with HO-1 expression.

  4. Canthin-6-one induces cell death, cell cycle arrest and differentiation in human myeloid leukemia cells.

    Science.gov (United States)

    Vieira Torquato, Heron F; Ribeiro-Filho, Antonio C; Buri, Marcus V; Araújo Júnior, Roberto T; Pimenta, Renata; de Oliveira, José Salvador R; Filho, Valdir C; Macho, Antonio; Paredes-Gamero, Edgar J; de Oliveira Martins, Domingos T

    2017-04-01

    Canthin-6-one is a natural product isolated from various plant genera and from fungi with potential antitumor activity. In the present study, we evaluate the antitumor effects of canthin-6-one in human myeloid leukemia lineages. Kasumi-1 lineage was used as a model for acute myeloid leukemia. Cells were treated with canthin-6-one and cell death, cell cycle and differentiation were evaluated in both total cells (Lin(+)) and leukemia stem cell population (CD34(+)CD38(-)Lin(-/low)). Among the human lineages tested, Kasumi-1 was the most sensitive to canthin-6-one. Canthin-6-one induced cell death with apoptotic (caspase activation, decrease of mitochondrial potential) and necrotic (lysosomal permeabilization, double labeling of annexin V/propidium iodide) characteristics. Moreover, canthin-6-one induced cell cycle arrest at G0/G1 (7μM) and G2 (45μM) evidenced by DNA content, BrdU incorporation and cyclin B1/histone 3 quantification. Canthin-6-one also promoted differentiation of Kasumi-1, evidenced by an increase in the expression of myeloid markers (CD11b and CD15) and the transcription factor PU.1. Furthermore, a reduction of the leukemic stem cell population and clonogenic capability of stem cells were observed. These results show that canthin-6-one can affect Kasumi-1 cells by promoting cell death, cell cycle arrest and cell differentiation depending on concentration used. Canthin-6-one presents an interesting cytotoxic activity against leukemic cells and represents a promising scaffold for the development of molecules for anti-leukemic applications, especially by its anti-leukemic stem cell activity. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Human embryonic stem cell derivation and directed differentiation.

    Science.gov (United States)

    Trounson, A

    2005-01-01

    Human embryonic stem cells (hESCs) are produced from normal, chromosomally aneuploid and mutant human embryos, which are available from in vitro fertilisation (IVF) for infertility or preimplantation diagnosis. These hESC lines are an important resource for functional genomics, drug screening and eventually cell and gene therapy. The methods for deriving hESCs are well established and repeatable, and are relatively successful, with a ratio of 1:10 to 1:2 hESC lines established to embryos used. hESCs can be formed from morula and blastocyst-stage embryos and from isolated inner cell mass cell (ICM) clusters. The hESCs can be formed and maintained on mouse or human somatic cells in serum-free conditions, and for several passages in cell-free cultures. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in culture while maintaining their original karyotype but this must be confirmed from time to time. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating attachment cultures and in unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes and characteristic morphology, and the culture thereafter enriched for further culture to more mature cell types. The most advanced directed differentiation pathways have been developed for neural cells and cardiac muscle cells, but many other cell types including haematopoietic progenitors, endothelial cells, lung alveoli, keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurones, hepatic progenitors and cells that have some markers of gut tissue and pancreatic cells have been produced. The prospects for regenerative medicine are significant and there is much

  6. Isolation, identification and differentiation of human embryonic cartilage stem cells.

    Science.gov (United States)

    Fu, Changhao; Yan, Zi; Xu, Hao; Zhang, Chen; Zhang, Qi; Wei, Anhui; Yang, Xi; Wang, Yi

    2015-07-01

    We isolated human embryonic cartilage stem cells (hECSCs), a novel stem cell population, from the articular cartilage of eight-week-old human embryos. These stem cells demonstrated a marker expression pattern and differentiation potential intermediate to those of human embryonic stem cells (hESCs) and human adult stem cells (hASCs). hECSCs expressed markers associated with both hESCs (OCT4, NANOG, SOX2, SSEA-3 and SSEA-4) and human adult stem cells (hASCs) (CD29, CD44, CD90, CD73 and CD10). These cells also differentiated into adipocytes, osteoblasts, chondrocytes, neurons and islet-like cells under specific inducing conditions. We identified N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) as an inducer of chondrogenic differentiation in hECSCs. Similar results using N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) were obtained for two other types of human embryonic tissue-derived stem cells, human embryonic hepatic stem cells (hEHSCs) and human embryonic amniotic fluid stem cells (hEASCs), both of which exhibited a marker expression pattern similar to that of hECSCs. The isolation of hECSCs and the discovery that N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) induces chondrogenic differentiation in different stem cell populations might aid the development of strategies in tissue engineering and cartilage repair.

  7. The microRNA-dependent cell fate of multipotent stromal cells differentiating to endothelial cells.

    Science.gov (United States)

    Cha, Min-Ji; Choi, Eunhyun; Lee, Seahyoung; Song, Byeong-Wook; Yoon, Cheesoon; Hwang, Ki-Chul

    2016-02-15

    In the endothelial recovery process, bone marrow-derived MSCs are a potential source of cells for both research and therapy, and their capacities to self-renew and to differentiate into all the cell types in the human body make them a promising therapeutic agent for remodeling cellular differentiation and a valuable resource for the treatment of many diseases. Based on the results provided in a miRNA database, we selected miRNAs with unique targets in cell fate-related signaling pathways. The tested miRNAs targeting GSK-3β (miR-26a), platelet-derived growth factor receptor, and CD133 (miR-26a and miR-29b) induced MSC differentiation into functional ECs, whereas miRNAs targeting VEGF receptor (miR-15, miR-144, miR-145, and miR-329) inhibited MSC differentiation into ECs through VEGF stimulation. In addition, the expression levels of these miRNAs were correlated with in vivo physiological endothelial recovery processes. These findings indicate that the miRNA expression profile is distinct for cells in different stages of differentiation from MSCs to ECs and that specific miRNAs can function as regulators of endothelialization.

  8. Gene Expression Analysis of Immunostained Endothelial Cells Isolated from Formaldehyde-fixated Paraffin Embedded Tumors Using Laser Capture Microdissection – a Technical Report

    Science.gov (United States)

    Kaneko, Tomoatsu; Okiji, Takashi; Kaneko, Reika; Suda, Hideaki; Nör, Jacques E.

    2009-01-01

    Laser capture microdissection (LCM) allows microscopic procurement of specific cell types from tissue sections that can then be used for gene expression analysis. In conventional LCM, frozen tissues stained with hematoxylin are normally used to the molecular analysis. Recent studies suggested that it is possible to carry out gene expression analysis of formaldehyde-fixated paraffin embedded (FFPE) tissues that were stained with hematoxylin. However, it is still unclear if quantitative gene expression analyses can be performed from LCM cells from FFPE tissues that were subjected to immunostaining to enhance identification of target cells. In this proof-of-principle study, we analyzed by RT-PCR and real time PCR the expression of genes in factor VIII immunostained human endothelial cells that were dissected from FFPE tissues by LCM. We observed that immunostaining should be performed at 4°C to preserve the mRNA from the cells. The expression of Bcl-2 in the endothelial cells was evaluated by RT-PCR and by real time PCR. GAPDH and 18S were used as house keeping genes for RT-PCR and real time PCR, respectively. This report unveils a method for quantitative gene expression analysis in cells that were identified by immunostaining and retrieved by LCM from FFPE tissues. This method is ideally suited for the analysis of relatively rare cell types within a tissue, and should improve on our ability to perform differential diagnosis of pathologies as compared to conventional LCM. PMID:19425073

  9. Interleukin-1 regulates proliferation and differentiation of oligodendrocyte progenitor cells.

    Science.gov (United States)

    Vela, José M; Molina-Holgado, Eduardo; Arévalo-Martín, Angel; Almazán, Guillermina; Guaza, Carmen

    2002-07-01

    Interleukin-1 (IL-1) is a pleiotropic cytokine expressed during normal CNS development and in inflammatory demyelinating diseases, but remarkably little is known about its effect on oligodendroglial cells. In this study we explored the role of IL-1beta in oligodendrocyte progenitors and differentiated oligodendrocytes. The effects of IL-1beta were compared to those of IL-1 receptor antagonist, the specific inhibitor of IL-1 activity, since progenitors and differentiated oligodendrocytes produce IL-1beta and express IL-1 receptors. Unlike other proinflammatory cytokines (TNFalpha and IFNgamma), IL-1beta was not toxic for oligodendrocyte lineage cells. However, this cytokine inhibited proliferation of oligodendrocyte progenitors in the presence of growth factors (PDGF plus bFGF). This was evidenced by a significant decrease in both cells incorporating bromodeoxyuridine (45%) and total cell numbers (57%) after 6 days of treatment. Interestingly, IL-1beta blocked proliferation at the late progenitor/prooligodendrocyte (O4+) stage but did not affect proliferation of early progenitors (A2B5+). Inhibition of proliferation paralleled with promotion of differentiation, as revealed by the increased percentage of R-mab+ cells (6.7-fold). Moreover, when oligodendrocyte progenitors were allowed to differentiate in the absence of growth factors, treatment with IL-1beta promoted maturation to the MBP+ stage (4.2-fold) and survival of differentiating oligodendrocytes (2.1-fold). Regarding intracellular signaling, IL-1beta activated the p38 mitogen-activated protein kinase (MAPK) but not the p42/p44 MAPK and, when combined with growth factors, intensified p38 activation but inhibited the growth-factor-induced p42/p44 activation. IL-1beta also induced a time-dependent inhibition of PFGF-Ralpha gene expression. These results support a role for IL-1beta in promoting mitotic arrest and differentiation of oligodendrocyte progenitors as well as maturation and survival of differentiating

  10. Advances and challenges in the differentiation of pluripotent stem cells into pancreatic β cells

    Institute of Scientific and Technical Information of China (English)

    Essam M Abdelalim; Mohamed M Emara

    2015-01-01

    Pluripotent stem cells (PSCs) are able to differentiate intoseveral cell types, including pancreatic β cells. Differentiationof pancreatic β cells depends on certain transcriptionfactors, which function in a coordinated way duringpancreas development. The existing protocols for in vitrodifferentiation produce pancreatic β cells, which are nothighly responsive to glucose stimulation except after theirtransplantation into immune-compromised mice and allowingseveral weeks for further differentiation to ensurethe maturation of these cells in vivo . Thus, although thesubstantial improvement that has been made for the differentiationof induced PSCs and embryonic stem cellstoward pancreatic β cells, several challenges still hinderingtheir full generation. Here, we summarize recent advancesin the differentiation of PSCs into pancreatic β cells anddiscuss the challenges facing their differentiation as wellas the different applications of these potential PSC-derivedβ cells.

  11. Metabolic profiling of hematopoietic stem and progenitor cells during proliferation and differentiation into red blood cells.

    Science.gov (United States)

    Daud, Hasbullah; Browne, Susan; Al-Majmaie, Rasoul; Murphy, William; Al-Rubeai, Mohamed

    2016-01-25

    An understanding of the metabolic profile of cell proliferation and differentiation should support the optimization of culture conditions for hematopoietic stem and progenitor cell (HSPC) proliferation, differentiation, and maturation into red blood cells. We have evaluated the key metabolic parameters during each phase of HSPC culture for red blood cell production in serum-supplemented (SS) and serum-free (SF) conditions. A simultaneous decrease in growth rate, total protein content, cell size, and the percentage of cells in the S/G2 phase of cell cycle, as well as an increase in the percentage of cells with a CD71(-)/GpA(+) surface marker profile, indicates HSPC differentiation into red blood cells. Compared with proliferating HSPCs, differentiating HSPCs showed significantly lower glucose and glutamine consumption rates, lactate and ammonia production rates, and amino acid consumption and production rates in both SS and SF conditions. Furthermore, extracellular acidification was associated with late proliferation phase, suggesting a reduced cellular metabolic rate during the transition from proliferation to differentiation. Under both SS and SF conditions, cells demonstrated a high metabolic rate with a mixed metabolism of both glycolysis and oxidative phosphorylation (OXPHOS) in early and late proliferation, an increased dependence on OXPHOS activity during differentiation, and a shift to glycolytic metabolism only during maturation phase. These changes indicate that cell metabolism may have an important impact on the ability of HSPCs to proliferate and differentiate into red blood cells.

  12. Energy Metabolism in Mesenchymal Stem Cells During Osteogenic Differentiation

    Science.gov (United States)

    Shum, Laura C.; White, Noelle S.; Mills, Bradley N.; de Mesy Bentley, Karen L.

    2016-01-01

    There is emerging interest in stem cell energy metabolism and its effect on differentiation. Bioenergetic changes in differentiating bone marrow mesenchymal stem cells (MSCs) are poorly understood and were the focus of our study. Using bioenergetic profiling and transcriptomics, we have established that MSCs activate the mitochondrial process of oxidative phosphorylation (OxPhos) during osteogenic differentiation, but they maintain levels of glycolysis similar to undifferentiated cells. Consistent with their glycolytic phenotype, undifferentiated MSCs have high levels of hypoxia-inducible factor 1 (HIF-1). Osteogenically induced MSCs downregulate HIF-1 and this downregulation is required for activation of OxPhos. In summary, our work provides important insights on MSC bioenergetics and proposes a HIF-based mechanism of regulation of mitochondrial OxPhos in MSCs. PMID:26487485

  13. Pericytes Stimulate Oligodendrocyte Progenitor Cell Differentiation during CNS Remyelination

    Directory of Open Access Journals (Sweden)

    Alerie Guzman De La Fuente

    2017-08-01

    Full Text Available The role of the neurovascular niche in CNS myelin regeneration is incompletely understood. Here, we show that, upon demyelination, CNS-resident pericytes (PCs proliferate, and parenchymal non-vessel-associated PC-like cells (PLCs rapidly develop. During remyelination, mature oligodendrocytes were found in close proximity to PCs. In Pdgfbret/ret mice, which have reduced PC numbers, oligodendrocyte progenitor cell (OPC differentiation was delayed, although remyelination proceeded to completion. PC-conditioned medium accelerated and enhanced OPC differentiation in vitro and increased the rate of remyelination in an ex vivo cerebellar slice model of demyelination. We identified Lama2 as a PC-derived factor that promotes OPC differentiation. Thus, the functional role of PCs is not restricted to vascular homeostasis but includes the modulation of adult CNS progenitor cells involved in regeneration.

  14. Mesenchymal stem cell ingrowth and differentiation on coralline hydroxyapatite scaffolds

    DEFF Research Database (Denmark)

    Mygind, Tina; Stiehler, Maik; Baatrup, Anette

    2007-01-01

    Culture of osteogenic cells on a porous scaffold could offer a new solution to bone grafting using autologous human mesenchymal stem cells (hMSC) from the patient. We compared coralline hydroxyapatite scaffolds with pore sizes of 200 and 500 microm for expansion and differentiation of hMSCs. We......MSC in a particular direction. We found that dynamic spinner flask cultivation of hMSC/scaffold constructs resulted in increased proliferation, differentiation and distribution of cells in scaffolds. Therefore, spinner flask cultivation is an easy-to-use inexpensive system for cultivating hMSCs on small...... cultivated the hMSC statically or in spinner flasks for 1, 7, 14 and 21 days and found that the 200-microm pore scaffolds exhibited a faster rate of osteogenic differentiation than did the 500-microm pore scaffolds as shown by an alkaline phosphatase activity assay and real-time reverse transcriptase...

  15. Geometric cues for directing the differentiation of mesenchymal stem cells

    Science.gov (United States)

    Kilian, Kristopher A.; Bugarija, Branimir; Lahn, Bruce T.; Mrksich, Milan

    2010-01-01

    Significant efforts have been directed to understanding the factors that influence the lineage commitment of stem cells. This paper demonstrates that cell shape, independent of soluble factors, has a strong influence on the differentiation of human mesenchymal stem cells (MSCs) from bone marrow. When exposed to competing soluble differentiation signals, cells cultured in rectangles with increasing aspect ratio and in shapes with pentagonal symmetry but with different subcellular curvature—and with each occupying the same area—display different adipogenesis and osteogenesis profiles. The results reveal that geometric features that increase actomyosin contractility promote osteogenesis and are consistent with in vivo characteristics of the microenvironment of the differentiated cells. Cytoskeletal-disrupting pharmacological agents modulate shape-based trends in lineage commitment verifying the critical role of focal adhesion and myosin-generated contractility during differentiation. Microarray analysis and pathway inhibition studies suggest that contractile cells promote osteogenesis by enhancing c-Jun N-terminal kinase (JNK) and extracellular related kinase (ERK1/2) activation in conjunction with elevated wingless-type (Wnt) signaling. Taken together, this work points to the role that geometric shape cues can play in orchestrating the mechanochemical signals and paracrine/autocrine factors that can direct MSCs to appropriate fates. PMID:20194780

  16. Derivation and differentiation of haploid human embryonic stem cells.

    Science.gov (United States)

    Sagi, Ido; Chia, Gloryn; Golan-Lev, Tamar; Peretz, Mordecai; Weissbein, Uri; Sui, Lina; Sauer, Mark V; Yanuka, Ofra; Egli, Dieter; Benvenisty, Nissim

    2016-04-07

    Diploidy is a fundamental genetic feature in mammals, in which haploid cells normally arise only as post-meiotic germ cells that serve to ensure a diploid genome upon fertilization. Gamete manipulation has yielded haploid embryonic stem (ES) cells from several mammalian species, but haploid human ES cells have yet to be reported. Here we generated and analysed a collection of human parthenogenetic ES cell lines originating from haploid oocytes, leading to the successful isolation and maintenance of human ES cell lines with a normal haploid karyotype. Haploid human ES cells exhibited typical pluripotent stem cell characteristics, such as self-renewal capacity and a pluripotency-specific molecular signature. Moreover, we demonstrated the utility of these cells as a platform for loss-of-function genetic screening. Although haploid human ES cells resembled their diploid counterparts, they also displayed distinct properties including differential regulation of X chromosome inactivation and of genes involved in oxidative phosphorylation, alongside reduction in absolute gene expression levels and cell size. Surprisingly, we found that a haploid human genome is compatible not only with the undifferentiated pluripotent state, but also with differentiated somatic fates representing all three embryonic germ layers both in vitro and in vivo, despite a persistent dosage imbalance between the autosomes and X chromosome. We expect that haploid human ES cells will provide novel means for studying human functional genomics and development.

  17. Surface topography during neural stem cell differentiation regulates cell migration and cell morphology.

    Science.gov (United States)

    Czeisler, Catherine; Short, Aaron; Nelson, Tyler; Gygli, Patrick; Ortiz, Cristina; Catacutan, Fay Patsy; Stocker, Ben; Cronin, James; Lannutti, John; Winter, Jessica; Otero, José Javier

    2016-12-01

    We sought to determine the contribution of scaffold topography to the migration and morphology of neural stem cells by mimicking anatomical features of scaffolds found in vivo. We mimicked two types of central nervous system scaffolds encountered by neural stem cells during development in vitro by constructing different diameter electrospun polycaprolactone (PCL) fiber mats, a substrate that we have shown to be topographically similar to brain scaffolds. We compared the effects of large fibers (made to mimic blood vessel topography) with those of small-diameter fibers (made to mimic radial glial process topography) on the migration and differentiation of neural stem cells. Neural stem cells showed differential migratory and morphological reactions with laminin in different topographical contexts. We demonstrate, for the first time, that neural stem cell biological responses to laminin are dependent on topographical context. Large-fiber topography without laminin prevented cell migration, which was partially reversed by treatment with rock inhibitor. Cell morphology complexity assayed by fractal dimension was inhibited in nocodazole- and cytochalasin-D-treated neural precursor cells in large-fiber topography, but was not changed in small-fiber topography with these inhibitors. These data indicate that cell morphology has different requirements on cytoskeletal proteins dependent on the topographical environment encountered by the cell. We propose that the physical structure of distinct scaffolds induces unique signaling cascades that regulate migration and morphology in embryonic neural precursor cells. J. Comp. Neurol. 524:3485-3502, 2016. © 2016 Wiley Periodicals, Inc.

  18. Differential expression of genes involved in the epigenetic regulation of cell identity in normal human mammary cell commitment and differentiation

    Institute of Scientific and Technical Information of China (English)

    Danila Coradini; Patrizia Boracchi; Saro Oriana; Elia Biganzoli; Federico Ambrogi

    2014-01-01

    The establishment and maintenance of mammary epithelial cell identity depends on the activity of a group of proteins, collectively called maintenance proteins, that act as epigenetic regulators of gene transcription through DNA methylation, histone modification, and chromatin remodeling. Increasing evidence indicates that dysregulation of these crucial proteins may disrupt epithelial cellintegrity and trigger breast tumor initiation. Therefore, we exploredin silico the expression pattern of a panel of 369 genes known to be involved in the establishment and maintenance of epithelial cellidentity and mammary gland remodeling in cell subpopulations isolated from normal human mammary tissue and selectively enriched in their content of bipotent progenitors, committed luminal progenitors, and differentiated myoepithelial or differentiated luminal cells. The results indicated that, compared to bipotent cells, differentiated myoepithelial and luminal subpopulations were both characterized by the differential expression of 4 genes involved in cell identity maintenance:CBX6 andPCGF2, encoding proteins belonging to the Polycomb group, andSMARCD3 andSMARCE1, encoding proteins belonging to the Trithorax group. In addition to these common genes, the myoepithelial phenotype was associated with the differential expression of HDAC1, which encodes histone deacetylase 1, whereas the luminal phenotype was associated with the differential expression ofSMARCA4 andHAT1, which encode a Trithorax protein and histone acetylase 1, respectively. The luminal compartment was further characterized by the overexpression ofALDH1A3 and GATA3, and the down-regulation ofNOTCH4and CCNB1, with the latter suggesting a block in cell cycle progression at the G2 phase. In contrast, myoepithelial differentiation was associated with the overexpression ofMYC and the down-regulation ofCCNE1, with the latter suggesting a block in cellcycle progression at the G1 phase.

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

    Science.gov (United States)

    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

  20. Globular adiponectin induces differentiation and fusion of skeletal muscle cells

    Institute of Scientific and Technical Information of China (English)

    Tania Fiaschi; Domenico Cirelli; Giuseppina Comito; Stefania Gelmini; Giampietro Ramponi; Maria Serio; Paola Chiarugi

    2009-01-01

    The growing interest in skeletal muscle regeneration is associated with the opening of new therapeutic strategies for muscle injury after trauma, as well as several muscular degenerative pathologies, including dystrophies, muscu-lar atrophy, and cachexia. Studies focused on the ability of extracellular factors to promote myogenesis are therefore highly promising. We now report that an adipocyte-derived factor, globular adiponectin (gAd), is able to induce mus-cle gene expression and cell differentiation, gAd, besides its well-known ability to regulate several metabolic func-tions in muscle, including glucose uptake and consumption and fatty acid catabolism, is able to block cell cycle entry of myoblasts, to induce the expression of specific skeletal muscle markers such as myosin heavy chain or eaveolin-3, as well as to provoke cell fusion into multinucleated syneytia and, finally, muscle fibre formation, gAd exerts its pro-differentiative activity through redox-dependent activation of p38, Akt and 5'-AMP-activated protein kinase path-ways. Interestingly, differentiating myoblasts are autocrine for adiponectiu, and the mimicking of pro-inflammatory settings or exposure to oxidative stress strongly increases the production of the hormone from differentiating cells. These data suggest a novel function of adiponectin, directly coordinating the myogenic differentiation program and serving an autocrine function during skeletal myogenesis.

  1. Cell Expansion During Directed Differentiation of Stem Cells Toward the Hepatic Lineage.

    Science.gov (United States)

    Raju, Ravali; Chau, David; Cho, Dong Seong; Park, Yonsil; Verfaillie, Catherine M; Hu, Wei-Shou

    2017-02-15

    The differentiation of human pluripotent stem cells toward the hepatocyte lineage can potentially provide an unlimited source of functional hepatocytes for transplantation and extracorporeal bioartificial liver applications. It is anticipated that the quantities of cells needed for these applications will be in the order of 10(9)-10(10) cells, because of the size of the liver. An ideal differentiation protocol would be to enable directed differentiation to the hepatocyte lineage with simultaneous cell expansion. We introduced a cell expansion stage after the commitment of human embryonic stem cells to the endodermal lineage, to allow for at least an eightfold increase in cell number, with continuation of cell maturation toward the hepatocyte lineage. The progressive changes in the transcriptome were measured by expression array, and the expression dynamics of certain lineage markers was measured by mass cytometry during the differentiation and expansion process. The findings revealed that while cells were expanding they were also capable of progressing in their differentiation toward the hepatocyte lineage. In addition, our transcriptome, protein and functional studies, including albumin secretion, drug-induced CYP450 expression and urea production, all indicated that the hepatocyte-like cells obtained with or without cell expansion are very similar. This method of simultaneous cell expansion and hepatocyte differentiation should facilitate obtaining large quantities of cells for liver cell applications.

  2. Structural properties of scaffolds: Crucial parameterstowards stem cells differentiation

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    Tissue engineering is a multidisciplinary field thatapplies the principles of engineering and life-sciencesfor regeneration of damaged tissues. Stem cells haveattracted much interest in tissue engineering as a cellsource due to their ability to proliferate in an undifferentiatedstate for prolonged time and capability ofdifferentiating to different cell types after induction.Scaffolds play an important role in tissue engineeringas a substrate that can mimic the native extracellularmatrix and the properties of scaffolds have been shownto affect the cell behavior such as the cell attachment,proliferation and differentiation. Here, we focus on therecent reports that investigated the various aspectsof scaffolds including the materials used for scaffoldfabrication, surface modification of scaffolds, topographyand mechanical properties of scaffolds towards stemcells differentiation effect. We will present a moredetailed overview on the effect of mechanical propertiesof scaffolds on stem cells fate.

  3. Motoneuron differentiation of immortalized human spinal cord cell lines.

    Science.gov (United States)

    Li, R; Thode, S; Zhou, J; Richard, N; Pardinas, J; Rao, M S; Sah, D W

    2000-02-01

    Human motoneuron cell lines will be valuable tools for spinal cord research and drug discovery. To create such cell lines, we immortalized NCAM(+)/neurofilament(+) precursors from human embryonic spinal cord with a tetracycline repressible v-myc oncogene. Clonal NCAM(+)/neurofilament(+) cell lines differentiated exclusively into neurons within 1 week. These neurons displayed extensive processes, exhibited immunoreactivity for mature neuron-specific markers such as tau and synaptophysin, and fired action potentials upon current injection. Moreover, a clonal precursor cell line gave rise to multiple types of spinal cord neurons, including ChAT(+)/Lhx3(+)/Lhx4(+) motoneurons and GABA(+) interneurons. These neuronal restricted precursor cell lines will expedite the elucidation of molecular mechanisms that regulate the differentiation, maturation and survival of specific subsets of spinal cord neurons, and the identification and validation of novel drug targets for motoneuron diseases and spinal cord injury.

  4. Rat Nasal Respiratory Mucosa-Derived Ectomesenchymal Stem Cells Differentiate into Schwann-Like Cells Promoting the Differentiation of PC12 Cells and Forming Myelin In Vitro

    Directory of Open Access Journals (Sweden)

    Jian Zhang

    2015-01-01

    Full Text Available Schwann cell (SC transplantation as a cell-based therapy can enhance peripheral and central nerve repair experimentally, but it is limited by the donor site morbidity for clinical application. We investigated weather respiratory mucosa stem cells (REMSCs, a kind of ectomesenchymal stem cells (EMSCs, isolated from rat nasal septum can differentiate into functional Schwann-like cells (SC-like cells. REMSCs proliferated quickly in vitro and expressed the neural crest markers (nestin, vimentin, SOX10, and CD44. Treated with a mixture of glial growth factors for 7 days, REMSCs differentiated into SC-like cells. The differentiated REMSCs (dREMSCs exhibited a spindle-like morphology similar to SC cells. Immunocytochemical staining and Western blotting indicated that SC-like cells expressed the glial markers (GFAP, S100β, Galc, and P75 and CNPase. When cocultured with dREMSCs for 5 days, PC12 cells differentiated into mature neuron-like cells with long neurites. More importantly, dREMSCs could form myelin structures with the neurites of PC12 cells at 21 days in vitro. Our data indicated that REMSCs, a kind of EMSCs, could differentiate into SC-like cells and have the ability to promote the differentiation of PC12 cells and form myelin in vitro.

  5. DIFFERENTIATION AND MALIGNANT SUPPRESSION INDUCED BY MOUSE ERYTHROID DIFFERENTIATION AND DENUCLEATION FACTOR ON MOUSE ERYTHROLEUKEMIA CELLS

    Institute of Scientific and Technical Information of China (English)

    韩代书; 赵青; 葛晔华; 周建平; 马静; 陈克铨; 薛社普

    2002-01-01

    Objective. To investigate the roles of mouse erythroid differentiation and denueleation factor (MEDDF), a novel factor cloned in our laboratory recently, in erythroid terminal differentiation.Methods. Mouse erythroleukemia (MEL) cells were transfected with eukaryotic expression plasmid pcD-NA-MEDDF. Then we investigated the changes on characteristics of cell growth by analyzing cells growth rate,mitotic index and colony-forming rate in semi-solid medium. The expressions of c-myc and β-globin genes were analysed by semi-quantitative RT-PCR.Results. MEL ceils transfected with pcDNA-MEDDF showed significant lower growth rate, mitotic index,and colony-forming rate in semi-solid medium ( P<0.01 ). The percentage of benzidine-positive cells was 32.8% after transfection. The expression of β-globin in cells transfected with pcDNA-MEDDF was 3.43 times higher than that of control (MEL transfected with blank vector, pcDNA3. 1 ), and the expression of c-myc decreased by 66.3%.Conclusions. MEDDF can induce differentiation of MEL cell and suppress its malignancy.

  6. Differentiation of Human Embryonic Stem Cells on Periodontal Ligament Fibroblasts.

    Science.gov (United States)

    Elçin, Y Murat; İnanç, Bülend; Elçin, A Eser

    2016-01-01

    Human embryonic stem cells' (hESCs) unlimited proliferative potential and differentiation capability to all somatic cell types makes them one of the potential cell sources in cell-based tissue engineering strategies as well as various experimental applications in fields such as developmental biology, pharmacokinetics, toxicology, and genetics. Periodontal tissue engineering is an approach to reconstitute the ectomesenchymally derived alveolar bone, periodontal ligament apparatus, and cementum tissues lost as a result of periodontal diseases. Cell-based therapies may offer potential advantage in overcoming the inherent limitations associated with contemporary regenerative procedures, such as dependency on defect type and size and the pool and capacity of progenitor cells resident in the wound area. Further elucidation of developmental mechanisms associated with tooth formation may also contribute to valuable knowledge based upon which the future therapies can be designed. Protocols for the differentiation of pluripotent hESCs into periodontal ligament fibroblastic cells (PDLF) as common progenitors for ligament, cementum, and alveolar bone tissue represent an initial step in developing hESC-based experimental and tissue engineering strategies. The present protocol describes methods associated with the guided differentiation of hESCs by the use of coculture with adult PDLFs and the resulting change of morphotype and phenotype of the pluripotent embryonic stem cells toward fibroblastic and osteoblastic lineages.

  7. Capturing power at higher voltages from arrays of microbial fuel cells without voltage reversal

    KAUST Repository

    Kim, Younggy

    2011-01-01

    Voltages produced by microbial fuel cells (MFCs) cannot be sustainably increased by linking them in series due to voltage reversal, which substantially reduces stack voltages. It was shown here that MFC voltages can be increased with continuous power production using an electronic circuit containing two sets of multiple capacitors that were alternately charged and discharged (every one second). Capacitors were charged in parallel by the MFCs, but linked in series while discharging to the circuit load (resistor). The parallel charging of the capacitors avoided voltage reversal, while discharging the capacitors in series produced up to 2.5 V with four capacitors. There were negligible energy losses in the circuit compared to 20-40% losses typically obtained with MFCs using DC-DC converters to increase voltage. Coulombic efficiencies were 67% when power was generated via four capacitors, compared to only 38% when individual MFCs were operated with a fixed resistance of 250 Ω. The maximum power produced using the capacitors was not adversely affected by variable performance of the MFCs, showing that power generation can be maintained even if individual MFCs perform differently. Longer capacitor charging and discharging cycles of up to 4 min maintained the average power but increased peak power by up to 2.6 times. These results show that capacitors can be used to easily obtain higher voltages from MFCs, allowing for more useful capture of energy from arrays of MFCs. © 2011 The Royal Society of Chemistry.

  8. Differential MDR in Breast Cancer Stem Cells

    Science.gov (United States)

    2006-05-01

    1977, Reya et al. 2001, Dick 2003, Al-Hajj et al. 2004, Donnenberg and Donnenberg, 2005, Dick and Lapidot, 2005, Wicha et al., 2006, Polyak and Hahn...Med. 341(7):491- 497, 1999. Polyak K, Hahn WC. Roots and stems: Stem cells in cancer. Nat Med 12, 296 – 300, 2006. Rendl M, Lewis L, Fuchs E...glycoprotein overexpression. Cancer Res. 1989;49:4542- 4549. 10. Doyle LA, Yang W, Abruzzo LV , et al. A multidrug resistance trans- porter from human

  9. MicroRNA in cell differentiation and development.

    Science.gov (United States)

    Shi, Yi; Jin, YouXin

    2009-03-01

    The regulation of gene expression by microRNAs (miRNAs) is a recently discovered pattern of gene regulation in animals and plants. MiRNAs have been implicated in various aspects of animal development and cell differentiation, such as early embryonic development, neuronal development, muscle development, and lymphocyte development, by the analysis of genetic deletions of individual miRNAs in mammals. These studies show that miRNAs are key regulators in animal development and are potential causes of human diseases. Here we review some recent discoveries about the functions of miRNAs in cell differentiation and development.

  10. MicroRNA in cell differentiation and development

    Institute of Scientific and Technical Information of China (English)

    SHI Yi; JIN YouXin

    2009-01-01

    The regulation of gene expression by microRNAs (miRNAs) Is a recently discovered pattern of gene regulation in animals and plants. MiRNAs have been implicated in various aspects of animal develop-ment and cell differentiation, such as early embryonic development, neuronal development, muscle development, and lymphocyte development, by the analysis of genetic deletions of individual miRNAs in mammals. These studies show that miRNAs are key regulators in animal development and are po-tential causes of human diseases. Here we review some recent discoveries about the functions of miRNAs in cell differentiation and development.

  11. MicroRNA in cell differentiation and development

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    The regulation of gene expression by microRNAs(miRNAs) is a recently discovered pattern of gene regulation in animals and plants.MiRNAs have been implicated in various aspects of animal development and cell differentiation,such as early embryonic development,neuronal development,muscle development,and lymphocyte development,by the analysis of genetic deletions of individual miRNAs in mammals.These studies show that miRNAs are key regulators in animal development and are potential causes of human diseases.Here we review some recent discoveries about the functions of miRNAs in cell differentiation and development.

  12. Lineage-specific interface proteins match up the cell cycle and differentiation in embryo stem cells

    DEFF Research Database (Denmark)

    Re, Angela; Workman, Christopher; Waldron, Levi

    2014-01-01

    The shortage of molecular information on cell cycle changes along embryonic stem cell (ESC) differentiation prompts an in silico approach, which may provide a novel way to identify candidate genes or mechanisms acting in coordinating the two programs. We analyzed germ layer specific gene expression...... interaction data. A new class of non-transcriptionally regulated genes was identified, encoding proteins which interact systematically with proteins corresponding to genes regulated during the cell cycle or cell differentiation, and which therefore can be seen as interface proteins coordinating the two...... changes during the cell cycle and ESC differentiation by combining four human cell cycle transcriptome profiles with thirteen in vitro human ESC differentiation studies. To detect cross-talk mechanisms we then integrated the transcriptome data that displayed differential regulation with protein...

  13. Differentiation of trophoblast cells from human embryonic stem cells: to be or not to be?

    Science.gov (United States)

    Roberts, R Michael; Loh, Kyle M; Amita, Mitsuyoshi; Bernardo, Andreia S; Adachi, Katsuyuki; Alexenko, Andrei P; Schust, Danny J; Schulz, Laura C; Telugu, Bhanu Prakash V L; Ezashi, Toshihiko; Pedersen, Roger A

    2014-05-01

    It is imperative to unveil the full range of differentiated cell types into which human pluripotent stem cells (hPSCs) can develop. The need is twofold: it will delimit the therapeutic utility of these stem cells and is necessary to place their position accurately in the developmental hierarchy of lineage potential. Accumulated evidence suggested that hPSC could develop in vitro into an extraembryonic lineage (trophoblast (TB)) that is typically inaccessible to pluripotent embryonic cells during embryogenesis. However, whether these differentiated cells are truly authentic TB has been challenged. In this debate, we present a case for and a case against TB differentiation from hPSCs. By analogy to other differentiation systems, our debate is broadly applicable, as it articulates higher and more challenging standards for judging whether a given cell type has been genuinely produced from hPSC differentiation.

  14. Redox environment in stem and differentiated cells: A quantitative approach

    Directory of Open Access Journals (Sweden)

    O.G. Lyublinskaya

    2017-08-01

    Full Text Available Stem cells are believed to maintain a specific intracellular redox status through a combination of enhanced removal capacity and limited production of ROS. In the present study, we challenge this assumption by developing a quantitative approach for the analysis of the pro- and antioxidant ability of human embryonic stem cells in comparison with their differentiated descendants, as well as adult stem and non-stem cells. Our measurements showed that embryonic stem cells are characterized by low ROS level, low rate of extracellular hydrogen peroxide removal and low threshold for peroxide-induced cytotoxicity. However, biochemical normalization of these parameters to cell volume/protein leads to matching of normalized values in stem and differentiated cells and shows that tested in the present study cells (human embryonic stem cells and their fibroblast-like progenies, adult mesenchymal stem cells, lymphocytes, HeLa maintain similar intracellular redox status. Based on these observations, we propose to use ROS concentration averaged over the cell volume instead of ROS level as a measure of intracellular redox balance. We show that attempts to use ROS level for comparative analysis of redox status of morphologically different cells could lead to false conclusions. Methods for the assessment of ROS concentration based on flow cytometry analysis with the use of H2DCFDA dye and HyPer, genetically encoded probe for hydrogen peroxide, are discussed.

  15. Defining Developmental Potency and Cell Lineage Trajectories by Expression Profiling of Differentiating Mouse Embryonic Stem Cells

    Science.gov (United States)

    Aiba, Kazuhiro; Nedorezov, Timur; Piao, Yulan; Nishiyama, Akira; Matoba, Ryo; Sharova, Lioudmila V.; Sharov, Alexei A.; Yamanaka, Shinya; Niwa, Hitoshi; Ko, Minoru S. H.

    2009-01-01

    Biologists rely on morphology, function and specific markers to define the differentiation status of cells. Transcript profiling has expanded the repertoire of these markers by providing the snapshot of cellular status that reflects the activity of all genes. However, such data have been used only to assess relative similarities and differences of these cells. Here we show that principal component analysis of global gene expression profiles map cells in multidimensional transcript profile space and the positions of differentiating cells progress in a stepwise manner along trajectories starting from undifferentiated embryonic stem (ES) cells located in the apex. We present three ‘cell lineage trajectories’, which represent the differentiation of ES cells into the first three lineages in mammalian development: primitive endoderm, trophoblast and primitive ectoderm/neural ectoderm. The positions of the cells along these trajectories seem to reflect the developmental potency of cells and can be used as a scale for the potential of cells. Indeed, we show that embryonic germ cells and induced pluripotent cells are mapped near the origin of the trajectories, whereas mouse embryo fibroblast and fibroblast cell lines are mapped near the far end of the trajectories. We suggest that this method can be used as the non-operational semi-quantitative definition of cell differentiation status and developmental potency. Furthermore, the global expression profiles of cell lineages provide a framework for the future study of in vitro and in vivo cell differentiation. PMID:19112179

  16. Small Buccal Fat Pad Cells Have High Osteogenic Differentiation Potential.

    Science.gov (United States)

    Tsurumachi, Niina; Akita, Daisuke; Kano, Koichiro; Matsumoto, Taro; Toriumi, Taku; Kazama, Tomohiko; Oki, Yoshinao; Tamura, Yoko; Tonogi, Morio; Isokawa, Keitaro; Shimizu, Noriyoshi; Honda, Masaki

    2016-03-01

    Dedifferentiated fat (DFAT) cells derived from mature adipocytes have mesenchymal stem cells' (MSCs) characteristics. Generally, mature adipocytes are 60-110 μm in diameter; however, association between adipocyte size and dedifferentiation efficiency is still unknown. This study, therefore, investigated the dedifferentiation efficiency of adipocytes based on cell diameter. Buccal fat pad was harvested from five human donors and dissociated by collagenase digestion. After exclusion of unwanted stromal cells by centrifugation, floating adipocytes were collected and their size distribution was analyzed. The floating adipocytes were then separated into two groups depending on cell size using 40- and 100-μm nylon mesh filters: cell diameters less than 40 μm (small adipocytes: S-adipocytes) and cell diameters of 40-100 μm (large adipocytes: L-adipocytes). Finally, we evaluated the efficiency of adipocyte dedifferentiation and then characterized the resultant DFAT cells. The S-adipocytes showed a higher capacity to dedifferentiate into DFAT cells (S-DFAT cells) compared to the L-adipocytes (L-DFAT cells). The S-DFAT cells also showed a relatively higher proportion of CD146-positive cells than L-DFAT cells, and exhibited more osteogenic differentiation ability based on the alkaline phosphatase activity and amount of calcium deposition. These results suggested that the S- and L-DFAT cells had distinct characteristics, and that the higher dedifferentiation potential of S-adipocytes compared to L-adipocytes gives the former group an advantage in yielding DFAT cells.

  17. A novel precision-engineered microfiltration device for capture and characterisation of bladder cancer cells in urine

    OpenAIRE

    2013-01-01

    Background: Sensitivity of standard urine cytology for detecting urothelial carcinoma of the bladder (UCB) is low, attributable largely to its inability to process entire samples, paucicellularity and presence of background cells. Objective: Evaluate performance and practical applicability of a novel portable microfiltration device for capture, enumeration and characterisation of exfoliated tumour cells in urine, and compare it with standard urine cytology for UCB detection. Meth...

  18. Interleukin-24 inhibits the plasma cell differentiation program in human germinal center B cells.

    Science.gov (United States)

    Maarof, Ghyath; Bouchet-Delbos, Laurence; Gary-Gouy, Hélène; Durand-Gasselin, Ingrid; Krzysiek, Roman; Dalloul, Ali

    2010-03-04

    Complex molecular mechanisms control B-cell fate to become a memory or a plasma cell. Interleukin-24 (IL-24) is a class II family cytokine of poorly understood immune function that regulates the cell cycle. We previously observed that IL-24 is strongly expressed in leukemic memory-type B cells. Here we show that IL-24 is also expressed in human follicular B cells; it is more abundant in CD27(+) memory B cells and CD5-expressing B cells, whereas it is low to undetectable in centroblasts and plasma cells. Addition of IL-24 to B cells, cultured in conditions shown to promote plasma cell differentiation, strongly inhibited plasma cell generation and immunoglobulin G (IgG) production. By contrast, IL-24 siRNA increased terminal differentiation of B cells into plasma cells. IL-24 is optimally induced by BCR triggering and CD40 engagement; IL-24 increased CD40-induced B-cell proliferation and modulated the transcription of key factors involved in plasma cell differentiation. It also inhibited activation-induced tyrosine phosphorylation of signal transducer and activator of transcription-3 (STAT-3), and inhibited the transcription of IL-10. Taken together, our results indicate that IL-24 is a novel cytokine involved in T-dependent antigen (Ag)-driven B-cell differentiation and suggest its physiologic role in favoring germinal center B-cell maturation in memory B cells at the expense of plasma cells.

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

    Science.gov (United States)

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

    2014-06-01

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

  20. Transcription factor interplay in T helper cell differentiation.

    Science.gov (United States)

    Evans, Catherine M; Jenner, Richard G

    2013-11-01

    The differentiation of CD4 helper T cells into specialized effector lineages has provided a powerful model for understanding immune cell differentiation. Distinct lineages have been defined by differential expression of signature cytokines and the lineage-specifying transcription factors necessary and sufficient for their production. The traditional paradigm of differentiation towards Th1 and Th2 subtypes driven by T-bet and GATA3, respectively, has been extended to incorporate additional T cell lineages and transcriptional regulators. Technological advances have expanded our view of these lineage-specifying transcription factors to the whole genome and revealed unexpected interplay between them. From these data, it is becoming clear that lineage specification is more complex and plastic than previous models might have suggested. Here, we present an overview of the different forms of transcription factor interplay that have been identified and how T cell phenotypes arise as a product of this interplay within complex regulatory networks. We also suggest experimental strategies that will provide further insight into the mechanisms that underlie T cell lineage specification and plasticity.

  1. Differentiation of human mesenchymal stem cell spheroids under microgravity conditions

    Directory of Open Access Journals (Sweden)

    Cerwinka Wolfgang H

    2012-06-01

    Full Text Available Abstract To develop and characterize a novel cell culture method for the generation of undifferentiated and differentiated human mesenchymal stem cell 3D structures, we utilized the RWV system with a gelatin-based scaffold. 3 × 106 cells generated homogeneous spheroids and maximum spheroid loading was accomplished after 3 days of culture. Spheroids cultured in undifferentiated spheroids of 3 and 10 days retained expression of CD44, without expression of differentiation markers. Spheroids cultured in adipogenic and osteogenic differentiation media exhibited oil red O staining and von Kossa staining, respectively. Further characterization of osteogenic lineage, showed that 10 day spheroids exhibited stronger calcification than any other experimental group corresponding with significant expression of vitamin D receptor, alkaline phosphatase, and ERp60 . In conclusion this study describes a novel RWV culture method that allowed efficacious engineering of undifferentiated human mesenchymal stem cell spheroids and rapid osteogenic differentiation. The use of gelatin scaffolds holds promise to design implantable stem cell tissue of various sizes and shapes for future regenerative treatment.

  2. The production and directed differentiation of human embryonic stem cells.

    Science.gov (United States)

    Trounson, Alan

    2006-04-01

    Human embryonic stem cells (hESCs) are being rapidly produced from chromosomally euploid, aneuploid, and mutant human embryos that are available from in vitro fertilization clinics treating patients for infertility or preimplantation genetic diagnosis. These hESC lines are an important resource for functional genomics, drug screening, and, perhaps eventually, cell and gene therapy. The methods for deriving hESCs are well established and repeatable and are relatively successful with a ratio of 1:10 to 1:2 new hESC lines produced from 4- to 8-d-old morula and blastocysts and from isolated inner cell mass cell clusters of human blastocysts. The hESCs can be formed and maintained on human somatic cells in humanized serum-free culture conditions and for several passages in cell-free culture systems. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in vitro while maintaining their original karyotype and epigenetic status, but this needs to be confirmed from time to time in long-term cultures. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating flat attachment cultures and unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes, and characteristic morphology, and the cells thereafter enriched for progenitor types and further culture to more mature cell types. Directed differentiation systems are well developed for ectodermal pathways that result in neural and glial cells and the mesendodermal pathway for cardiac muscle cells and many other cell types including hematopoietic progenitors and endothelial cells. Directed differentiation into endoderm has been more difficult to achieve, perhaps because of the lack of markers of

  3. NLRP3 inflammasome activation in mesenchymal stem cells inhibits osteogenic differentiation and enhances adipogenic differentiation.

    Science.gov (United States)

    Wang, Linghao; Chen, Ke; Wan, Xinxing; Wang, Fang; Guo, Zi; Mo, Zhaohui

    2017-03-18

    Osteoporosis is one of the most common skeletal disease featured by osteopenia and adipose accumulation in bone tissue. NLRP3 inflammasome activation is an essential player in aging-related chronic diseases like osteoporosis, particularly due to the causal caspase-1 activation and its correlation to adipose accumulation in bone tissue. Moreover, the expression of anti-aging/senescence SIRT1 was reported to decline along with aging. As the major cellular contributor of bone formation, mesenchymal stem cells (MSCs) are multipotent stem cells processing mutually exclusive differentiatability toward osteocytes or adipocytes. Therefore, we hypothesized that NLRP3 inflammasome activation promotes adipogenesis and repress osteogenesis in MSCs via inhibiting SIRT1 expression. We activated NLRP3 inflammasome in human MSCs via lipopolysaccharide and palmitic acid (LPS/PA) treatment for self-renewal maintenance, adipogenic differentiation or osteogenic differentiation. LPS/PA treatment significantly increased NLRP3 expression, decreased SIRT1 expression and promoted caspase-1 activity in MSCs. LPS/PA treatment also boosted adipogenesis of MSCs and suppressed osteogenesis. Moreover, inhibition of caspase-1 activity repressed adipogenic differentiation and partially improved osteogenic differentiation of MSCs with LPS/PA treatment. Our study demonstrated the pivotal roles of NLRP3 inflammasome and downstream mediator caspase-1 for the progress of osteo-differentiation MSCs, and offered novel therapeutic target of treatment for osteoporosis.

  4. O2 level controls hematopoietic circulating progenitor cells differentiation into endothelial or smooth muscle cells.

    Directory of Open Access Journals (Sweden)

    Nicolas Berthelemy

    Full Text Available BACKGROUND: Recent studies showed that progenitor cells could differentiate into mature vascular cells. The main physiological factors implicated in cell differentiation are specific growth factors. We hypothesized that simply by varying the oxygen content, progenitor cells can be differentiated either in mature endothelial cells (ECs or contractile smooth muscle cells (SMCs while keeping exactly the same culture medium. METHODOLOGY/PRINCIPAL FINDINGS: Mononuclear cells were isolated by density gradient were cultivated under hypoxic (5% O2 or normoxic (21% O2 environment. Differentiated cells characterization was performed by confocal microscopy examination and flow cytometry analyses. The phenotype stability over a longer time period was also performed. The morphological examination of the confluent obtained cells after several weeks (between 2 and 4 weeks showed two distinct morphologies: cobblestone shape in normoxia and a spindle like shape in hypoxia. The cell characterization showed that cobblestone cells were positive to ECs markers while spindle like shape cells were positive to contractile SMCs markers. Moreover, after several further amplification (until 3(rd passage in hypoxic or normoxic conditions of the previously differentiated SMC, immunofluorescence studies showed that more than 80% cells continued to express SMCs markers whatever the cell environmental culture conditions with a higher contractile markers expression compared to control (aorta SMCs signature of phenotype stability. CONCLUSION/SIGNIFICANCE: We demonstrate in this paper that in vitro culture of peripheral blood mononuclear cells with specific angiogenic growth factors under hypoxic conditions leads to SMCs differentiation into a contractile phenotype, signature of their physiological state. Moreover after amplification, the differentiated SMC did not reverse and keep their contractile phenotype after the 3rd passage performed under hypoxic and normoxic conditions

  5. Decreased Intracellular pH Induced by Cariporide Differentially Contributes to Human Umbilical Cord-Derived Mesenchymal Stem Cells Differentiation

    Directory of Open Access Journals (Sweden)

    Wei Gao

    2014-01-01

    Full Text Available Background/Aims: Na+/H+ exchanger 1 (NHE1 is an important regulator of intracellular pH (pHi. High pHi is required for cell proliferation and differentiation. Our previous study has proven that the pHi of mesenchymal stem cells is higher than that of normal differentiated cells and similar to tumor cells. NHE1 is highly expressed in both mesenchymal stem cells and tumor cells. Targeted inhibition of NHE1 could induce differentiation of K562 leukemia cells. In the present paper we explored whether inhibition of NHE1 could induce differentiation of mesenchymal stem cells. Methods: MSCs were obtained from human umbilical cord and both the surface phenotype and functional characteristics were analyzed. Selective NHE1 inhibitor cariporide was used to treat human umbilical cord-derived mesenchymal stem cells (hUC-MSCs. The pHi and the differentiation of hUC-MSCs were compared upon cariporide treatment. The putative signaling pathway involved was also explored. Results: The pHi of hUC-MSCs was decreased upon cariporide treatment. Cariporide up-regulated the osteogenic differentiation of hUC-MSCs while the adipogenic differentiation was not affected. For osteogenic differentiation, β-catenin expression was up-regulated upon cariporide treatment. Conclusion: Decreased pHi induced by cariporide differentially contributes to hUC-MSCs differentiation.

  6. Globoside accelerates the differentiation of dental epithelial cells into ameloblasts

    Institute of Scientific and Technical Information of China (English)

    Takashi Nakamura; Yuta Chiba; Masahiro Naruse; Kan Saito; Hidemitsu Harada; Satoshi Fukumoto

    2016-01-01

    Tooth crown morphogenesis is tightly regulated by the proliferation and differentiation of dental epithelial cells. Globoside (Gb4), a globo-series glycosphingolipid, is highly expressed during embryogenesis as well as organogenesis, including tooth development. We previously reported that Gb4 is dominantly expressed in the neutral lipid fraction of dental epithelial cells. However, because its functional role in tooth development remains unknown, we investigated the involvement of Gb4 in dental epithelial cell differentiation. The expression of Gb4 was detected in ameloblasts of postnatal mouse molars and incisors. A cell culture analysis using HAT-7 cells, a rat-derived dental epithelial cell line, revealed that Gb4 did not promote dental epithelial cell proliferation. Interestingly, exogenous administration of Gb4 enhanced the gene expression of enamel extracellular matrix proteins such as ameloblastin, amelogenin, and enamelin in dental epithelial cells as well as in developing tooth germs. Gb4 also induced the expression of TrkB, one of the key receptors required for ameloblast induction in dental epithelial cells. In contrast, Gb4 downregulated the expression of p75, a receptor for neurotrophins (including neurotrophin-4) and a marker of undifferentiated dental epithelial cells. In addition, we found that exogenous administration of Gb4 to dental epithelial cells stimulated the extracellular signal-regulated kinase and p38 mitogen-activated protein kinase signalling pathways. Furthermore, Gb4 induced the expression of epiprofin and Runx2, the positive regulators for ameloblastin gene transcription. Thus, our results suggest that Gb4 contributes to promoting the differentiation of dental epithelial cells into ameloblasts.

  7. Optical Imaging for Stem Cell Differentiation to Neuronal Lineage

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Do Won; Lee, Dong Soo [Seoul National Univ., Seoul (Korea, Republic of)

    2012-03-15

    In regenerative medicine, the prospect of stem cell therapy hold great promise for the recovery of injured tissues and effective treatment of intractable diseases. Tracking stem cell fate provides critical information to understand and evaluate the success of stem cell therapy. The recent emergence of in vivo noninvasive molecular imaging has enabled assessment of the behavior of grafted stem cells in living subjects. In this review, we provide an overview of current optical imaging strategies based on cell or tissue specific reporter gene expression and of in vivo methods to monitor stem cell differentiation into neuronal lineages. These methods use optical reporters either regulated by neuron-specific promoters or containing neuron-specific microRNA binding sites. Both systems revealed dramatic changes in optical reporter imaging signals in cells differentiating a yeast GAL4 amplification system or an engineering-enhanced luciferase reported gene. Furthermore, we propose an advanced imaging system to monitor neuronal differentiation during neurogenesis that uses in vivo multiplexed imaging techniques capable of detecting several targets simultaneously.

  8. Correlation between membrane fluidity cellular development and stem cell differentiation

    KAUST Repository

    Noutsi, Pakiza

    2016-12-01

    Cell membranes are made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as neuronal differentiation, cell membranes undergo dramatic structural changes induced by proteins such as ARC and Cofilin among others in the case of synaptic modification. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. As expected, NIH3T3 cells have more rigid membrane at earlier stages of their development. On the other hand neurons tend to have the highest membrane fluidity early in their development emphasizing its correlation with plasticity and the need for this malleability during differentiation. This study sheds light on the involvement of membrane fluidity during neuronal differentiation and development of other cell lines.

  9. Optical imaging for stem cell differentiation to neuronal lineage.

    Science.gov (United States)

    Hwang, Do Won; Lee, Dong Soo

    2012-03-01

    In regenerative medicine, the prospect of stem cell therapy holds great promise for the recovery of injured tissues and effective treatment of intractable diseases. Tracking stem cell fate provides critical information to understand and evaluate the success of stem cell therapy. The recent emergence of in vivo noninvasive molecular imaging has enabled assessment of the behavior of grafted stem cells in living subjects. In this review, we provide an overview of current optical imaging strategies based on cell- or tissue-specific reporter gene expression and of in vivo methods to monitor stem cell differentiation into neuronal lineages. These methods use optical reporters either regulated by neuron-specific promoters or containing neuron-specific microRNA binding sites. Both systems revealed dramatic changes in optical reporter imaging signals in cells differentiating into a neuronal lineage. The detection limit of weak promoters or reporter genes can be greatly enhanced by adopting a yeast GAL4 amplification system or an engineering-enhanced luciferase reporter gene. Furthermore, we propose an advanced imaging system to monitor neuronal differentiation during neurogenesis that uses in vivo multiplexed imaging techniques capable of detecting several targets simultaneously.

  10. Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin.

    Science.gov (United States)

    Dorn, Isabel; Klich, Katharina; Arauzo-Bravo, Marcos J; Radstaak, Martina; Santourlidis, Simeon; Ghanjati, Foued; Radke, Teja F; Psathaki, Olympia E; Hargus, Gunnar; Kramer, Jan; Einhaus, Martin; Kim, Jeong Beom; Kögler, Gesine; Wernet, Peter; Schöler, Hans R; Schlenke, Peter; Zaehres, Holm

    2015-01-01

    Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34(+) hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34(+) hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34(+) hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34(+) cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential.

  11. Delivery of differentiation factors by mesoporous silica particles assists advanced differentiation of transplanted murine embryonic stem cells

    DEFF Research Database (Denmark)

    Garcia-Bennett, Alfonso E; Kozhevnikova, Mariya; König, Niclas;

    2013-01-01

    Stem cell transplantation holds great hope for the replacement of damaged cells in the nervous system. However, poor long-term survival after transplantation and insufficiently robust differentiation of stem cells into specialized cell types in vivo remain major obstacles for clinical application...... neurotrophic factor and glial cell line-derived neurotrophic factor, respectively, with these particles enabled not only robust functional differentiation of motor neurons from transplanted embryonic stem cells but also their long-term survival in vivo. We propose that the delivery of growth factors...... by mesoporous nanoparticles is a potentially versatile and widely applicable strategy for efficient differentiation and functional integration of stem cell derivatives upon transplantation....

  12. New method of immobilization of microbial cells by capture on the surface of insoluble pyridinium-type resin.

    Science.gov (United States)

    Kawabata, N; Nishimura, S; Yoshimura, T

    1990-04-25

    A new method for the immobilization of microbial cells has been developed. Whole cells of Escherichia coli with aspartase activity were immobilized by capture on the surface of cross-linked poly(N-benzyl-4-vinylpyridinium bromide) containing styrene (BVPS resin), an insoluble pyridinium-type resin. When a suspension of the bacterial cells in buffer solution was passed through a glass column containing beads of BVPS resin, the cells were captured on the resin surface and formed an immobilized cell system. A fixed-bed column reactor containing 300 mg of the bacterial cells immobilized by capture on 10 g of BVPS resin beads was used for the preparation of L-aspartic acid from ammonium fumarate. Continuous operation of tne bioreactor produced L-aspartic acid in a quantitative yield when the influent substrate concentration was 0.1M and the flow rate was 0.41-0.83 bed volumes per hour at pH 7.4-7.7 at 30 degrees C.

  13. Differentiation state determines neural effects on microvascular endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Muffley, Lara A., E-mail: muffley@u.washington.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Pan, Shin-Chen, E-mail: pansc@mail.ncku.edu.tw [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Smith, Andria N., E-mail: gnaunderwater@gmail.com [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Ga, Maricar, E-mail: marga16@uw.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Hocking, Anne M., E-mail: ahocking@u.washington.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States); Gibran, Nicole S., E-mail: nicoleg@u.washington.edu [University of Washington, Campus Box 359796, 300 9th Avenue, Seattle, WA 98104 (United States)

    2012-10-01

    Growing evidence indicates that nerves and capillaries interact paracrinely in uninjured skin and cutaneous wounds. Although mature neurons are the predominant neural cell in the skin, neural progenitor cells have also been detected in uninjured adult skin. The aim of this study was to characterize differential paracrine effects of neural progenitor cells and mature sensory neurons on dermal microvascular endothelial cells. Our results suggest that neural progenitor cells and mature sensory neurons have unique secretory profiles and distinct effects on dermal microvascular endothelial cell proliferation, migration, and nitric oxide production. Neural progenitor cells and dorsal root ganglion neurons secrete different proteins related to angiogenesis. Specific to neural progenitor cells were dipeptidyl peptidase-4, IGFBP-2, pentraxin-3, serpin f1, TIMP-1, TIMP-4 and VEGF. In contrast, endostatin, FGF-1, MCP-1 and thrombospondin-2 were specific to dorsal root ganglion neurons. Microvascular endothelial cell proliferation was inhibited by dorsal root ganglion neurons but unaffected by neural progenitor cells. In contrast, microvascular endothelial cell migration in a scratch wound assay was inhibited by neural progenitor cells and unaffected by dorsal root ganglion neurons. In addition, nitric oxide production by microvascular endothelial cells was increased by dorsal root ganglion neurons but unaffected by neural progenitor cells. -- Highlights: Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate microvascular endothelial cell proliferation. Black-Right-Pointing-Pointer Neural progenitor cells, not dorsal root ganglion neurons, regulate microvascular endothelial cell migration. Black-Right-Pointing-Pointer Neural progenitor cells and dorsal root ganglion neurons do not effect microvascular endothelial tube formation. Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate

  14. ROS-mediated redox signaling during cell differentiation in plants.

    Science.gov (United States)

    Schmidt, Romy; Schippers, Jos H M

    2015-08-01

    Reactive oxygen species (ROS) have emerged in recent years as important regulators of cell division and differentiation. The cellular redox state has a major impact on cell fate and multicellular organism development. However, the exact molecular mechanisms through which ROS manifest their regulation over cellular development are only starting to be understood in plants. ROS levels are constantly monitored and any change in the redox pool is rapidly sensed and responded upon. Different types of ROS cause specific oxidative modifications, providing the basic characteristics of a signaling molecule. Here we provide an overview of ROS sensors and signaling cascades that regulate transcriptional responses in plants to guide cellular differentiation and organ development. Although several redox sensors and cascades have been identified, they represent only a first glimpse on the impact that redox signaling has on plant development and growth. We provide an initial evaluation of ROS signaling cascades involved in cell differentiation in plants and identify potential avenues for future studies. This article is part of a Special Issue entitled Redox regulation of differentiation and de-differentiation. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Hematopoietic Pyk2 regulates migration of differentiated HL-60 cells

    Directory of Open Access Journals (Sweden)

    Duan Yingli

    2010-05-01

    Full Text Available Abstract Background Pyk2 is a non-receptor cytoplasmic tyrosine kinase that belongs to the focal adhesion kinase family and has been implicated in neutrophil spreading and respiratory burst activity caused by TNF-α. However, the role of Pyk2 in neutrophil migration is incompletely defined. In this study, we tested the hypothesis that Pyk2 regulates the migration of neutrophil-like differentiated HL-60 cells subsequent to β2-integrin mediated cell adhesion. Methods HL-60 cells were induced to differentiate into neutrophil-like cells (dHL60 by incubation in medium containing 1.25% DMSO for up to 4 days. Pyk2 expression and tyrosine phosphorylation was measured by Western blot analysis. Adhesion of dHL60 cells to plated fibrinogen was measured by residual myeloperoxidase activity. dHL60 cell migration was evaluated using a 96-well chemoTx chamber. Results Western blot analysis demonstrated that hematopoietic Pyk2 was predominantly expressed after HL60 cell differentiation. Pyk2 was tyrosine phosphorylated upon adhesion of dHL60 cells to plated fibrinogen in the presence of fMLP. By contrast, tyrosine phosphorylation of Pyk2 was insignificant in dHL60 cells treated in suspension with fMLP. Antibodies against CD18 blocked both phosphorylation of Pyk2 and adhesion of dHL60 cells to fibrinogen, demonstrating that phosphorylation of Pyk2 was β2-integrin dependent. TAT-Pyk2-CT, a dominant negative fusion protein in which the TAT protein transduction domain was fused to the c-terminal Pyk2, attenuated fMLP-stimulated spreading, migration and phosphorylation of endogenous Pyk2 without blocking adhesion of dHL-60 cells to fibrinogen. Similarly, silencing of Pyk2 expression by siRNA in dHL60 cells also attenuated dHL60 cell migration caused by fMLP. Phospho-Pyk2 was evenly distributed around cell membrane circumferentially in unstimulated dHL-60 cells adherent to plated fibrinogen. In dHL60 cells treated with fMLP to cause cell spreading and polarization

  16. The integration of cell division, growth and differentiation.

    Science.gov (United States)

    Harashima, Hirofumi; Schnittger, Arp

    2010-02-01

    The development of a multicellular organism such as a flowering plant relies on the patterned control of cell proliferation, differentiation, and growth. Research in the recent years has revealed that the control of cell-cycle progression and growth in plants is distinct from the regulation found in yeast or metazoans. Understanding these plant-specific regulators and networks, in which they act, is key for the understanding of plant development and is of current global importance as a basis for breeding of energy crops as well as the breeding of plants adapted for changing environmental conditions. However, the production of cells and their specification and differentiation overlap in time and space and build an intricate interrelationship of dependencies and feedback loops. In this network, the developmental context and the generation of specific cell types and tissues are often decisive. Copyright 2009 Elsevier Ltd. All rights reserved.

  17. Arsenic inhibits hedgehog signaling during P19 cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Jui Tung [Environmental Toxicology Program, Clemson University, 132 Long Hall, Clemson, SC 29634 (United States); Bain, Lisa J., E-mail: lbain@clemson.edu [Environmental Toxicology Program, Clemson University, 132 Long Hall, Clemson, SC 29634 (United States); Department of Biological Sciences, Clemson University, 132 Long Hall, Clemson, SC 29634 (United States)

    2014-12-15

    Arsenic is a toxicant found in ground water around the world, and human exposure mainly comes from drinking water or from crops grown in areas containing arsenic in soils or water. Epidemiological studies have shown that arsenic exposure during development decreased intellectual function, reduced birth weight, and altered locomotor activity, while in vitro studies have shown that arsenite decreased muscle and neuronal cell differentiation. The sonic hedgehog (Shh) signaling pathway plays an important role during the differentiation of both neurons and skeletal muscle. The purpose of this study was to investigate whether arsenic can disrupt Shh signaling in P19 mouse embryonic stem cells, leading to changes muscle and neuronal cell differentiation. P19 embryonic stem cells were exposed to 0, 0.25, or 0.5 μM of sodium arsenite for up to 9 days during cell differentiation. We found that arsenite exposure significantly reduced transcript levels of genes in the Shh pathway in both a time and dose-dependent manner. This included the Shh ligand, which was decreased 2- to 3-fold, the Gli2 transcription factor, which was decreased 2- to 3-fold, and its downstream target gene Ascl1, which was decreased 5-fold. GLI2 protein levels and transcriptional activity were also reduced. However, arsenic did not alter GLI2 primary cilium accumulation or nuclear translocation. Moreover, additional extracellular SHH rescued the inhibitory effects of arsenic on cellular differentiation due to an increase in GLI binding activity. Taken together, we conclude that arsenic exposure affected Shh signaling, ultimately decreasing the expression of the Gli2 transcription factor. These results suggest a mechanism by which arsenic disrupts cell differentiation. - Highlights: • Arsenic exposure decreases sonic hedgehog pathway-related gene expression. • Arsenic decreases GLI2 protein levels and transcriptional activity in P19 cells. • Arsenic exposure does not alter the levels of SHH

  18. Plant Phosphoglycerolipids: The Gatekeepers of Vascular Cell Differentiation.

    Science.gov (United States)

    Gujas, Bojan; Rodriguez-Villalon, Antia

    2016-01-01

    In higher plants, the plant vascular system has evolved as an inter-organ communication network essential to deliver a wide range of signaling factors among distantly separated organs. To become conductive elements, phloem and xylem cells undergo a drastic differentiation program that involves the degradation of the majority of their organelles. While the molecular mechanisms regulating such complex process remain poorly understood, it is nowadays clear that phosphoglycerolipids display a pivotal role in the regulation of vascular tissue formation. In animal cells, this class of lipids is known to mediate acute responses as signal transducers and also act as constitutive signals that help defining organelle identity. Their rapid turnover, asymmetrical distribution across subcellular compartments as well as their ability to rearrange cytoskeleton fibers make phosphoglycerolipids excellent candidates to regulate complex morphogenetic processes such as vascular differentiation. Therefore, in this review we aim to summarize, emphasize and connect our current understanding about the involvement of phosphoglycerolipids in phloem and xylem differentiation.

  19. Plant phosphoglycerolipids: the gatekeepers of vascular cell differentiation

    Directory of Open Access Journals (Sweden)

    Bojan eGujas

    2016-02-01

    Full Text Available In higher plants, the plant vascular system has evolved as an inter-organ communication network essential to deliver a wide range of signaling factors among distantly separated organs. To become conductive elements, phloem and xylem cells undergo a drastic differentiation program that involves the degradation of the majority of their organelles. While the molecular mechanisms regulating such complex process remain poorly understood, it is nowadays clear that phosphoglycerolipids display a pivotal role in the regulation of vascular formation. In animal cells, this class of lipids is known to mediate acute responses as signal transducers and also act as constitutive signals that help defining organelle identity. Their rapid turnover, asymmetrical distribution across subcellular compartments as well as their ability to rearrange cytoskeleton fibers make phosphoglycerolipids excellent candidates to regulate complex morphogenetic processes such as vascular differentiation. Therefore, in this review we aim to summarize, emphasize and connect our current understanding about the involvement of phosphoglycerolipids in phloem and xylem differentiation.

  20. Functionalization and cellular uptake of boron carbide nanoparticles. The first step toward T cell-guided boron neutron capture therapy.

    Science.gov (United States)

    Mortensen, M W; Björkdahl, O; Sørensen, P G; Hansen, T; Jensen, M R; Gundersen, H J G; Bjørnholm, T

    2006-01-01

    In this paper we present surface modification strategies of boron carbide nanoparticles, which allow for bioconjugation of the transacting transcriptional activator (TAT) peptide and fluorescent dyes. Coated nanoparticles can be translocated into murine EL4 thymoma cells and B16 F10 malignant melanoma cells in amounts as high as 0.3 wt. % and 1 wt. %, respectively. Neutron irradiation of a test system consisting of untreated B16 cells mixed with B16 cells loaded with boron carbide nanoparticles were found to inhibit the proliferative capacity of untreated cells, showing that cells loaded with boron-containing nanoparticles can hinder the growth of neighboring cells upon neutron irradiation. This could provide the first step toward a T cell-guided boron neutron capture therapy.

  1. Inorganic arsenic impairs differentiation and functions of human dendritic cells

    Energy Technology Data Exchange (ETDEWEB)

    Macoch, Mélinda; Morzadec, Claudie [UMR INSERM U1085, Institut de Recherche sur la Santé, l' Environnement et le Travail (IRSET), Université de Rennes 1, 2 avenue du Professeur Léon Bernard, 35043 Rennes (France); Fardel, Olivier [UMR INSERM U1085, Institut de Recherche sur la Santé, l' Environnement et le Travail (IRSET), Université de Rennes 1, 2 avenue du Professeur Léon Bernard, 35043 Rennes (France); Pôle Biologie, Centre Hospitalier Universitaire (CHU) Rennes, 2 rue Henri Le Guilloux, 35033 Rennes (France); Vernhet, Laurent, E-mail: laurent.vernhet@univ-rennes1.fr [UMR INSERM U1085, Institut de Recherche sur la Santé, l' Environnement et le Travail (IRSET), Université de Rennes 1, 2 avenue du Professeur Léon Bernard, 35043 Rennes (France)

    2013-01-15

    Experimental studies have demonstrated that the antileukemic trivalent inorganic arsenic prevents the development of severe pro-inflammatory diseases mediated by excessive Th1 and Th17 cell responses. Differentiation of Th1 and Th17 subsets is mainly regulated by interleukins (ILs) secreted from dendritic cells (DCs) and the ability of inorganic arsenic to impair interferon-γ and IL-17 secretion by interfering with the physiology of DCs is unknown. In the present study, we demonstrate that high concentrations of sodium arsenite (As(III), 1–2 μM) clinically achievable in plasma of arsenic-treated patients, block differentiation of human peripheral blood monocytes into immature DCs (iDCs) by inducing their necrosis. Differentiation of monocytes in the presence of non-cytotoxic concentrations of As(III) (0.1 to 0.5 μM) only slightly impacts endocytotic activity of iDCs or expression of co-stimulatory molecules in cells activated with lipopolysaccharide. However, this differentiation in the presence of As(III) strongly represses secretion of IL-12p70 and IL-23, two major regulators of Th1 and Th17 activities, from iDCs stimulated with different toll-like receptor (TLR) agonists in metalloid-free medium. Such As(III)-exposed DCs also exhibit reduced mRNA levels of IL12A and/or IL12B genes when activated with TLR agonists. Finally, differentiation of monocytes with non-cytotoxic concentrations of As(III) subsequently reduces the ability of activated DCs to stimulate the release of interferon-γ and IL-17 from Th cells. In conclusion, our results demonstrate that clinically relevant concentrations of inorganic arsenic markedly impair in vitro differentiation and functions of DCs, which may contribute to the putative beneficial effects of the metalloid towards inflammatory autoimmune diseases. Highlights: ► Inorganic arsenic impairs differentiation and functions of human dendritic cells (DCs) ► Arsenite (> 1 μM) blocks differentiation of dendritic cells by

  2. Zfp423 promotes adipogenic differentiation of bovine stromal vascular cells.

    Directory of Open Access Journals (Sweden)

    Yan Huang

    Full Text Available Intramuscular fat or marbling is critical for the palatability of beef. In mice, very recent studies show that adipocytes and fibroblasts share a common pool of progenitor cells, with Zinc finger protein 423 (Zfp423 as a key initiator of adipogenic differentiation. To evaluate the role of Zfp423 in intramuscular adipogenesis and marbling in beef cattle, we sampled beef muscle for separation of stromal vascular cells. These cells were immortalized with pCI neo-hEST2 and individual clones were selected by G418. A total of 288 clones (3×96 well plates were isolated and induced to adipogenesis. The presence of adipocytes was assessed by Oil-Red-O staining. Three clones with high and low adipogenic potential respectively were selected for further analyses. In addition, fibro/adipogenic progenitor cells were selected using a surface marker, platelet derived growth factor receptor (PDGFR α. The expression of Zfp423 was much higher (307.4±61.9%, P<0.05 in high adipogenic cells, while transforming growth factor (TGF-β was higher (156.1±48.7%, P<0.05 in low adipogenic cells. Following adipogenic differentiation, the expression of peroxisome proliferator-activated receptor γ (PPARγ and CCAAT/enhancer binding protein α (C/EBPα were much higher (239.4±84.1% and 310.7±138.4%, respectively, P<0.05 in high adipogenic cells. Over-expression of Zfp423 in stromal vascular cells and cloned low adipogenic cells dramatically increased their adipogenic differentiation, accompanied with the inhibition of TGF-β expression. Zfp423 knockdown by shRNA in high adipogenic cells largely prevented their adipogenic differentiation. The differential regulation of Zfp423 and TGF-β between low and high adipogenic cells is associated with the DNA methylation in their promoters. In conclusion, data show that Zfp423 is a critical regulator of adipogenesis in stromal vascular cells of bovine muscle, and Zfp423 may provide a molecular target for enhancing intramuscular

  3. Surface Curvature Differentially Regulates Stem Cell Migration and Differentiation via Altered Attachment Morphology and Nuclear Deformation

    Science.gov (United States)

    Werner, Maike; Blanquer, Sébastien B. G.; Haimi, Suvi P.; Korus, Gabriela; Dunlop, John W. C.; Duda, Georg N.; Grijpma, Dirk. W.

    2016-01-01

    Signals from the microenvironment around a cell are known to influence cell behavior. Material properties, such as biochemical composition and substrate stiffness, are today accepted as significant regulators of stem cell fate. The knowledge of how cell behavior is influenced by 3D geometric cues is, however, strongly limited despite its potential relevance for the understanding of tissue regenerative processes and the design of biomaterials. Here, the role of surface curvature on the migratory and differentiation behavior of human mesenchymal stem cells (hMSCs) has been investigated on 3D surfaces with well‐defined geometric features produced by stereolithography. Time lapse microscopy reveals a significant increase of cell migration speed on concave spherical compared to convex spherical structures and flat surfaces resulting from an upward‐lift of the cell body due to cytoskeletal forces. On convex surfaces, cytoskeletal forces lead to substantial nuclear deformation, increase lamin‐A levels and promote osteogenic differentiation. The findings of this study demonstrate a so far missing link between 3D surface curvature and hMSC behavior. This will not only help to better understand the role of extracellular matrix architecture in health and disease but also give new insights in how 3D geometries can be used as a cell‐instructive material parameter in the field of biomaterial‐guided tissue regeneration.

  4. Trichomes as models for studying plant cell differentiation.

    Science.gov (United States)

    Yang, Changxian; Ye, Zhibiao

    2013-06-01

    Trichomes, originating from epidermal cells, are present on nearly all terrestrial plants. They exist in diverse forms, are readily accessible, and serve as an excellent model system for analyzing the molecular mechanisms in plant cell differentiation, including cell fate choices, cell cycle control, and cell morphogenesis. In Arabidopsis, two regulatory models have been identified that function in parallel in trichome formation; the activator-inhibitor model and the activator-depletion model. Cotton fiber, a similar unicellular structure, is controlled by some functional homologues of Arabidopsis trichome-patterning genes. Multicellular trichomes, as in tobacco and tomato, may form through a distinct pathway from unicellular trichomes. Recent research has shown that cell cycle control participates in trichome formation. In this review, we summarize the molecular mechanisms involved in the formation of unicellular and multicellular trichomes, and discuss the integration of the cell cycle in its initiation and morphogenesis.

  5. Ultrastructure of neuronal-like cells differentiated from adult adipose-derived stromal cells

    Institute of Scientific and Technical Information of China (English)

    Changqing Ye; Xiaodong Yuan; Hui Liu; Yanan Cai; Ya Ou

    2010-01-01

    β-mercaptoethanol induces in vitro adult adipose-derived stromal cells (ADSCs) to differentiate into neurons. However, the ultrastructural features of the differentiated neuronal-like cells remain unknown. In the present study, inverted phase contrast microscopy was utilized to observe β-mercaptcethanol-induced differentiation of neuronal-like cells from human ADSCs, and immunocytochemistry and real-time polymerase chain reaction were employed to detect expression of a neural stem cells marker (nestin), a neuronal marker (neuron-specific enolase), and a glial marker (glial fibrillary acidic protein). In addition, ultrastructure of neuronal-like cells was observed by transmission election microscopy. Results revealed highest expression rate of nestin and neuron-specific enolase at 3 and 5 hours following induced differentiation; cells in the 5-hour induction group exhibited a neuronal-specific structure, i.e., Nissl bodies. However, when induction solution was replaced by complete culture medium after 8-hour induction, the differentiated cells reverted to the fibroblast-like morphology from day 1. These results demonstrate that β-mercaptoethanol-induced ADSCs induced differentiation into neural stem cells, followed by morphology of neuronal-like cells. However, this differentiation state was not stable.

  6. Differentiation of Mouse Embryonic Stem Cells into Ventral Foregut Precursors

    DEFF Research Database (Denmark)

    Rothová, Michaela; Hölzenspies, Jurriaan J; Livigni, Alessandra

    2016-01-01

    endoderm with pronounced anterior character. ADE-containing cultures can be produced in vitro by suspension (embryoid body) culture or in a serum-free adherent monolayer culture. ESC-derived ADE cells are committed to endodermal fates and can undergo further differentiation in vitro towards ventral foregut...

  7. Ghrelin promotes differentiation of human embryonic stem cells into cardiomyocytes

    Institute of Scientific and Technical Information of China (English)

    Jin YANG; Guo-qiang LIU; Rui WEI; Wen-fang HOU; Mei-juan GAO; Ming-xia ZHU; Hai-ning WANG; Gui-an CHEN; Tian-pei HONG

    2011-01-01

    Aim:Ghrelin is involved in regulating the differentiation of mesoderm-derived precursor cells.The aim of this study was to investigate whether ghrelin modulated the differentiation of human embryonic stem (hES) cells into cardiomyocytes and,if so,whether the effect was mediated by growth hormone secretagogue receptor 1α (GHS-R1α).Methods:Cardiomyocyte differentiation from hES cells was performed according to an embryoid body (EB)-based protocol.The cumulative percentage of beating EBs was calculated.The expression of cardiac-specific markers including cardiac troponin Ⅰ (cTnl) and α-myosin heavy chain (α-MHC) was detected using RT-PCR,real-time PCR and Western blot.The dispersed beating EBs were examined using immunofluorescent staining.Results:The percentage of beating EBs and the expression of cTnl were significantly increased after ghrelin (0.1 and 1 nmol/L) added into the differentiation medium.From 6 to 18 d of differentiation,the increased expression of cTnl and α-MHC by ghrelin (1 nmol/L)was time-dependent,and in line with the alteration of the percentages of beating EBs.Furthermore,the dispersed beating EBs were double-positively immunostained with antibodies against cTnl and α-actinin.However,blockage of GHS-R1α with its specific antagonist D-[lys3]-GHRP-6 (1 μmol/L) did not alter the effects of ghrelin on cardiomyocyte differentiation.Conclusion:Our data show that ghrelin enhances the generation of cardiomyocytes from hES cells,which is not mediated via GHS-R1α.

  8. Epigenetic dysregulation in mesenchymal stem cell aging and spontaneous differentiation.

    Directory of Open Access Journals (Sweden)

    Zhilong Li

    Full Text Available BACKGROUND: Mesenchymal stem cells (MSCs hold great promise for the treatment of difficult diseases. As MSCs represent a rare cell population, ex vivo expansion of MSCs is indispensable to obtain sufficient amounts of cells for therapies and tissue engineering. However, spontaneous differentiation and aging of MSCs occur during expansion and the molecular mechanisms involved have been poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Human MSCs in early and late passages were examined for their expression of genes involved in osteogenesis to determine their spontaneous differentiation towards osteoblasts in vitro, and of genes involved in self-renewal and proliferation for multipotent differentiation potential. In parallel, promoter DNA methylation and hostone H3 acetylation levels were determined. We found that MSCs underwent aging and spontaneous osteogenic differentiation upon regular culture expansion, with progressive downregulation of TERT and upregulation of osteogenic genes such as Runx2 and ALP. Meanwhile, the expression of genes associated with stem cell self-renewal such as Oct4 and Sox2 declined markedly. Notably, the altered expression of these genes were closely associated with epigenetic dysregulation of histone H3 acetylation in K9 and K14, but not with methylation of CpG islands in the promoter regions of most of these genes. bFGF promoted MSC proliferation and suppressed its spontaneous osteogenic differentiation, with corresponding changes in histone H3 acetylation in TERT, Oct4, Sox2, Runx2 and ALP genes. CONCLUSIONS/SIGNIFICANCE: Our results indicate that histone H3 acetylation, which can be modulated by extrinsic signals, plays a key role in regulating MSC aging and differentiation.

  9. Auxin regulates distal stem cell differentiation in Arabidopsis roots.

    Science.gov (United States)

    Ding, Zhaojun; Friml, Jirí

    2010-06-29

    The stem cell niche in the root meristem is critical for the development of the plant root system. The plant hormone auxin acts as a versatile trigger in many developmental processes, including the regulation of root growth, but its role in the control of the stem cell activity remains largely unclear. Here we show that local auxin levels, determined by biosynthesis and intercellular transport, mediate maintenance or differentiation of distal stem cells in the Arabidopsis thaliana roots. Genetic analysis shows that auxin acts upstream of the major regulators of the stem cell activity, the homeodomain transcription factor WOX5, and the AP-2 transcription factor PLETHORA. Auxin signaling for differentiation of distal stem cells requires the transcriptional repressor IAA17/AXR3 as well as the ARF10 and ARF16 auxin response factors. ARF10 and ARF16 activities repress the WOX5 transcription and restrict it to the quiescent center, where WOX5, in turn, is needed for the activity of PLETHORA. Our investigations reveal that long-distance auxin signals act upstream of the short-range network of transcriptional factors to mediate the differentiation of distal stem cells in roots.

  10. Selenoprotein O deficiencies suppress chondrogenic differentiation of ATDC5 cells.

    Science.gov (United States)

    Yan, Jidong; Fei, Yao; Han, Yan; Lu, Shemin

    2016-10-01

    Selenoprotein O (Sel O) is a selenium-containing protein, but its function is still unclear. In the present study, we observed that the mRNA and protein expression levels of Sel O increased during chondrogenic induction of ATDC5 cells. The effects of Sel O on chondrocyte differentiation were then examined through shRNA-mediated gene silencing technique. The expression of Sel O was significantly suppressed at both mRNA and protein levels in a stable cell line transfected with a Sel O-specific target shRNA construct. Thereafter, we demonstrated that Sel O deficiencies suppress chondrogenic differentiation of ATDC5 cells. Sel O deficiencies inhibited expression of chondrogenic gene Sox9, Col II, and aggrecan. Sel O-deficient cells also accumulated a few cartilage glycosaminoglycans (GAGs) and decreased the activity of alkaline phosphatase (ALP). In addition, Sel O deficiencies inhibited chondrocyte proliferation through delayed cell cycle progression by suppression of cyclin D1 expression. Moreover, Sel O deficiencies induced chondrocyte death through cell apoptosis. In summary, we describe the expression patterns and the essential roles of Sel O in chondrocyte viability, proliferation, and chondrogenic differentiation. Additionally, Sel O deficiency-mediated impaired chondrogenesis may illustrate the mechanisms of Se deficiency in the pathophysiological process of the endemic osteoarthropathy.

  11. Silk scaffolds with tunable mechanical capability for cell differentiation.

    Science.gov (United States)

    Bai, Shumeng; Han, Hongyan; Huang, Xiaowei; Xu, Weian; Kaplan, David L; Zhu, Hesun; Lu, Qiang

    2015-07-01

    Bombyx mori silk fibroin is a promising biomaterial for tissue regeneration and is usually considered an "inert" material with respect to actively regulating cell differentiation due to few specific cell signaling peptide domains in the primary sequence and the generally stiffer mechanical properties due to crystalline content formed in processing. In the present study, silk fibroin porous 3D scaffolds with nanostructures and tunable stiffness were generated via a silk fibroin nanofiber-assisted lyophilization process. The silk fibroin nanofibers with high β-sheet content were added into the silk fibroin solutions to modulate the self-assembly, and to directly induce water-insoluble scaffold formation after lyophilization. Unlike previously reported silk fibroin scaffold formation processes, these new scaffolds had lower overall β-sheet content and softer mechanical properties for improved cell compatibility. The scaffold stiffness could be further tuned to match soft tissue mechanical properties, which resulted in different differentiation outcomes with rat bone marrow-derived mesenchymal stem cells toward myogenic and endothelial cells, respectively. Therefore, these silk fibroin scaffolds regulate cell differentiation outcomes due to their mechanical features.

  12. Human pluripotent stem cell differentiation into authentic striatal projection neurons.

    Science.gov (United States)

    Delli Carri, Alessia; Onorati, Marco; Castiglioni, Valentina; Faedo, Andrea; Camnasio, Stefano; Toselli, Mauro; Biella, Gerardo; Cattaneo, Elena

    2013-08-01

    Here we present the principles and steps of a protocol that we have recently developed for the differentiation of hES/iPS cells into the authentic human striatal projection medium spiny neurons (MSNs) that die in Huntington's Disease (HD). Authenticity is judged by the convergence of multiple features within individual cells. Our procedure lasts 80 days and couples neural induction via BMP/TGF-β inhibition with exposure to the developmental factors sonic hedgehog (SHH) and dickkopf1 (DKK-1) to drive ventral telencephalic specification, followed by terminal differentiation [1]. Authenticity of the resulting neuronal population is monitored by the appearance of FOXG1(+)/GSX2(+) progenitor cells of the lateral ganglionic eminence (LGE) at day 15-25 of differentiation, followed by appearance of CTIP2-, FOXP1- and FOXP2-positive cells at day 45. These precursor cells then mature into MAP2(+)/GABA(+) neurons with 20 % of them ultimately co-expressing the DARPP-32 and CTIP2 diagnostic markers and carrying electrophysiological properties expected for fully functional MSNs.The protocol is characterized by its replicability in at least three human pluripotent cell lines. Altogether this protocol defines a useful platform for in vitro developmental neurobiology studies, drug screening, and regenerative medicine approaches.

  13. Diacylglycerols mimic phorbol diester induction of leukemic cell differentiation.

    Science.gov (United States)

    Ebeling, J G; Vandenbark, G R; Kuhn, L J; Ganong, B R; Bell, R M; Niedel, J E

    1985-01-01

    Activation of cellular protein kinase C appears to be involved in the mechanism by which phorbol diesters induce differentiation of human myeloid leukemia cells (HL-60). Protein kinase C is thought to be physiologically activated by diacylglycerol derived from receptor-mediated phosphatidylinositol hydrolysis. sn-1,2-diacylglycerols with short saturated acyl side chains (C4-C10) were synthesized and found to be potent activators of protein kinase C partially purified from HL-60 cells. These diacylglycerols were also competitive inhibitors of [3H]phorbol dibutyrate binding to the soluble phorbol diester receptor. The most potent diacylglycerol, sn-1,2-dioctanoylglycerol, displaced greater than 90% of [3H]phorbol dibutyrate from the phorbol diester receptor of intact HL-60 cells. Because of probable cellular metabolism of sn-1,2-dioctanoylglycerol, hourly doses were required to maintain persistent occupancy of the phorbol diester binding site. Treatment of HL-60 cells with either phorbol 12-myristate 13-acetate or sn-1,2-dioctanoylglycerol produced identical phosphoprotein changes. Finally, sn-1,2-dioctanoylglycerol induced differentiation of the HL-60 cells into cells with morphologic characteristics of macrophages. Substitution of the hydroxyl group at position 3 with a hydrogen, chloro, or sulfhydryl moiety inactivated sn-1,2-dioctanoylglycerol. These data strengthen the hypothesis that protein kinase C activation plays a role in macrophage differentiation. Images PMID:3156372

  14. Citalopram increases the differentiation efifcacy of bone marrow mesenchymal stem cells into neuronal-like cells

    Institute of Scientific and Technical Information of China (English)

    Javad Verdi; Seyed Abdolreza Mortazavi-Tabatabaei; Shiva Sharif; Hadi Verdi; Alireza Shoae-Hassani

    2014-01-01

    Several studies have demonstrated that selective serotonin reuptake inhibitor antidepressants can promote neuronal cell proliferation and enhance neuroplasticity both in vitro and in vivo. It is hypothesized that citalopram, a selective serotonin reuptake inhibitor, can promote the neuronal differentiation of adult bone marrow mesenchymal stem cells. Citalopram strongly enhanced neuronal characteristics of the cells derived from bone marrow mesenchymal stem cells. The rate of cell death was decreased in citalopram-treated bone marrow mesenchymal stem cells than in control cells in neurobasal medium. In addition, the cumulative population doubling level of the citalopram-treated cells was signiifcantly increased compared to that of control cells. Also BrdU incorporation was elevated in citalopram-treated cells. These ifndings suggest that citalopram can improve the neuronal-like cell differentiation of bone marrow mesenchymal stem cells by increasing cell proliferation and survival while maintaining their neuronal characteristics.

  15. Citalopram increases the differentiation efficacy of bone marrow mesenchymal stem cells into neuronal-like cells.

    Science.gov (United States)

    Verdi, Javad; Mortazavi-Tabatabaei, Seyed Abdolreza; Sharif, Shiva; Verdi, Hadi; Shoae-Hassani, Alireza

    2014-04-15

    Several studies have demonstrated that selective serotonin reuptake inhibitor antidepressants can promote neuronal cell proliferation and enhance neuroplasticity both in vitro and in vivo. It is hypothesized that citalopram, a selective serotonin reuptake inhibitor, can promote the neuronal differentiation of adult bone marrow mesenchymal stem cells. Citalopram strongly enhanced neuronal characteristics of the cells derived from bone marrow mesenchymal stem cells. The rate of cell death was decreased in citalopram-treated bone marrow mesenchymal stem cells than in control cells in neurobasal medium. In addition, the cumulative population doubling level of the citalopram-treated cells was significantly increased compared to that of control cells. Also BrdU incorporation was elevated in citalopram-treated cells. These findings suggest that citalopram can improve the neuronal-like cell differentiation of bone marrow mesenchymal stem cells by increasing cell proliferation and survival while maintaining their neuronal characteristics.

  16. Differentiation: A Central Topic in Developmental and Cell Biology

    Science.gov (United States)

    Müller, W. A.

    The concept of "differentiation" encompasses all processes leading to differently specialized cell types, beginning with the progressive divergence of developmental pathways and ending with the successive programming and final elaboration of each particular cell type. Guidance and positional information are provided by external cues, by differentially allotted cytoplasmic determinants such as mRNA for transcription factors, and by cascades of intercellular signals. Eventually cell type specific selector genes, such as the muscle cell determining MyoD/myogenin genes and neural key genes (e.g., achaete scute-C, neurogenin), are switched on which control entire sets of subordinate effector genes. In multiplying cells "cell heredity" based on an epigenetic cellular memory enables transmission of the cell type determining program from parental to daughter cells. This memory can be based on autocatalytic self-activation of cell type specific selector genes and on the enduring action of gene groups such as the Polycomb and thrithorax complexes that code for proteins which bind to DNA sequences called cellular memory modules. These modules confer permanent accessibility (potentiation) or inaccessibility (silencing) upon many different gene loci on the chromosomes.

  17. In vitro differentiation potential of human haematopoietic CD34(+) cells towards pancreatic β-cells.

    Science.gov (United States)

    Sunitha, Manne Mudhu; Srikanth, Lokanathan; Santhosh Kumar, Pasupuleti; Chandrasekhar, Chodimella; Sarma, Potukuchi Venkata Gurunadha Krishna

    2016-10-01

    Haematopoietic stem cells (HSCs) possess multipotent ability to differentiate into various types of cells on providing appropriate niche. In the present study, the differentiating potential of human HSCs into β-cells of islets of langerhans was explored. Human HSCs were apheretically isolated from a donor and cultured. Phenotypic characterization of CD34 glycoprotein in the growing monolayer HSCs was confirmed by immunocytochemistry and flow cytometry techniques. HSCs were induced by selection with beta cell differentiating medium (BDM), which consists of epidermal growth factor (EGF), fibroblast growth factor (FGF), transferrin, Triiodo-l-Tyronine, nicotinamide and activin A. Distinct morphological changes of differentiated cells were observed on staining with dithizone (DTZ) and expression of PDX1, insulin and synaptophysin was confirmed by immunocytochemistry. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed distinct expression of specific β-cell markers, pancreatic and duodenal homeobox-1 (PDX1), glucose transporter-2 (GLUT-2), synaptophysin (SYP) and insulin (INS) in these differentiated cells compared to HSCs. Further, these cells exhibited elevated expression of INS gene at 10 mM glucose upon inducing with different glucose concentrations. The prominent feature of the obtained β-cells was the presence of glucose sensors, which was determined by glucokinase activity and high glucokinase activity compared with CD34(+) stem cells. These findings illustrate the differentiation of CD34(+) HSCs into β-cells of islets of langerhans.

  18. Enhancement of Capture Sensitivity for Circulating Tumor Cells in a Breast Cancer Patient's Blood by Silicon Nanowire Platform.

    Science.gov (United States)

    Kim, Dong-Joo; Choi, Mun-Ki; Jeong, Jin-Tak; Lim, Jung-Taek; Lee, Han-Byoel; Han, Wonshik; Lee, Sang-Kwon

    2016-04-01

    The separation of circulating tumor cells (CTCs) from the blood of cancer patients with high sensitivity is an essential technique for selecting chemotherapeutic agents at a patient-by-patient level. Recently, various research groups have reported a nanostructure-based platform for rare cell capture due to its high surface area and 3D nanotopographic features. However, evaluation of capture sensitivity based on chemical modification of the nanostructure surface has not yet been performed. Here, we evaluated the capture sensitivity for CTCs from the blood of three patients diagnosed with stage IV metastatic breast cancer by using the following three platforms: streptavidin-conjugated silicon nanowire (STR-SiNW), poly-l-lysine-coated silicon nanowire (PLL-SiNW), and poly-l-lysine-coated glass (PLL-glass). The number of evaluated CTCs on STR-SiNW, PLL-SiNW, and PLL-glass were 16.2 ± 5.5 cells, 7.3 ± 2.9 cells, and 4.7 ± 1.5 cells, respectively, per 0.5 ml. Therefore, we suggest that the STR-SiNW platform is highly adaptable for the quantitative evaluation of CTCs from the blood of cancer patients in the clinical setting.

  19. Capturing CD4 cells using a functionalized circular microfluidic device and glutaraldehyde as biolinker for tuberculosis detection and diagnosis

    Science.gov (United States)

    Shih, Yeu-Farn; Huang, Nien-Tsu; Lee, Chih-Kung

    2015-03-01

    It is estimated that about one-third of the world's population has already been infected by tuberculosis. Mycobacterium tuberculosis, in general, can result in an active case of tuberculosis in approximately 5%-10% of those who suffer from latent tuberculosis and the chance of becoming ill is the highest within one of year of getting the disease. Although a newly developed methods called interferon gamma release assay (IGRA) can monitor CD4 cells secreted cytokine to diagnose tuberculosis (TB) condition. However, it is difficult to count total numbers of cytokine secreted CD4 cells, which make the diagnosis less accurate. Therefore, we develop a functionalized polydimethylsiloxane (PDMS) device using glutaraldehyde to capture CD4 cells. To enhance the capture efficiency, we use COMSOL simulation to optimize the arrangement of PDMS micro pillars to make cells uniformly distributed in the device. Our preliminary data showed the microfluidic configuration in a circular shape with HCP patterned micro pillars turned 30 degrees offers the highest cell capture rate.

  20. D609 induces vascular endothelial cells and marrow stromal cells differentiation into neuron-like cells

    Institute of Scientific and Technical Information of China (English)

    Nan WANG; Chun-qing DU; Shao-shan WANG; Kun XIE; Shang-li ZHANG; Jun-ying MIAO

    2004-01-01

    AIM: To investigate the effect of tricyclodecane-9-yl-xanthogenate (D609) on cell differentiation in vascular endothelial cells (VECs) and marrow stromal cells (MSCs). METHODS: Morphological changes were observed under phase contrast microscope. Electron microscope and immunostaining were used for VECs identification. The expressions of neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) were examined by immunohistochemistry. RESULTS: After 6 h of induction with D609, some VECs showed morphological changes characteristic of neurones. 9 h later, more VECs became neuron-like cells. About 30.8 % of VECs displayed positive NSE (P<0.01), while the expression of GFAP was negative. When MSCs were exposed to D609, the cells displayed neuronal morphologies, such as pyramidal cell bodies and processes formed extensive networks at 3 h. 6 h later, almost all of the cells exhibited a typical neuronal appearance, and 85.6 % of MSCs displayed intensive positive NSE, but GFAP did not express. CONCLUSION: D609 induces VECs and MSCs differentiation into neuron-like cells.

  1. miRNA-720 controls stem cell phenotype, proliferation and differentiation of human dental pulp cells.

    Directory of Open Access Journals (Sweden)

    Emilio Satoshi Hara

    Full Text Available Dental pulp cells (DPCs are known to be enriched in stem/progenitor cells but not well characterized yet. Small non-coding microRNAs (miRNAs have been identified to control protein translation, mRNA stability and transcription, and have been reported to play important roles in stem cell biology, related to cell reprogramming, maintenance of stemness and regulation of cell differentiation. In order to characterize dental pulp stem/progenitor cells and its mechanism of differentiation, we herein sorted stem-cell-enriched side population (SP cells from human DPCs and periodontal ligament cells (PDLCs, and performed a locked nucleic acid (LNA-based miRNA array. As a result, miR-720 was highly expressed in the differentiated main population (MP cells compared to that in SP cells. In silico analysis and a reporter assay showed that miR-720 targets the stem cell marker NANOG, indicating that miR-720 could promote differentiation of dental pulp stem/progenitor cells by repressing NANOG. Indeed, gain-and loss-of-function analyses showed that miR-720 controls NANOG transcript and protein levels. Moreover, transfection of miR-720 significantly decreased the number of cells positive for the early stem cell marker SSEA-4. Concomitantly, mRNA levels of DNA methyltransferases (DNMTs, which are known to play crucial factors during stem cell differentiation, were also increased by miR-720 through unknown mechanism. Finally, miR-720 decreased DPC proliferation as determined by immunocytochemical analysis against ki-67, and promoted odontogenic differentiation as demonstrated by alizarin red staining, as well as alkaline phosphatase and osteopontin mRNA levels. Our findings identify miR-720 as a novel miRNA regulating the differentiation of DPCs.

  2. Increased differentiation of Th22 cells in Hashimoto's thyroiditis.

    Science.gov (United States)

    Bai, Xue; Sun, Jie; Wang, Weiwei; Shan, Zhongyan; Zheng, Hongzhi; Li, Yushu; Zhao, Yuhang; Gong, Ming; Teng, Weiping

    2014-01-01

    As Th22 subsets are identified, their involvement in the pathogenesis of numerous autoimmune diseases has become apparent. In this study, we investigated differentiation of Th22 cells in the autoimmune thyroid diseases including Hashimoto's thyroiditis (HT) and Graves' disease (GD). Besides, we also explored the involvement of Th22 cells in an iodine-induced autoimmune thyroiditis (AIT) model (i.e., NOD.H-2(h4) mice). In HT patients, we showed the level of circulating Th22 cells correlated with the level of serum IL-22, and was significantly higher than in GD patients and healthy control subjects. Levels of serum IL-6, a major Th22 cell differentiation effector, were also higher in HT, and correlated with Th22 cells concentration. Peripheral blood mononuclear cells isolated from HT patients produced larger amounts of IL-6 in vitro than did those isolated from other groups. Furthermore, unlike those from GD patients, T lymphocytes from HT patients showed an enhanced differentiation in vitro into Th22 cells in the presence of recombinant IL-6 and TNF-α. In addition, levels of circulating Th22 cells and titers of thyroid peroxidase antibody were positively correlated in HT patients. In NOD.H-2(h4) mice, higher numbers of Th22 cells were observed in the spleens of the AIT group, while splenocytes of this group also produced larger amounts of IL-6 and IL-22 in vitro compared with the control. Intra-thyroid infiltrating IL-22+ lymphocytes were significantly increased in mice of the AIT group compared with the control. Our results indicate that Th22 cells may contribute to the pathogenesis of HT.

  3. The Vast Universe of T Cell Diversity: Subsets of Memory Cells and Their Differentiation.

    Science.gov (United States)

    Jandus, Camilla; Usatorre, Amaia Martínez; Viganò, Selena; Zhang, Lianjun; Romero, Pedro

    2017-01-01

    The T cell receptor confers specificity for antigen recognition to T cells. By the first encounter with the cognate antigen, reactive T cells initiate a program of expansion and differentiation that will define not only the ultimate quantity of specific cells that will be generated, but more importantly their quality and functional heterogeneity. Recent achievements using mouse model infection systems have helped to shed light into the complex network of factors that dictate and sustain memory T cell differentiation, ranging from antigen load, TCR signal strength, metabolic fitness, transcriptional programs, and proliferative potential. The different models of memory T cell differentiation are discussed in this chapter, and key phenotypic and functional attributes of memory T cell subsets are presented, both for mouse and human cells. Therapeutic manipulation of memory T cell generation is expected to provide novel unique ways to optimize current immunotherapies, both in infection and cancer.

  4. Limbal Stromal Tissue Specific Stem Cells and Their Differentiation Potential to Corneal Epithelial Cells.

    Science.gov (United States)

    Katikireddy, Kishore Reddy; Jurkunas, Ula V

    2016-01-01

    From the derivation of the first human embryonic stem (hES) cell line to the development of induced pluripotent stem (iPS) cells; it has become evident that tissue specific stem cells are able to differentiate into a specific somatic cell types. The understanding of key processes such as the signaling pathways and the role of the microenvironment in epidermal/epithelial development has provided important clues for the derivation of specific epithelial cell types.Various differentiation protocols/methods were used to attain specific epithelial cell types. Here, we describe in detail the procedure to follow for isolation of tissue specific stem cells, mimicking their microenvironment to attain stem cell characteristics, and their potential differentiation to corneal epithelial cells.

  5. Lignin Induces ES Cells to Differentiate into Neuroectodermal Cells through Mediation of the Wnt Signaling Pathway.

    Directory of Open Access Journals (Sweden)

    Yu Inoue

    Full Text Available Embryonic stem cells (ES cells are characterized by their pluripotency and infinite proliferation potential. Ever since ES cells were first established in 1981, there have been a growing number of studies aimed at clinical applications of ES cells. In recent years, various types of differentiation inducement systems using ES cells have been established. Further studies have been conducted to utilize differentiation inducement systems in the field of regenerative medicine. For cellular treatments using stem cells including ES cells, differentiation induction should be performed in a sufficient manner to obtain the intended cell lineages. Lignin is a high-molecular amorphous material that forms plants together with cellulose and hemicelluloses, in which phenylpropane fundamental units are complexly condensed. Lignin derivatives have been shown to have several bioactive functions. In spite of these findings, few studies have focused on the effects of lignin on stem cells. Our study aimed to develop a novel technology using lignin to effectively induce ES cells to differentiate into neuroectodermal cells including ocular cells and neural cells. Since lignin can be produced at a relatively low cost in large volumes, its utilization is expected for more convenient differentiation induction technologies and in the field of regenerative medicine in the future.

  6. Differentiation of Human Cord Blood and Stromal Derived Stem Cells into Neuron Cells

    Directory of Open Access Journals (Sweden)

    Özlem Pamukçu Baran

    2007-01-01

    Full Text Available The most basic properties of stem cells are the capacities to self-renew indefinitely and to differentiate into multiple cell or tissue types. Umbilical cord blood has been utilized for human hematopoietic stem cell transplantation as an alternative source to bone marrow.The experiments show that Wharton’s jelly cells are easily attainable and can be expanded in vitro, maintained in culture, and induced to differentiate into neural cells. Almost recent studies it has been discovered that the cord blood-derived cells can differantiate not only to blood cells but also to various somatic cells like neuron or muscle cell with the signals taken from the envoirenment.Interestingly, neural cells obtained from umbilical cord blood show a relatively high spontaneous differentiation into oligodendrocytes, Embryonic stem cells proliferate indefinitely and can differentiate spontaneously into all tissue types.It has been shown that embryonic stem cells can be induced to differentiate into neurons and glia by treatment with retinoic acid or basic fibroblast growth factor. It has been studied that the diseases as Motor Neuron Disease, Parkinson, Alzheimer and degeneration of medulla spinalis and also paralysises could be treated with transplantation of cord blood-dericed stem cells.

  7. Induced Pluripotent Stem (iPS) Cell Culture Methods and Induction of Differentiation into Endothelial Cells

    Science.gov (United States)

    Chatterjee, Ishita; Li, Fei; Kohler, Erin E.; Rehman, Jalees; Malik, Asrar B.; Wary, Kishore K.

    2015-01-01

    Summary The studies of stem cell behavior and differentiation in a developmental context is complex, time-consuming and expensive, and for this reason, cell culture remains a method of choice for developmental and regenerative biology and mechanistic studies. Similar to ES cells, iPS cells have the ability to differentiate into endothelial cells (ECs), and the route for differentiation appears to mimic the developmental process that occurs during the formation of an embryo. Traditional EC induction methods from embryonic stem (ES) cells rely mostly on the formation the embryoid body (EB), which employs feeder or feeder-free conditions in the presence or absence of supporting cells. Similar to ES cells, iPS cells can be cultured in feeder-layer or feeder-free conditions. Here, we describe the iPS cell culture methods and induction differentiation of these cells into ECs. We use anti-mouse Flk1 and anti-mouse VE-cadherin to isolate and characterize mouse ECs, because these antibodies are commercially available and their use has been described in the literature, including by our group. The ECs produced by this method have been used by our laboratory, and we have demonstrated their in vivo potential. We also discuss how iPS cells differ in their ability to differentiate into endothelial cells in culture. PMID:25687301

  8. Induced Pluripotent Stem (iPS) Cell Culture Methods and Induction of Differentiation into Endothelial Cells.

    Science.gov (United States)

    Chatterjee, Ishita; Li, Fei; Kohler, Erin E; Rehman, Jalees; Malik, Asrar B; Wary, Kishore K

    2016-01-01

    The study of stem cell behavior and differentiation in a developmental context is complex, time-consuming, and expensive, and for this reason, cell culture remains a method of choice for developmental and regenerative biology and mechanistic studies. Similar to ES cells, iPS cells have the ability to differentiate into endothelial cells (ECs), and the route for differentiation appears to mimic the developmental process that occurs during the formation of an embryo. Traditional EC induction methods from embryonic stem (ES) cells rely mostly on the formation of embryoid body (EB), which employs feeder or feeder-free conditions in the presence or absence of supporting cells. Similar to ES cells, iPS cells can be cultured in feeder layer or feeder-free conditions. Here, we describe the iPS cell culture methods and induction differentiation of these cells into ECs. We use anti-mouse Flk1 and anti-mouse VE-cadherin to isolate and characterize mouse ECs, because these antibodies are commercially available and their use has been described in the literature, including by our group. The ECs produced by this method have been used by our laboratory, and we have demonstrated their in vivo potential. We also discuss how iPS cells differ in their ability to differentiate into endothelial cells in culture.

  9. Evaluation of a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay (SOT)

    Science.gov (United States)

    The Embryonic Stem Cell Test (EST) has been used to evaluate the effects of xenobiotics using three endpoints, stem cell differentiation, stem cell viability and 3T3-cell viability. Our research goal is to establish amodel system that would evaluate chemical effects using a singl...

  10. Differentiation and functional regulation of human fetal NK cells.

    Science.gov (United States)

    Ivarsson, Martin A; Loh, Liyen; Marquardt, Nicole; Kekäläinen, Eliisa; Berglin, Lena; Björkström, Niklas K; Westgren, Magnus; Nixon, Douglas F; Michaëlsson, Jakob

    2013-09-01

    The human fetal immune system is naturally exposed to maternal allogeneic cells, maternal antibodies, and pathogens. As such, it is faced with a considerable challenge with respect to the balance between immune reactivity and tolerance. Here, we show that fetal natural killer (NK) cells differentiate early in utero and are highly responsive to cytokines and antibody-mediated stimulation but respond poorly to HLA class I-negative target cells. Strikingly, expression of killer-cell immunoglobulin-like receptors (KIRs) did not educate fetal NK cells but rendered them hyporesponsive to target cells lacking HLA class I. In addition, fetal NK cells were highly susceptible to TGF-β-mediated suppression, and blocking of TGF-β signaling enhanced fetal NK cell responses to target cells. Our data demonstrate that KIR-mediated hyporesponsiveness and TGF-β-mediated suppression are major factors determining human fetal NK cell hyporesponsiveness to HLA class I-negative target cells and provide a potential mechanism for fetal-maternal tolerance in utero. Finally, our results provide a basis for understanding the role of fetal NK cells in pregnancy complications in which NK cells could be involved, for example, during in utero infections and anti-RhD-induced fetal anemia.

  11. Chemo-mechanical control of neural stem cell differentiation

    Science.gov (United States)

    Geishecker, Emily R.

    Cellular processes such as adhesion, proliferation, and differentiation are controlled in part by cell interactions with the microenvironment. Cells can sense and respond to a variety of stimuli, including soluble and insoluble factors (such as proteins and small molecules) and externally applied mechanical stresses. Mechanical properties of the environment, such as substrate stiffness, have also been suggested to play an important role in cell processes. The roles of both biochemical and mechanical signaling in fate modification of stem cells have been explored independently. However, very few studies have been performed to study well-controlled chemo-mechanotransduction. The objective of this work is to design, synthesize, and characterize a chemo-mechanical substrate to encourage neuronal differentiation of C17.2 neural stem cells. In Chapter 2, Polyacrylamide (PA) gels of varying stiffnesses are functionalized with differing amounts of whole collagen to investigate the role of protein concentration in combination with substrate stiffness. As expected, neurons on the softest substrate were more in number and neuronal morphology than those on stiffer substrates. Neurons appeared locally aligned with an expansive network of neurites. Additional experiments would allow for statistical analysis to determine if and how collagen density impacts C17.2 differentiation in combination with substrate stiffness. Due to difficulties associated with whole protein approaches, a similar platform was developed using mixed adhesive peptides, derived from fibronectin and laminin, and is presented in Chapter 3. The matrix elasticity and peptide concentration can be individually modulated to systematically probe the effects of chemo-mechanical signaling on differentiation of C17.2 cells. Polyacrylamide gel stiffness was confirmed using rheological techniques and found to support values published by Yeung et al. [1]. Cellular growth and differentiation were assessed by cell counts

  12. Bladder Smooth Muscle Cells Differentiation from Dental Pulp Stem Cells: Future Potential for Bladder Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Bing Song

    2016-01-01

    Full Text Available Dental pulp stem cells (DPSCs are multipotent cells capable of differentiating into multiple cell lines, thus providing an alternative source of cell for tissue engineering. Smooth muscle cell (SMC regeneration is a crucial step in tissue engineering of the urinary bladder. It is known that DPSCs have the potential to differentiate into a smooth muscle phenotype in vitro with differentiation agents. However, most of these studies are focused on the vascular SMCs. The optimal approaches to induce human DPSCs to differentiate into bladder SMCs are still under investigation. We demonstrate in this study the ability of human DPSCs to differentiate into bladder SMCs in a growth environment containing bladder SMCs-conditioned medium with the addition of the transforming growth factor beta 1 (TGF-β1. After 14 days of exposure to this medium, the gene and protein expression of SMC-specific marker (α-SMA, desmin, and calponin increased over time. In particular, myosin was present in differentiated cells after 11 days of induction, which indicated that the cells differentiated into the mature SMCs. These data suggested that human DPSCs could be used as an alternative and less invasive source of stem cells for smooth muscle regeneration, a technology that has applications for bladder tissue engineering.

  13. Natural Killer Cells Differentiate Human Adipose-Derived Stem Cells and Modulate Their Adipogenic Potential.

    Science.gov (United States)

    Rezzadeh, Kameron S; Hokugo, Akishige; Jewett, Anahid; Kozlowska, Anna; Segovia, Luis Andres; Zuk, Patricia; Jarrahy, Reza

    2015-09-01

    Natural killer cells are thought to represent more than 30 percent of all lymphocytes within the stromal vascular fraction of lipoaspirates. However, their physiologic interaction with adipocytes and their precursors has never been specifically examined. The authors hypothesized that natural killer cells, by means of cytokine secretion, are capable of promoting the differentiation of adipose-derived stem cells. Human natural killer cells purified from healthy donors' peripheral blood mononuclear cells were activated with a combination of interleukin-2 and anti-CD16 monoclonal antibody; natural killer cell supernatant was collected. Adipose-derived stem cells isolated from raw human lipoaspirates from healthy patients were treated with growth media, growth media with natural killer cell supernatant, adipogenic media, and adipogenic media with natural killer cells supernatant. Flow cytometric analysis was performed on cells using antibodies against B7H1, CD36, CD44, CD34, CD29, and MHC-1. Adipogenic-related gene expression (PPAR-γ, LPL, GPD-1, and aP2) was assessed. Oil Red O staining was performed as a functional assay of adipocyte differentiation and adipogenesis. Adipose-derived stem cells maintained in growth media with natural killer cell supernatant lost markers of "stemness," including CD44, CD34, and CD29; and expressed markers of differentiation, including B7H1 and MHC-1. Adipose-derived stem cells treated with natural killer cell supernatant accumulated small amounts of lipid after 10 days of natural killer cell supernatant treatment. Adipose-derived stem cells treated with natural killer cell supernatant showed altered expression of adipogenesis-associated genes compared with cells maintained in growth media. Adipose-derived stem cells maintained in adipogenic media with natural killer cell supernatant accumulated less lipid than those cells in adipogenic media alone. The authors demonstrate that, through secreted factors, natural killer cells are capable

  14. Spatial coordination between stem cell activity and cell differentiation in the root meristem.

    Science.gov (United States)

    Moubayidin, Laila; Di Mambro, Riccardo; Sozzani, Rosangela; Pacifici, Elena; Salvi, Elena; Terpstra, Inez; Bao, Dongping; van Dijken, Anja; Dello Ioio, Raffaele; Perilli, Serena; Ljung, Karin; Benfey, Philip N; Heidstra, Renze; Costantino, Paolo; Sabatini, Sabrina

    2013-08-26

    A critical issue in development is the coordination of the activity of stem cell niches with differentiation of their progeny to ensure coherent organ growth. In the plant root, these processes take place at opposite ends of the meristem and must be coordinated with each other at a distance. Here, we show that in Arabidopsis, the gene SCR presides over this spatial coordination. In the organizing center of the root stem cell niche, SCR directly represses the expression of the cytokinin-response transcription factor ARR1, which promotes cell differentiation, controlling auxin production via the ASB1 gene and sustaining stem cell activity. This allows SCR to regulate, via auxin, the level of ARR1 expression in the transition zone where the stem cell progeny leaves the meristem, thus controlling the rate of differentiation. In this way, SCR simultaneously controls stem cell division and differentiation, ensuring coherent root growth. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Differential regulation of the histone chaperone HIRA during muscle cell differentiation by a phosphorylation switch.

    Science.gov (United States)

    Yang, Jae-Hyun; Song, Tae-Yang; Jo, Chanhee; Park, Jinyoung; Lee, Han-Young; Song, Ilang; Hong, Suji; Jung, Kwan Young; Kim, Jaehoon; Han, Jeung-Whan; Youn, Hong-Duk; Cho, Eun-Jung

    2016-08-12

    Replication-independent incorporation of variant histone H3.3 has a profound impact on chromatin function and numerous cellular processes, including the differentiation of muscle cells. The histone chaperone HIRA and H3.3 have essential roles in MyoD regulation during myoblast differentiation. However, the precise mechanism that determines the onset of H3.3 deposition in response to differentiation signals is unclear. Here we show that HIRA is phosphorylated by Akt kinase, an important signaling modulator in muscle cells. By generating a phosphospecific antibody, we found that a significant amount of HIRA was phosphorylated in myoblasts. The phosphorylation level of HIRA and the occupancy of phosphorylated protein on muscle genes gradually decreased during cellular differentiation. Remarkably, the forced expression of the phosphomimic form of HIRA resulted in reduced H3.3 deposition and suppressed the activation of muscle genes in myotubes. Our data show that HIRA phosphorylation limits the expression of myogenic genes, while the dephosphorylation of HIRA is required for proficient H3.3 deposition and gene activation, demonstrating that the phosphorylation switch is exploited to modulate HIRA/H3.3-mediated muscle gene regulation during myogenesis.

  16. Large-scale differential display analysis of T helper cell differentiation.

    Science.gov (United States)

    Ojala, Pekka; Virtanen, Eveliina; Chen, Zhi

    2007-03-01

    We have developed a novel large-scale multicapillary fluorescent differential display (FDD) platform amenable to further automation. The power of the method is demonstrated by the analysis of T helper cell differentiation. Eight RNA samples from wild type, Stat4 knockout and Stat6 knockout mice were analyzed with 16 anchoring primers and 24 arbitrary primers, resulting in 285 294 sample peaks. Visually selected patterns of differential expression suggest two major regulatory mechanisms: activation and Stat4 genotype. A subset of the findings is reproduced in the confirmatory differential display (DD) that included technical and biological replicates. In a small fragment identification pilot study, we identify Ifi27 and Cct8 to be up-regulated by T cell activation. We present a method for the analysis of electropherogram similarity across large datasets, based on correlation of low-resolution representations of electrophoretic data. We show how it can be applied to analyze experimental and technical variables. Using this method, we demonstrate the effect of activation and genotype. In addition, agreement of our real experimental data to the theoretical basis of DD, as well as issues in anchoring primer selectivity, are studied.

  17. Photoresist Derived Carbon for Growth and Differentiation of Neuronal Cells

    Directory of Open Access Journals (Sweden)

    Tie Zou

    2007-08-01

    Full Text Available Apoptosis or necrosis of neurons in the central nervous system (CNS is thehallmark of many neurodegenerative diseases and Traumatic Brain Injury (TBI. Theinability to regenerate in CNS offers little hope for naturally repairing the damagedneurons. However, with the rapid development of new technologies, regenerative medicineoffers great promises to patients with these disorders. Among many events for furtheradvancement of regenerative medicine, extracellular matrix (ECM plays a critical role forcellular migration and differentiation. To develop a biocompatible and electricallyconductive substrate that can be potentially used to promote growth and regeneration ofneurons and to record intracellular and multisite signals from brain as a probe, a polymericprecursor – SPR 220.7 was fabricated by pyrolysis at temperatures higher than 700 oC.Human Neuroblastoma cells - SK-N-MC, SY5Y, mouse teratocarcinoma cells P-19 and ratPC12 cells were found to attach and proliferate on photoresist derived carbon film.Significantly, neuronal differentiation of PC12 cells induced by NGF was demonstrated byobserving cell shape and size, and measuring the length of neurites under SEM. Our resultsindicated that fabricated carbon could potentially be explored in regenerative medicine forpromoting neuronal growth and differentiation in CNS with neurodegeneration.

  18. Biological characteristics of breast carcinomas with neuroendocrine cell differentiation

    Institute of Scientific and Technical Information of China (English)

    姚根有; 周吉林; 赵仲生; 阮俊

    2004-01-01

    Background The aim of this study was to investigate DNA content and expression of c-erbB-2, PS2, and prostate-specific antigen (PSA) proteins in breast carcinomas with neuroendocrine (NE) cell differentiation.Methods Chromogranin, c-erbB-2, PS2, and PSA in 131 samples of breast cancer were detected immunohistochemically. Classic Feulgen staining image analysis techniques were used to quantify DNA content in 81 of the breast cancer samples.Results The c-erbB-2 positive rate in breast carcinoma samples containing neuroendocrine cells was 37.5% and the rate of high expression of c-erbB-2 (++ or +++) was 33.3%, both significantly lower than that in breast carcinomas without neuroendocrine cells (62.6% and 68.7%, respectively, P 5c aneuploidy cells, and rate of aneuploidy among cells were all lower than that in NE (-) breast carcinomas (P<0.01). In NE (+) grade I or II breast carcinomas, these indices were also all lower than that in the NE (-) breast carcinoma samples (P<0.01).Conclusion Breast carcinomas with neuroendocrine differentiation have a lower rate of malignancy. Neuroendocrine differentiation could serve as a prognostic marker in clinical practice.

  19. FTIR Spectroscopic and Molecular Analysis during Differentiation of Pluripotent Stem Cells to Pancreatic Cells.

    Science.gov (United States)

    Vazquez-Zapien, Gustavo Jesus; Mata-Miranda, Monica Maribel; Sanchez-Monroy, Virginia; Delgado-Macuil, Raul Jacobo; Perez-Ishiwara, David Guillermo; Rojas-Lopez, Marlon

    2016-01-01

    Some of the greatest challenges in stem cells (SCs) biology and regenerative medicine are differentiation control of SCs and ensuring the purity of differentiated cells. In this work, we differentiated mouse pluripotent stem cells (mPSCs) toward pancreatic cells characterizing this differentiation process by molecular and spectroscopic technics. Both mPSCs and Differentiated Pancreatic Cells (DPCs) were subjected to a genetic, phenotypic, and biochemical analysis by real-time quantitative PCR (RT-qPCR), immunocytochemistry, and Fourier Transform Infrared (FTIR) spectroscopy. Cultured mPCSs expressed pluripotent genes and proteins (Nanog and SOX2). DPCs expressed endodermal genes (SOX17 and Pdx1) at day 11, an inductor gene of embryonic pancreas development (Pdx1) at day 17 and pancreas genes and proteins (Insulin and Glucagon) at day 21 of differentiation. Likewise, FTIR spectra of mPSCs and DPCs at different maturation stages (11, 17, and 21 days) were obtained and showed absorption bands related with different types of biomolecules. These FTIR spectra exhibited significant spectral changes agreeing with the differentiation process, particularly in proteins and nucleic acids bands. In conclusion, the obtained DPCs passed through the chronological stages of embryonic pancreas development and FTIR spectra provide a new biophysical parameter based on molecular markers indicating the differentiation process of mPSCs to specialized cells.

  20. FTIR Spectroscopic and Molecular Analysis during Differentiation of Pluripotent Stem Cells to Pancreatic Cells

    Directory of Open Access Journals (Sweden)

    Gustavo Jesus Vazquez-Zapien

    2016-01-01

    Full Text Available Some of the greatest challenges in stem cells (SCs biology and regenerative medicine are differentiation control of SCs and ensuring the purity of differentiated cells. In this work, we differentiated mouse pluripotent stem cells (mPSCs toward pancreatic cells characterizing this differentiation process by molecular and spectroscopic technics. Both mPSCs and Differentiated Pancreatic Cells (DPCs were subjected to a genetic, phenotypic, and biochemical analysis by real-time quantitative PCR (RT-qPCR, immunocytochemistry, and Fourier Transform Infrared (FTIR spectroscopy. Cultured mPCSs expressed pluripotent genes and proteins (Nanog and SOX2. DPCs expressed endodermal genes (SOX17 and Pdx1 at day 11, an inductor gene of embryonic pancreas development (Pdx1 at day 17 and pancreas genes and proteins (Insulin and Glucagon at day 21 of differentiation. Likewise, FTIR spectra of mPSCs and DPCs at different maturation stages (11, 17, and 21 days were obtained and showed absorption bands related with different types of biomolecules. These FTIR spectra exhibited significant spectral changes agreeing with the differentiation process, particularly in proteins and nucleic acids bands. In conclusion, the obtained DPCs passed through the chronological stages of embryonic pancreas development and FTIR spectra provide a new biophysical parameter based on molecular markers indicating the differentiation process of mPSCs to specialized cells.

  1. FTIR Spectroscopic and Molecular Analysis during Differentiation of Pluripotent Stem Cells to Pancreatic Cells

    Science.gov (United States)

    Mata-Miranda, Monica Maribel; Sanchez-Monroy, Virginia; Delgado-Macuil, Raul Jacobo; Perez-Ishiwara, David Guillermo

    2016-01-01

    Some of the greatest challenges in stem cells (SCs) biology and regenerative medicine are differentiation control of SCs and ensuring the purity of differentiated cells. In this work, we differentiated mouse pluripotent stem cells (mPSCs) toward pancreatic cells characterizing this differentiation process by molecular and spectroscopic technics. Both mPSCs and Differentiated Pancreatic Cells (DPCs) were subjected to a genetic, phenotypic, and biochemical analysis by real-time quantitative PCR (RT-qPCR), immunocytochemistry, and Fourier Transform Infrared (FTIR) spectroscopy. Cultured mPCSs expressed pluripotent genes and proteins (Nanog and SOX2). DPCs expressed endodermal genes (SOX17 and Pdx1) at day 11, an inductor gene of embryonic pancreas development (Pdx1) at day 17 and pancreas genes and proteins (Insulin and Glucagon) at day 21 of differentiation. Likewise, FTIR spectra of mPSCs and DPCs at different maturation stages (11, 17, and 21 days) were obtained and showed absorption bands related with different types of biomolecules. These FTIR spectra exhibited significant spectral changes agreeing with the differentiation process, particularly in proteins and nucleic acids bands. In conclusion, the obtained DPCs passed through the chronological stages of embryonic pancreas development and FTIR spectra provide a new biophysical parameter based on molecular markers indicating the differentiation process of mPSCs to specialized cells. PMID:27651798

  2. Pre-Meiotic Anther Development: Cell Fate Specification and Differentiation.

    Science.gov (United States)

    Walbot, Virginia; Egger, Rachel L

    2016-04-29

    Research into anther ontogeny has been an active and developing field, transitioning from a strictly lineage-based view of cellular differentiation events to a more complex understanding of cell fate specification. Here we describe the modern interpretation of pre-meiotic anther development, from the earliest cell specifications within the anther lobes through SPL/NZZ-, MSP1-, and MEL1-dependent pathways as well as the initial setup of the abaxial and adaxial axes and outgrowth of the anther lobes. We then continue with a look at the known information regarding further differentiation of the somatic layers of the anther (the epidermis, endothecium, middle layer, and tapetum), with an emphasis on male-sterile mutants identified as defective in somatic cell specification. We also describe the differences in developmental stages among species and use this information to discuss molecular studies that have analyzed transcriptome, proteome, and small-RNA information in the anther.

  3. The AP-1 transcription factor Fra1 inhibits follicular B cell differentiation into plasma cells.

    Science.gov (United States)

    Grötsch, Bettina; Brachs, Sebastian; Lang, Christiane; Luther, Julia; Derer, Anja; Schlötzer-Schrehardt, Ursula; Bozec, Aline; Fillatreau, Simon; Berberich, Ingolf; Hobeika, Elias; Reth, Michael; Wagner, Erwin F; Schett, Georg; Mielenz, Dirk; David, Jean-Pierre

    2014-10-20

    The cornerstone of humoral immunity is the differentiation of B cells into antibody-secreting plasma cells. This process is tightly controlled by a regulatory gene network centered on the transcriptional repressor B lymphocyte-induced maturation protein 1 (Blimp1). Proliferation of activated B cells is required to foster Blimp1 expression but needs to be terminated to avoid overshooting immune reactions. Activator protein 1 (AP-1) transcription factors become quickly up-regulated upon B cell activation. We demonstrate that Fra1, a Fos member of AP-1, enhances activation-induced cell death upon induction in activated B cells. Moreover, mice with B cell-specific deletion of Fra1 show enhanced plasma cell differentiation and exacerbated antibody responses. In contrast, transgenic overexpression of Fra1 blocks plasma cell differentiation and immunoglobulin production, which cannot be rescued by Bcl2. On the molecular level, Fra1 represses Blimp1 expression and interferes with binding of the activating AP-1 member c-Fos to the Blimp1 promoter. Conversely, overexpression of c-Fos in Fra1 transgenic B cells releases Blimp1 repression. As Fra1 lacks transcriptional transactivation domains, we propose that Fra1 inhibits Blimp1 expression and negatively controls plasma cell differentiation through binding to the Blimp1 promoter. In summary, we demonstrate that Fra1 negatively controls plasma cell differentiation by repressing Blimp1 expression.

  4. Poorly Differentiated Squamous Cell Carcinoma Arising in Tattooed Skin

    Directory of Open Access Journals (Sweden)

    Deba P. Sarma

    2010-01-01

    Full Text Available Introduction. Tattoos have increasingly become accepted by mainstream Western society. As a result, the incidence of tattoo-associated dermatoses is on the rise. The presence of a poorly differentiated squamous cell carcinoma in an old tattooed skin is of interest as it has not been previously documented. Case Presentation. A 79-year-old white homeless man of European descent presented to the dermatology clinic with a painless raised nodule on his left forearm arising in a tattooed area. A biopsy of the lesion revealed a poorly differentiated squamous cell carcinoma infiltrating into a tattoo. The lesion was completely excised and the patient remains disease-free one year later. Conclusion. All previous reports of squamous cell carcinomas arising in tattoos have been well-differentiated low-grade type or keratoacanthoma-type and are considered to be coincidental rather than related to any carcinogenic effect of the tattoo pigments. Tattoo-associated poorly differentiated invasive carcinoma appears to be extremely rare.

  5. Poorly differentiated squamous cell carcinoma arising in tattooed skin.

    Science.gov (United States)

    Sarma, Deba P; Dentlinger, Renee B; Forystek, Amanda M; Stevens, Todd; Huerter, Christopher

    2010-01-01

    Introduction. Tattoos have increasingly become accepted by mainstream Western society. As a result, the incidence of tattoo-associated dermatoses is on the rise. The presence of a poorly differentiated squamous cell carcinoma in an old tattooed skin is of interest as it has not been previously documented. Case Presentation. A 79-year-old white homeless man of European descent presented to the dermatology clinic with a painless raised nodule on his left forearm arising in a tattooed area. A biopsy of the lesion revealed a poorly differentiated squamous cell carcinoma infiltrating into a tattoo. The lesion was completely excised and the patient remains disease-free one year later. Conclusion. All previous reports of squamous cell carcinomas arising in tattoos have been well-differentiated low-grade type or keratoacanthoma-type and are considered to be coincidental rather than related to any carcinogenic effect of the tattoo pigments. Tattoo-associated poorly differentiated invasive carcinoma appears to be extremely rare.

  6. Organ-derived dendritic cells have differential effects on alloreactive T cells

    OpenAIRE

    Kim, Theo D.; Terwey, Theis H.; Zakrzewski, Johannes L; Suh, David; Kochman, Adam A.; Chen, Megan E.; King, Chris G.; Borsotti, Chiara; Grubin, Jeremy; Smith, Odette M.; Heller, Glenn; Liu, Chen; Murphy, George F.; Alpdogan, Onder; Marcel R. M. van den Brink

    2008-01-01

    Dendritic cells (DCs) are considered critical for the induction of graft-versus-host disease (GVHD) after bone marrow transplantation (BMT). In addition to their priming function, dendritic cells have been shown to induce organ-tropism through induction of specific homing molecules on T cells. Using adoptive transfer of CFSE-labeled cells, we first demonstrated that alloreactive T cells differentially up-regulate specific homing molecules in vivo. Host-type dendritic cells from the GVHD targe...

  7. Induced Differentiation of Adipose-derived Stromal Cells into Myoblasts

    Institute of Scientific and Technical Information of China (English)

    吴桂珠; 郑秀; 江忠清; 王金华; 宋岩峰

    2010-01-01

    This study aimed to induce the differentiation of isolated and purified adipose-derived stromal cells(ADSCs) into myoblasts,which may provide a new strategy for tissue engineering in patients with stress urinary incontinence(SUI).ADSCs,isolated and cultured ex vivo,were identified by flow cytometry and induced to differentiate into myoblasts in the presence of an induction solution consisting of DMEM supplemented with 5-azacytidine(5-aza),5% FBS,and 5% horse serum.Cellular morphology was observed under an i...

  8. Stem cells in light of a new concept for cell differentiation.

    Science.gov (United States)

    Kristeva, Marlene Anastassova

    2008-10-01

    My concept of cell differentiation involves genetic information from DNA being transcribed into mRNA proteins-morphogenes (mRNAs+ homeodomain proteins)-and stored in the ovoplasm as maternal inheritance, or cytoplasmic genetic memory. Feedback mechanism(s) allow these morphogenes to selectively unlock new genes, regulating the development of the embryo. The blastomeres and the embryonic pluripotent cells of the inner cell mass of early (5 day) blastocysts are loaded with morphogenes which hamper the production of cell lines and are responsible for the formation of embryoid bodies in vitro and teratomas in vivo. There are therefore legitimate concerns as to proposals to use embryonic pluripotent cells for cell therapy and regenerative medicine. An alternative cell therapy would involve the production of tailored growth-related genes-morphogenes-and hence selective in vitro differentiation of adult de-differentiated cells.

  9. Endothelial cell high-enrichment from endovascular biopsy sample by laser capture microdissection and fluorescence activated cell sorting.

    Science.gov (United States)

    Sun, Zhengda; Su, Hua; Long, Brian; Sinclair, Elizabeth; Hetts, Steven W; Higashida, Randall T; Dowd, Christopher F; Halbach, Van V; Cooke, Daniel L

    2014-12-20

    Endovascular sampling and characterization from patients can provide very useful information about the pathogenesis of different vascular diseases, but it has been limited by the lack of an effective method of endothelial cell (EC) enrichment. We optimized the EC yield and enrichment from conventional guide wires by laser capture microdissection (LCM) and fluorescence activated cell sorting (FACS) technique, and addressed the feasibility of using these enriched ECs for downstream gene expression detection. Iliac artery endovascular samples from 10 patients undergoing routine catheter angiography were collected using conventional 0.038 in. J-shape guide wires. Each of these samples was equally divided into two parts, which were respectively used for EC enrichment by immunocytochemistry-coupled LCM or multiple color FACS. After RNA extraction and reverse transcription, the amplified cDNA was used for quantitative polymerase chain reaction (qPCR). Fixed ECs, with positive CD31 or vWF fluorescent signal and endothelial like nucleus, were successfully separated by LCM and live single ECs were sorted on FACS by a seven color staining panel. EC yields by LCM and FACS were 51 ± 22 and 149 ± 56 respectively (P < 0.001). The minimum number of fixed ECs from ICC-coupled LCM for acceptable qPCR results of endothelial marker genes was 30, while acceptable qPCR results as enriched by FACS were attainable from a single live EC. Both LCM and FACS can be used to enrich ECs from conventional guide wires and the enriched ECs can be used for downstream gene expression detection. FACS generated a higher EC yield and the sorted live ECs may be used for single cell gene expression detection.

  10. Endothelial cell high-enrichment from endovascular biopsy sample by laser capture microdissection and fluorescence activated cell sorting

    Science.gov (United States)

    Sun, Zhengda; Su, Hua; Long, Brian; Sinclair, Elizabeth; Hetts, Steven W.; Higashida, Randall T.; Dowd, Christopher F.; Halbach, Van V.; Cooke, Daniel L.

    2015-01-01

    Background and purpose Endovascular sampling and characterization from patients can provide very useful information about the pathogenesis of different vascular diseases, but it has been limited by the lack of an effective method of endothelial cell (EC) enrichment. We optimized the EC yield and enrichment from conventional guide wires by laser capture microdissection (LCM) and fluorescence activated cell sorting (FACS) technique, and addressed the feasibility of using these enriched ECs for downstream gene expression detection. Methods Iliac artery endovascular samples from 10 patients undergoing routine catheter angiography were collected using conventional 0.038 in. J-shape guide wires. Each of these samples was equally divided into two parts, which were respectively used for EC enrichment by immunocytochemistry-coupled LCM or multiple color FACS. After RNA extraction and reverse transcription, the amplified cDNA was used for quantitative polymerase chain reaction (qPCR). Results Fixed ECs, with positive CD31 or vWF fluorescent signal and endothelial like nucleus, were successfully separated by LCM and live single ECs were sorted on FACS by a seven color staining panel. EC yields by LCM and FACS were 51 ± 22 and 149 ± 56 respectively (P < 0.001). The minimum number of fixed ECs from ICC-coupled LCM for acceptable qPCR results of endothelial marker genes was 30, while acceptable qPCR results as enriched by FACS were attainable from a single live EC. Conclusion Both LCM and FACS can be used to enrich ECs from conventional guide wires and the enriched ECs can be used for downstream gene expression detection. FACS generated a higher EC yield and the sorted live ECs may be used for single cell gene expression detection. PMID:25450638

  11. Efficient Differentiation of Mouse Embryonic Stem Cells into Insulin-Producing Cells

    Directory of Open Access Journals (Sweden)

    Szu-Hsiu Liu

    2012-01-01

    Full Text Available Embryonic stem (ES cells are a potential source of a variety of differentiated cells for cell therapy, drug discovery, and toxicology screening. Here, we present an efficacy strategy for the differentiation of mouse ES cells into insulin-producing cells (IPCs by a two-step differentiation protocol comprising of (i the formation of definitive endoderm in monolayer culture by activin A, and (ii this monolayer endoderm being induced to differentiate into IPCs by nicotinamide, insulin, and laminin. Differentiated cells can be obtained within approximately 7 days. The differentiation IPCs combined application of RT-PCR, ELISA, and immunofluorescence to characterize phenotypic and functional properties. In our study, we demonstrated that IPCs produced pancreatic transcription factors, endocrine progenitor marker, definitive endoderm, pancreatic β-cell markers, and Langerhans α and δ cells. The IPCs released insulin in a manner that was dose dependent upon the amount of glucose added. These techniques may be able to be applied to human ES cells, which would have very important ramifications for treating human disease.

  12. Myoepithelial cell differentiation markers in ductal carcinoma in situ progression.

    Science.gov (United States)

    Russell, Tanya D; Jindal, Sonali; Agunbiade, Samiat; Gao, Dexiang; Troxell, Megan; Borges, Virginia F; Schedin, Pepper

    2015-11-01

    We describe a preclinical model that investigates progression of early-stage ductal carcinoma in situ (DCIS) and report that compromised myoepithelial cell differentiation occurs before transition to invasive disease. Human breast cancer MCF10DCIS.com cells were delivered into the mouse mammary teat by intraductal injection in the absence of surgical manipulations and accompanying wound-healing confounders. DCIS-like lesions developed throughout the mammary ducts with full representation of human DCIS histologic patterns. Tumor cells were incorporated into the normal mammary epithelium, developed ductal intraepithelial neoplasia and DCIS, and progressed to invasive carcinoma, suggesting the model provides a rigorous approach to study early stages of breast cancer progression. Mammary glands were evaluated for myoepithelium integrity with immunohistochemical assays. Progressive loss of the myoepithelial cell differentiation markers p63, calponin, and α-smooth muscle actin was observed in the mouse myoepithelium surrounding DCIS-involved ducts. p63 loss was an early indicator, calponin loss intermediate, and α-smooth muscle actin a later indicator of compromised myoepithelium. Loss of myoepithelial calponin was specifically associated with gain of the basal marker p63 in adjacent tumor cells. In single time point biopsies obtained from 16 women diagnosed with pure DCIS, a similar loss in myoepithelial cell markers was observed. These results suggest that further research is warranted into the role of myoepithelial cell p63 and calponin expression on DCIS progression to invasive disease.

  13. Senescence from glioma stem cell differentiation promotes tumor growth

    Energy Technology Data Exchange (ETDEWEB)

    Ouchi, Rie [Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550 (Japan); Laboratory of Molecular Target Therapy of Cancer, Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550 (Japan); Okabe, Sachiko; Migita, Toshiro [Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550 (Japan); Nakano, Ichiro [Department of Neurosurgery, Comprehensive Cancer Center, University of Alabama at Birmingham, 1824 6th Avenue South, Birmingham, AL 35233 (United States); Seimiya, Hiroyuki, E-mail: hseimiya@jfcr.or.jp [Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550 (Japan); Laboratory of Molecular Target Therapy of Cancer, Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550 (Japan)

    2016-02-05

    Glioblastoma (GBM) is a lethal brain tumor composed of heterogeneous cellular populations including glioma stem cells (GSCs) and differentiated non-stem glioma cells (NSGCs). While GSCs are involved in tumor initiation and propagation, NSGCs' role remains elusive. Here, we demonstrate that NSGCs undergo senescence and secrete pro-angiogenic proteins, boosting the GSC-derived tumor formation in vivo. We used a GSC model that maintains stemness in neurospheres, but loses the stemness and differentiates into NSGCs upon serum stimulation. These NSGCs downregulated telomerase, shortened telomeres, and eventually became senescent. The senescent NSGCs released pro-angiogenic proteins, including vascular endothelial growth factors and senescence-associated interleukins, such as IL-6 and IL-8. Conditioned medium from senescent NSGCs promoted proliferation of brain microvascular endothelial cells, and mixed implantation of GSCs and senescent NSGCs into mice enhanced the tumorigenic potential of GSCs. The senescent NSGCs seem to be clinically relevant, because both clinical samples and xenografts of GBM contained tumor cells that expressed the senescence markers. Our data suggest that senescent NSGCs promote malignant progression of GBM in part via paracrine effects of the secreted proteins. - Highlights: • Non-stem glioma cells (NSGCs) lose telomerase and eventually become senescent. • Senescent NSGCs secrete pro-angiogenic proteins, such as VEGFs, IL-6, and IL-8. • Senescent NSGCs enhance the growth of brain microvascular endothelial cells. • Senescent NSGCs enhance the tumorigenic potential of glioma stem cells in vivo.

  14. Cell fate and cell differentiation status in the Arabidopsis root

    NARCIS (Netherlands)

    Scheres, B.J.G.; Berg, C. van den; Weisbeek, P.

    1998-01-01

    Post-embryonic development in plants is mainly achieved by its meristems. Within the Arabidopsis root meristem, both the fate and origin of its cells can be predicted with high accuracy. Mutants defective in the determination of root cell fates show that the corresponding genes are first required

  15. Interleukin 4: signalling mechanisms and control of T cell differentiation.

    Science.gov (United States)

    Paul, W E

    1997-01-01

    Interleukin 4 (IL-4) is a pleiotropic type I cytokine that controls both growth and differentiation among haemopoietic and non-haemopoietic cells. Its receptor is a heterodimer. One chain, the IL-4R alpha chain, binds IL-4 with high affinity and determines the nature of the biochemical signals that are induced. The second chain, gamma c, is required for the induction of such signals. IL-4-mediated growth depends upon activation events that involve phosphorylation of Y497 of IL-4R alpha, leading to the binding and phosphorylation of 4PS/IRS-2 in haemopoietic cells and of IRS-1 in non-haemopoietic cells. By contrast, IL-4-mediated differentiation events depend upon more distal regions of the IL-4R alpha chain that include a series of STAT-6 binding sites. The distinctive roles of these receptor domains was verified by receptor-reconstruction experiments. The 'growth' and 'differentiation' domains of the IL-4R alpha chain, independently expressed as chimeric structures with a truncated version of the IL-2R beta chain, were shown to convey their functions to the hybrid receptor. The critical role of STAT-6 in IL-4-mediated gene activation and differentiation was made clear by the finding that lymphocytes from STAT-6 knockout mice are strikingly deficient in these functions but have retained the capacity to grow, at least partially, in response to IL-4. IL-4 plays a central role in determining the phenotype of naive CD4+ T cells. In the presence of IL-4, newly primed naive T cells develop into IL-4 producers while in its absence they preferentially become gamma-interferon (IFN-gamma) producers. Recently, a specialized subpopulation of T cells, CD4+/NK1.1+ cells, has been shown to produce large amounts of IL-4 upon stimulation. Two examples of mice with deficiencies in these cells are described--beta 2-microglobulin knockout mice and SJL mice. Both show defects in the development of IL-4-producing cells and in the increase in serum IgE in response to stimulation with the

  16. Analysis of oocyte-like cells differentiated from porcine fetal skin-derived stem cells.

    Science.gov (United States)

    Dyce, Paul W; Shen, Wei; Huynh, Evanna; Shao, Hua; Villagómez, Daniel A F; Kidder, Gerald M; King, W Allan; Li, Julang

    2011-05-01

    We previously reported the differentiation of cells derived from porcine female fetal skin into cells resembling germ cells and oocytes. A subpopulation of these cells expressed germ cell markers and formed aggregates resembling cumulus-oocyte complexes. Some of these aggregates extruded large oocyte-like cells (OLCs) that expressed markers consistent with those of oocytes. The objective of the current study was to further characterize OLCs differentiated from porcine skin-derived stem cells. Reverse transcriptase (RT)-polymerase chain reaction and Western blot revealed the expression of connexin37 and connexin43, both of which are characteristic of ovarian follicles. The expression of meiosis markers DMC1 and synaptonemal complex protein, but not STRA8 and REC8, was detected in the OLC cultures. Immunofluorescence with an antibody against synaptonemal complex protein on chromosome spreads revealed a very small subpopulation of stained OLCs that had a similar pattern to leptotene, zytotene, or pachytene nuclei during prophase I of meiosis. Sodium bisulfite sequencing of the differentially methylated region of H19 indicated that this region is almost completely demethylated in OLCs, similar to in vivo-derived oocytes. We also investigated the differentiation potential of male skin-derived stem cells in the same differentiation medium. Large cells with oocyte morphology were generated in the male stem cell differentiation cultures. These OLCs expressed oocyte genes such as octamer-binding transcription factor 4 (OCT4), growth differentiation factor-9b (GDF9B), deleted in azoospermia-like (DAZL), VASA, zona pellucida B (ZPB), and zona pellucida C (ZPC). It was concluded that skin-derived stem cells from both male and female porcine fetuses are capable of entering an oocyte differentiation pathway, but the culture system currently in place is inadequate to support the complete development of competent oocytes.

  17. PAX4 enhances beta-cell differentiation of human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Chee Gee Liew

    Full Text Available BACKGROUND: Human embryonic stem cells (HESC readily differentiate into an apparently haphazard array of cell types, corresponding to all three germ layers, when their culture conditions are altered, for example by growth in suspension as aggregates known as embryoid bodies (EBs. However, this diversity of differentiation means that the efficiency of producing any one particular cell type is inevitably low. Although pancreatic differentiation has been reported from HESC, practicable applications for the use of beta-cells derived from HESC to treat diabetes will only be possible once techniques are developed to promote efficient differentiation along the pancreatic lineages. METHODS AND FINDINGS: Here, we have tested whether the transcription factor, Pax4 can be used to drive the differentiation of HESC to a beta-cell fate in vitro. We constitutively over-expressed Pax4 in HESCs by stable transfection, and used Q-PCR analysis, immunocytochemistry, ELISA, Ca(2+ microfluorimetry and cell imaging to assess the role of Pax4 in the differentiation and intracellular Ca(2+ homeostasis of beta-cells developing in embryoid bodies produced from such HESC. Cells expressing key beta-cell markers were isolated by fluorescence-activated cell sorting after staining for high zinc content using the vital dye, Newport Green. CONCLUSION: Constitutive expression of Pax4 in HESC substantially enhances their propensity to form putative beta-cells. Our findings provide a novel foundation to study the mechanism of pancreatic beta-cells differentiation during early human development and to help evaluate strategies for the generation of purified beta-cells for future clinical applications.

  18. Autocrine VEGF isoforms differentially regulate endothelial cell behavior

    Directory of Open Access Journals (Sweden)

    Hideki Yamamoto

    2016-09-01

    Full Text Available Vascular endothelial growth factor A (VEGF is involved in all the essential biology of endothelial cells, from proliferation to vessel function, by mediating intercellular interactions and monolayer integrity. It is expressed as three major alternative spliced variants. In mice, these are VEGF120, VEGF164, and VEGF188, each with different affinities for extracellular matrices and cell surfaces, depending on the inclusion of heparin-binding sites, encoded by exons 6 and 7. To determine the role of each VEGF isoform in endothelial homeostasis, we compared phenotypes of primary endothelial cells isolated from lungs of mice expressing single VEGF isoforms in normoxic and hypoxic conditions. The differential expression and distribution of VEGF isoforms affect endothelial cell functions, such as proliferation, adhesion, migration and integrity, which are dependent on the stability of and affinity to VEGF receptor 2 (VEGFR2. We found a correlation between autocrine VEGF164 and VEGFR2 stability, which is also associated with increased expression of proteins involved in cell adhesion. Endothelial cells expressing only VEGF188, which localizes to extracellular matrices or cell surfaces, presented a mesenchymal morphology and weakened monolayer integrity. Cells expressing only VEGF120 lacked stable VEGFR2 and dysfunctional downstream processes, rendering the cells unviable. Endothelial cells expressing these different isoforms in isolation also had differing rates of apoptosis, proliferation, and signaling via nitric oxide (NO synthesis. These data indicate that autocrine signaling of each VEGF isoform has unique functions on endothelial homeostasis and response to hypoxia, due to both distinct VEGF distribution and VEGFR2 stability, which appears to be, at least partly, affected by differential NO production. This study demonstrates that each autocrine VEGF isoform has a distinct effect on downstream functions, namely VEGFR2-regulated endothelial cell

  19. Biophysical characteristics reveal neural stem cell differentiation potential.

    Directory of Open Access Journals (Sweden)

    Fatima H Labeed

    Full Text Available BACKGROUND: Distinguishing human neural stem/progenitor cell (huNSPC populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers. METHODOLOGY/PRINCIPAL FINDINGS: We used dielectrophoresis (DEP to analyze the biophysical, specifically electrophysiological, properties of cortical human and mouse NSPCs that vary in differentiation potential. Our data demonstrate that the electrophysiological property membrane capacitance inversely correlates with the neurogenic potential of NSPCs. Furthermore, as huNSPCs are continually passaged they decrease neuron generation and increase membrane capacitance, confirming that this parameter dynamically predicts and negatively correlates with neurogenic potential. In contrast, differences in membrane conductance between NSPCs do not consistently correlate with the ability of the cells to generate neurons. DEP crossover frequency, which is a quantitative measure of cell behavior in DEP, directly correlates with neuron generation of NSPCs, indicating a potential mechanism to separate stem cells biased to particular differentiated cell fates. CONCLUSIONS/SIGNIFICANCE: We show here that whole cell membrane capacitance, but not membrane conductance, reflects and predicts the neurogenic potential of human and mouse NSPCs. Stem cell biophysical characteristics therefore provide a completely novel and quantitative measure of stem cell fate potential and a label-free means to identify neuron- or glial-biased progenitors.

  20. Human Liver Stem Cells Suppress T-Cell Proliferation, NK Activity, and Dendritic Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Stefania Bruno

    2016-01-01

    Full Text Available Human liver stem cells (HLSCs are a mesenchymal stromal cell-like population resident in the adult liver. Preclinical studies indicate that HLSCs could be a good candidate for cell therapy. The aim of the present study was to evaluate the immunogenicity and the immunomodulatory properties of HLSCs on T-lymphocytes, natural killer cells (NKs, and dendritic cells (DCs in allogeneic experimental settings. We found that HLSCs inhibited T-cell proliferation by a mechanism independent of cell contact and dependent on the release of prostaglandin E2 (PGE2 and on indoleamine 2,3-dioxygenase activity. When compared with mesenchymal stromal cells (MSCs, HLSCs were more efficient in inhibiting T-cell proliferation. At variance with MSCs, HLSCs did not elicit NK degranulation. Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release. When tested on DC generation from monocytes, HLSCs were found to impair DC differentiation and DCs ability to induce T-cell proliferation through PGE2. This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response.

  1. Human Liver Stem Cells Suppress T-Cell Proliferation, NK Activity, and Dendritic Cell Differentiation.

    Science.gov (United States)

    Bruno, Stefania; Grange, Cristina; Tapparo, Marta; Pasquino, Chiara; Romagnoli, Renato; Dametto, Ennia; Amoroso, Antonio; Tetta, Ciro; Camussi, Giovanni

    2016-01-01

    Human liver stem cells (HLSCs) are a mesenchymal stromal cell-like population resident in the adult liver. Preclinical studies indicate that HLSCs could be a good candidate for cell therapy. The aim of the present study was to evaluate the immunogenicity and the immunomodulatory properties of HLSCs on T-lymphocytes, natural killer cells (NKs), and dendritic cells (DCs) in allogeneic experimental settings. We found that HLSCs inhibited T-cell proliferation by a mechanism independent of cell contact and dependent on the release of prostaglandin E2 (PGE2) and on indoleamine 2,3-dioxygenase activity. When compared with mesenchymal stromal cells (MSCs), HLSCs were more efficient in inhibiting T-cell proliferation. At variance with MSCs, HLSCs did not elicit NK degranulation. Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release. When tested on DC generation from monocytes, HLSCs were found to impair DC differentiation and DCs ability to induce T-cell proliferation through PGE2. This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response.

  2. Vascular Cell Induction Culture System Using Arabidopsis Leaves (VISUAL) Reveals the Sequential Differentiation of Sieve Element-Like Cells

    Science.gov (United States)

    Kondo, Yuki; Nurani, Alif Meem; Saito, Chieko; Ichihashi, Yasunori; Saito, Masato; Yamazaki, Kyoko; Mitsuda, Nobutaka; Ohme-Takagi, Masaru; Fukuda, Hiroo

    2016-01-01

    Cell differentiation is a complex process involving multiple steps, from initial cell fate specification to final differentiation. Procambial/cambial cells, which act as vascular stem cells, differentiate into both xylem and phloem cells during vascular development. Recent studies have identified regulatory cascades for xylem differentiation. However, the molecular mechanism underlying phloem differentiation is largely unexplored due to technical challenges. Here, we established an ectopic induction system for phloem differentiation named Vascular Cell Induction Culture System Using Arabidopsis Leaves (VISUAL). Our results verified similarities between VISUAL-induced Arabidopsis thaliana phloem cells and in vivo sieve elements. We performed network analysis using transcriptome data with VISUAL to dissect the processes underlying phloem differentiation, eventually identifying a factor involved in the regulation of the master transcription factor gene APL. Thus, our culture system opens up new avenues not only for genetic studies of phloem differentiation, but also for future investigations of multidirectional differentiation from vascular stem cells. PMID:27194709

  3. Differentiation of neuroepithelial stem cells into functional dopaminergic neurons in 3D microfluidic cell culture.

    Science.gov (United States)

    Moreno, Edinson Lucumi; Hachi, Siham; Hemmer, Kathrin; Trietsch, Sebastiaan J; Baumuratov, Aidos S; Hankemeier, Thomas; Vulto, Paul; Schwamborn, Jens C; Fleming, Ronan M T

    2015-06-07

    A hallmark of Parkinson's disease is the progressive loss of nigrostriatal dopaminergic neurons. We derived human neuroepithelial cells from induced pluripotent stem cells and successfully differentiated them into dopaminergic neurons within phase-guided, three-dimensional microfluidic cell culture bioreactors. After 30 days of differentiation within the microfluidic bioreactors, in situ morphological, immunocytochemical and calcium imaging confirmed the presence of dopaminergic neurons that were spontaneously electrophysiologically active, a characteristic feature of nigrostriatal dopaminergic neurons in vivo. Differentiation was as efficient as in macroscopic culture, with up to 19% of differentiated neurons immunoreactive for tyrosine hydroxylase, the penultimate enzyme in the synthesis of dopamine. This new microfluidic cell culture model integrates the latest innovations in developmental biology and microfluidic cell culture to generate a biologically realistic and economically efficient route to personalised drug discovery for Parkinson's disease.

  4. Mesenchymal stem cell ingrowth and differentiation on coralline hydroxyapatite scaffolds.

    Science.gov (United States)

    Mygind, Tina; Stiehler, Maik; Baatrup, Anette; Li, Haisheng; Zou, Xuenong; Flyvbjerg, Allan; Kassem, Moustapha; Bünger, Cody

    2007-02-01

    Culture of osteogenic cells on a porous scaffold could offer a new solution to bone grafting using autologous human mesenchymal stem cells (hMSC) from the patient. We compared coralline hydroxyapatite scaffolds with pore sizes of 200 and 500 microm for expansion and differentiation of hMSCs. We cultivated the hMSC statically or in spinner flasks for 1, 7, 14 and 21 days and found that the 200-microm pore scaffolds exhibited a faster rate of osteogenic differentiation than did the 500-microm pore scaffolds as shown by an alkaline phosphatase activity assay and real-time reverse transcriptase polymerase chain reaction for 10 osteogenic markers. The 500-microm scaffolds had increased proliferation rates and accommodated a higher number of cells (shown by DNA content, scanning electron microscopy and fluorescence microscopy). Thus the porosity of a 3D microporous biomaterial may be used to steer hMSC in a particular direction. We found that dynamic spinner flask cultivation of hMSC/scaffold constructs resulted in increased proliferation, differentiation and distribution of cells in scaffolds. Therefore, spinner flask cultivation is an easy-to-use inexpensive system for cultivating hMSCs on small to intermediate size 3D scaffolds.

  5. Directed Differentiation of Human Pluripotent Stem Cells to Microglia

    Directory of Open Access Journals (Sweden)

    Panagiotis Douvaras

    2017-06-01

    Full Text Available Microglia, the immune cells of the brain, are crucial to proper development and maintenance of the CNS, and their involvement in numerous neurological disorders is increasingly being recognized. To improve our understanding of human microglial biology, we devised a chemically defined protocol to generate human microglia from pluripotent stem cells. Myeloid progenitors expressing CD14/CX3CR1 were generated within 30 days of differentiation from both embryonic and induced pluripotent stem cells (iPSCs. Further differentiation of the progenitors resulted in ramified microglia with highly motile processes, expressing typical microglial markers. Analyses of gene expression and cytokine release showed close similarities between iPSC-derived (iPSC-MG and human primary microglia as well as clear distinctions from macrophages. iPSC-MG were able to phagocytose and responded to ADP by producing intracellular Ca2+ transients, whereas macrophages lacked such response. The differentiation protocol was highly reproducible across several pluripotent stem cell lines.

  6. Paraoxon and Pyridostigmine Interfere with Neural Stem Cell Differentiation

    Science.gov (United States)

    Berríos, Verónica O.; Boukli, Nawal M.; Rodriguez, Jose W.; Negraes, Priscilla D.; Schwindt, Telma T.; Trujillo, Cleber A.; Oliveira, Sophia L. B.; Cubano, Luis A.; Ferchmin, P. A.; Eterovic, Vesna A.; Ulrich, Henning; Martins, Antonio H.

    2015-01-01

    Acetylcholinesterase (AChE) inhibition has been described as the main mechanism of organophosphate (OP)-evoked toxicity. OPs represent a human health threat, because chronic exposure to low doses can damage the developing brain, and acute exposure can produce long-lasting damage to adult brains, despite post-exposure medical countermeasures. Although the main mechanism of OP toxicity is AChE inhibition, several lines of evidence suggest that OPs also act by other mechanisms. We hypothesized that rat neural progenitor cells extracted on embryonic day 14.5 would be affected by constant inhibition of AChE from chronic exposure to OP or pyri-dostigmine (a reversible AChE blocker) during differentiation. In this work, the OP paraoxon decreased cell viability in concentrations >50 μM, as measured with the MTT assay; however, this effect was not dose-dependent. Reduced viability could not be attributed to blockade of AChE activity, since treatment with 200 μM pyri-dostigmine did not affect cell viability, even after 6 days. Although changes in protein expression patterns were noted in both treatments, the distribution of differentiated phenotypes, such as the percentages of neurons and glial cells, was not altered, as determined by flow cytometry. Since paraoxon and pyridostigmine each decreased neurite outgrowth (but did not prevent differentiation), we infer that developmental patterns may have been affected. PMID:25758980

  7. Enhancement of cardiomyocyte differentiation from human embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Several approaches have been used to encourage the differentiation of cardiomyocytes from human embryonic stem cells.However,the differentiation efficiency is low,and appropriate culture protocols are needed to produce adequate numbers of cardiomyocytes for therapeutic cell transplantation.This study investigated the effects of serum on cardiomyocyte differentiation in suspension culture medium during embryoid body(EB) formation by human embryonic stem cells.The addition of ascorbic acid,dimethylsulfoxide and 5-aza-2’-deoxycytidine during days 5-7 at the EB-forming stage resulted in an increase in the numbers of rhythmically contracting clusters of derived cardiomyocytes.Treatment with 0.1 mmol L-1 ascorbic acid alone,or more notably in combination with 10 μmol L-1 5-aza-2’-deoxycytidine,induced the formation of beating cells within EBs.Most of the beating clusters had spontaneous contraction rates similar to those found in human adults,and their contractile ac-tivity lasted for up to 194 days.

  8. The Hematopoietic Differentiation and Production of Mature Myeloid Cells from Human Pluripotent Stem Cells

    OpenAIRE

    Choi, Kyung-Dal; Vodyanik, Maxim; Slukvin, Igor I.

    2011-01-01

    Here we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (hPSCs) and generation of mature myeloid cells from hPSCs through expansion and differentiation of hPSC-derived lin-CD34+CD43+CD45+ multipotent progenitors. The protocol is comprised of three major steps: (i) induction of hematopoietic differentiation by coculture of hPSCs with OP9 bone marrow stromal cells, (ii) short-term expansion of multipotent myeloid progenitors with a high dose of GM-CSF, and ...

  9. Cell population structure prior to bifurcation predicts efficiency of directed differentiation in human induced pluripotent cells

    Science.gov (United States)

    Bargaje, Rhishikesh; Trachana, Kalliopi; Shelton, Martin N.; McGinnis, Christopher S.; Zhou, Joseph X.; Chadick, Cora; Cook, Savannah; Cavanaugh, Christopher; Huang, Sui; Hood, Leroy

    2017-01-01

    Steering the differentiation of induced pluripotent stem cells (iPSCs) toward specific cell types is crucial for patient-specific disease modeling and drug testing. This effort requires the capacity to predict and control when and how multipotent progenitor cells commit to the desired cell fate. Cell fate commitment represents a critical state transition or “tipping point” at which complex systems undergo a sudden qualitative shift. To characterize such transitions during iPSC to cardiomyocyte differentiation, we analyzed the gene expression patterns of 96 developmental genes at single-cell resolution. We identified a bifurcation event early in the trajectory when a primitive streak-like cell population segregated into the mesodermal and endodermal lineages. Before this branching point, we could detect the signature of an imminent critical transition: increase in cell heterogeneity and coordination of gene expression. Correlation analysis of gene expression profiles at the tipping point indicates transcription factors that drive the state transition toward each alternative cell fate and their relationships with specific phenotypic readouts. The latter helps us to facilitate small molecule screening for differentiation efficiency. To this end, we set up an analysis of cell population structure at the tipping point after systematic variation of the protocol to bias the differentiation toward mesodermal or endodermal cell lineage. We were able to predict the proportion of cardiomyocytes many days before cells manifest the differentiated phenotype. The analysis of cell populations undergoing a critical state transition thus affords a tool to forecast cell fate outcomes and can be used to optimize differentiation protocols to obtain desired cell populations. PMID:28167799

  10. Upconversion nanoparticles for differential imaging of plant cells and detection of fluorescent dyes

    Institute of Scientific and Technical Information of China (English)

    吴笑峰; 刘云新; 胡盼; 胡仕刚; 陈增辉; 严焕元; 唐志军; 席在芳; 余意; 戴港涛

    2016-01-01

    Upconversion NaLuF4 nanoparticles were synthesized by the solvothermal method which could emit multicolor visible light under the excitation of 980 nm near-infrared (NIR) photons. These upconversion nanoparticles (UCNPs) with an acidic ligand could rapidly capture the basic rhodamine-B (RB) in plant cells to generate a close UCNPs@RB system. RB could efficiently absorb the green fluorescence from NaLuF4:18 mol.%Yb3+,2 mol.%Er3+ UCNPs and then emitted red light in the UCNPs@RB system by a robust luminescence resonance energy transfer (LRET) from UCNPs to RB. The detection limit of RB with these upconversion fluo-rescent nanoprobes could reach 0.25μg/cm3 in plant cell even under an ultra low excitation power source of 0.2 W/mm2. This LRET phenomenon was also extended to NaLuF4:18 mol.%Yb3+,0.5 mol.%Tm3+@Sodium fluorescein (SF) system. In addition, the differ-ential imaging could be achieved by successively incubating plant cells with fluorescent dyes and UCNPs. The fluorescent dyes ag-gregated in cell wall while UCNPs with surface modification distributed both in cell wall and cytoplasm, so that UCNPs@Dyes formed in cell walls which could emit multicolor light by LRET which was different from the emission in cytoplasm with only UCNPs.

  11. [Differentiation of functional cells from iPS cells by efficient gene transfer].

    Science.gov (United States)

    Kawabata, Kenji; Tashiro, Katsuhisa; Mizuguchi, Hiroyuki

    2010-11-01

    Induced pluripotent stem (iPS) cells, which are generated from somatic cells by transducing four genes, are expected to have broad application to regenerative medicine. Although establishment of an efficient gene transfer system for iPS cells is considered to be essential for differentiating them into functional cells, the detailed transduction characteristics of iPS cells have not been examined. By using an adenovirus (Ad) vector containing the cytomegalovirus enhancer/beta-actin (CA) promoters, we have developed an efficient transduction system for mouse mesenchymal stem cells and embryonic stem (ES) cells. Also, we applied our transduction system to mouse iPS cells and investigated whether efficient differentiation could be achieved by Ad vector-mediated transduction of a functional gene. As in the case of ES cells, the Ad vector could efficiently transduce transgenes into mouse iPS cells. We found that the CA promoter had potent transduction ability in iPS cells. Moreover, exogenous expression of a PPARγ gene or a Runx2 gene into mouse iPS cells by an optimized Ad vector enhanced adipocyte or osteoblast differentiation, respectively. These results suggest that Ad vector-mediated transient transduction is sufficient to promote cellular differentiation and that our transduction methods would be useful for therapeutic applications based on iPS cells.

  12. Planar Cell Polarity Controls Pancreatic Beta Cell Differentiation and Glucose Homeostasis

    DEFF Research Database (Denmark)

    Cortijo, Cedric; Gouzi, Mathieu; Tissir, Fadel

    2012-01-01

    Planar cell polarity (PCP) refers to the collective orientation of cells within the epithelial plane. We show that progenitor cells forming the ducts of the embryonic pancreas express PCP proteins and exhibit an active PCP pathway. Planar polarity proteins are acquired at embryonic day 11.......5 synchronously to apicobasal polarization of pancreas progenitors. Loss of function of the two PCP core components Celsr2 and Celsr3 shows that they control the differentiation of endocrine cells from polarized progenitors, with a prevalent effect on insulin-producing beta cells. This results in a decreased...... glucose clearance. Loss of Celsr2 and 3 leads to a reduction of Jun phosphorylation in progenitors, which, in turn, reduces beta cell differentiation from endocrine progenitors. These results highlight the importance of the PCP pathway in cell differentiation in vertebrates. In addition, they reveal...

  13. Mitochondria in mesenchymal stem cell biology and cell therapy: From cellular differentiation to mitochondrial transfer.

    Science.gov (United States)

    Hsu, Yi-Chao; Wu, Yu-Ting; Yu, Ting-Hsien; Wei, Yau-Huei

    2016-04-01

    Mesenchymal stem cells (MSCs) are characterized to have the capacity of self-renewal and the potential to differentiate into mesoderm, ectoderm-like and endoderm-like cells. MSCs hold great promise for cell therapies due to their multipotency in vitro and therapeutic advantage of hypo-immunogenicity and lower tumorigenicity. Moreover, it has been shown that MSCs can serve as a vehicle to transfer mitochondria into cells after cell transplantation. Mitochondria produce most of the energy through oxidative phosphorylation in differentiated cells. It has been increasingly clear that the switch of energy supply from glycolysis to aerobic metabolism is essential for successful differentiation of MSCs. Post-translational modifications of proteins have been established to regulate mitochondrial function and metabolic shift during MSCs differentiation. In this article, we review and provide an integrated view on the roles of different protein kinases and sirtuins in the maintenance and differentiation of MSCs. Importantly, we provide evidence to suggest that alteration in the expression of Sirt3 and Sirt5 and relative changes in the acylation levels of mitochondrial proteins might be involved in the activation of mitochondrial function and adipogenic differentiation of adipose-derived MSCs. We summarize their roles in the regulation of mitochondrial biogenesis and metabolism, oxidative responses and differentiation of MSCs. On the other hand, we discuss recent advances in the study of mitochondrial dynamics and mitochondrial transfer as well as their roles in the differentiation and therapeutic application of MSCs to improve cell function in vitro and in animal models. Accumulating evidence has substantiated that the therapeutic potential of MSCs is conferred not only by cell replacement and paracrine effects but also by transferring mitochondria into injured tissues or cells to modulate the cellular metabolism in situ. Therefore, elucidation of the underlying mechanisms

  14. Equine induced pluripotent stem cells have a reduced tendon differentiation capacity compared to embryonic stem cells

    Directory of Open Access Journals (Sweden)

    Emma Patricia Bavin

    2015-11-01

    Full Text Available Tendon injuries occur commonly in horses and their repair through scar tissue formation predisposes horses to a high rate of re-injury. Pluripotent stem cells may provide a cell replacement therapy to improve tendon tissue regeneration and lower the frequency of re-injury. We have previously demonstrated that equine embryonic stem cells (ESCs differentiate into the tendon cell lineage upon injection into the damaged horse tendon and can differentiate into functional tendon cells in vitro to generate artificial tendons. Induced pluripotent stem cells (iPSCs have now been derived from horses but, to date, there are no reports on their ability to differentiate into tendon cells. As iPSCs can be produced from adult cell types, they provide a more accessible source of cells than ESCs, which require the use of horse embryos. The aim of this study was to compare tendon differentiation by ESCs and iPSCs produced through two independent methods. In 2-dimensional differentiation assays the iPSCs expressed tendon associated genes and proteins, which were enhanced by the presence of transforming growth factor-β3. However, in 3-dimensional differentiation assays the iPSCs failed to differentiate into functional tendon cells and generate artificial tendons. These results demonstrate the utility of the 3-dimensional in vitro tendon assay for measuring tendon differentiation and the need for more detailed studies to be performed on equine iPSCs to identify and understand their epigenetic differences from pluripotent ESCs prior to their clinical application.

  15. The use of Nanotrap particles technology in capturing HIV-1 virions and viral proteins from infected cells.

    Directory of Open Access Journals (Sweden)

    Elizabeth Jaworski

    Full Text Available HIV-1 infection results in a chronic but incurable illness since long-term HAART can keep the virus to an undetectable level. However, discontinuation of therapy rapidly increases viral burden. Moreover, patients under HAART frequently develop various metabolic disorders and HIV-associated neuronal disease. Today, the main challenge of HIV-1 research is the elimination of the residual virus in infected individuals. The current HIV-1 diagnostics are largely comprised of serological and nucleic acid based technologies. Our goal is to integrate the nanotrap technology into a standard research tool that will allow sensitive detection of HIV-1 infection. This study demonstrates that majority of HIV-1 virions in culture supernatants and Tat/Nef proteins spiked in culture medium can be captured by nanotrap particles. To determine the binding affinities of different baits, we incubated target molecules with nanotrap particles at room temperature. After short sequestration, materials were either eluted or remained attached to nanotrap particles prior to analysis. The unique affinity baits of nanotrap particles preferentially bound HIV-1 materials while excluded albumin. A high level capture of Tat or Tat peptide by NT082 and NT084 particles was measured by western blot (WB. Intracellular Nef protein was captured by NT080, while membrane-associated Nef was captured by NT086 and also detected by WB. Selective capture of HIV-1 particles by NT073 and NT086 was measured by reverse transcriptase assay, while capture of infectious HIV-1 by these nanoparticles was demonstrated by functional transactivation in TZM-bl cells. We also demonstrated specific capture of HIV-1 particles and exosomes-containing TAR-RNA in patients' serum by NT086 and NT082 particles, respectively, using specific qRT-PCR. Collectively, our data indicate that certain types of nanotrap particles selectively capture specific HIV-1 molecules, and we propose to use this technology as a

  16. The Use of Nanotrap Particles Technology in Capturing HIV-1 Virions and Viral Proteins from Infected Cells

    Science.gov (United States)

    Sampey, Gavin; Shafagati, Nazly; Van Duyne, Rachel; Iordanskiy, Sergey; Kehn-Hall, Kylene; Liotta, Lance; Petricoin, Emanuel; Young, Mary; Lepene, Benjamin; Kashanchi, Fatah

    2014-01-01

    HIV-1 infection results in a chronic but incurable illness since long-term HAART can keep the virus to an undetectable level. However, discontinuation of therapy rapidly increases viral burden. Moreover, patients under HAART frequently develop various metabolic disorders and HIV-associated neuronal disease. Today, the main challenge of HIV-1 research is the elimination of the residual virus in infected individuals. The current HIV-1 diagnostics are largely comprised of serological and nucleic acid based technologies. Our goal is to integrate the nanotrap technology into a standard research tool that will allow sensitive detection of HIV-1 infection. This study demonstrates that majority of HIV-1 virions in culture supernatants and Tat/Nef proteins spiked in culture medium can be captured by nanotrap particles. To determine the binding affinities of different baits, we incubated target molecules with nanotrap particles at room temperature. After short sequestration, materials were either eluted or remained attached to nanotrap particles prior to analysis. The unique affinity baits of nanotrap particles preferentially bound HIV-1 materials while excluded albumin. A high level capture of Tat or Tat peptide by NT082 and NT084 particles was measured by western blot (WB). Intracellular Nef protein was captured by NT080, while membrane-associated Nef was captured by NT086 and also detected by WB. Selective capture of HIV-1 particles by NT073 and NT086 was measured by reverse transcriptase assay, while capture of infectious HIV-1 by these nanoparticles was demonstrated by functional transactivation in TZM-bl cells. We also demonstrated specific capture of HIV-1 particles and exosomes-containing TAR-RNA in patients' serum by NT086 and NT082 particles, respectively, using specific qRT-PCR. Collectively, our data indicate that certain types of nanotrap particles selectively capture specific HIV-1 molecules, and we propose to use this technology as a platform to enhance HIV-1

  17. mTOR and the differentiation of mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Xinxin Xiang; Jing Zhao; Geyang Xu; Yin Li; Weizhen Zhang

    2011-01-01

    The mammalian target of rapamycin (mTOR), an evolutionarily conserved serine-threonine protein kinase,belongs to the phosphoinositide 3-kinase (PI3K)-related kinase family, which contains a lipid kinase-like domain within their C-terminal region. Recent studies have revealed that mTOR as a critical intracellular molecule can sense the extracellular energy status and regulate the cell growth and proliferation in a variety of cells and tissues. This review summarizes our current understanding about the effects of mTOR on cell differentiation and tissue development, with an emphasis on the lineage determination of mesenchymal stem cells, mTOR can promote adipogenesis in white adipocytes, brown adipocytes, and muscle satellite cells, while rapamycin inhibits the adipogenic function of mTOR. mTOR signaling may function to affect osteoblast proliferation and differentiation, however, rapamycin has been reported to either inhibit or promote osteogenesis. Although the precise mechanism remains unclear, mTOR is indispensable for myogenesis. Depending on the cell type, rapamycin has been reported to inhibit, promote, or have no effect on myogenesis.

  18. ATML1 promotes epidermal cell differentiation in Arabidopsis shoots.

    Science.gov (United States)

    Takada, Shinobu; Takada, Nozomi; Yoshida, Ayaka

    2013-05-01

    Molecular mechanisms that generate distinct tissue layers in plant shoots are not well understood. ATML1, an Arabidopsis homeobox gene, is expressed in the outermost cell layer, beginning at an early stage of development. The promoters of many epidermis-specific genes, including ATML1, contain an ATML1-binding site called an L1 box, suggesting that ATML1 regulates epidermal cell fate. Here, we show that overexpression of ATML1 was sufficient to activate the expression of epidermal genes and to induce epidermis-related traits such as the formation of stomatal guard cells and trichome-like cells in non-epidermal seedling tissues. Detailed observation of the division planes of these ectopic stomatal cells suggested that a near-surface position, as well as epidermal cell identity, were required for regular anticlinal cell division, as seen in wild-type epidermis. Moreover, analyses of a loss-of-function mutant and overexpressors implied that differentiation of epidermal cells was associated with repression of mesophyll cell fate. Collectively, our studies contribute new information about the molecular basis of cell fate determination in different layers of plant aerial organs.

  19. Isolation and differentiation of medaka embryonic stem cells.

    Science.gov (United States)

    Hong, Yunhan; Schartl, Manfred

    2006-01-01

    Medaka is a small laboratory fish that daily produces eggs easily controllable by light cycles. This fish represents a unique lower vertebrate compared to mammals, in which embryonic stem (ES) cell lines can be derived from midblastula embryos (MBEs). Like mouse ES cells, medaka ES cells most resemble the totipotent embryonic cells at the blastula stage. Medaka ES cells retain a diploid karyotype, pluripotency in vitro, and chimera competence in vivo. They give rise to high efficiencies of transient and stable gene transfer and maintain their pluripotency after long-term drug selection for transgene integration. They can also be directed to differentiate into particular cell types. Medaka is the most distantly related vertebrate to mammals, and its ES cell lines provide an ideal reference to mammalian ES cells for the molecular analysis of stemness. More important, medaka ES cell lines on their own offer an excellent tool for studying stem cell biology in vitro and in vivo because production and observation of ES-derived chimeras as well as phenotypic analyses are very easy because of its external, transparent, and temperature-adjustable embryology.

  20. Snai1 represses Nanog to promote embryonic stem cell differentiation

    Directory of Open Access Journals (Sweden)

    F. Galvagni

    2015-06-01

    Full Text Available Embryonic stem cell (ESC self-renewal and pluripotency is maintained by an external signaling pathways and intrinsic regulatory networks involving ESC-specific transcriptional complexes (mainly formed by OCT3/4, Sox2 and Nanog proteins, the Polycomb repressive complex 2 (PRC2 and DNA methylation [1–8]. Among these, Nanog represents the more ESC specific factor and its repression correlates with the loss of pluripotency and ESC differentiation [9–11]. During ESC early differentiation, many development-associated genes become upregulated and although, in general, much is known about the pluripotency self-renewal circuitry, the molecular events that lead ESCs to exit from pluripotency and begin differentiation are largely unknown. Snai1 is one the most early induced genes during ESC differentiation in vitro and in vivo [12,13]. Here we show that Snai1 is able to directly repress several stemness-associated genes including Nanog. We use a ESC stable-line expressing a inducible Snai1 protein. We here show microarray analysis of embryonic stem cells (ESC expressing Snail-ER at various time points of induction with 4-OH. Data were deposited in Gene Expression Omnibus (GEO datasets under reference GSE57854 and here: http://epigenetics.hugef-research.org/data.php.

  1. Molecular Analysis of Stromal Cells-Induced Neural Differentiation of Mouse Embryonic Stem Cells.

    Science.gov (United States)

    Joshi, Ramila; Buchanan, James Carlton; Paruchuri, Sailaja; Morris, Nathan; Tavana, Hossein

    2016-01-01

    Deriving specific neural cells from embryonic stem cells (ESCs) is a promising approach for cell replacement therapies of neurodegenerative diseases. When co-cultured with certain stromal cells, mouse ESCs (mESCs) differentiate efficiently to neural cells. In this study, a comprehensive gene and protein expression analysis of differentiating mESCs is performed over a two-week culture period to track temporal progression of cells from a pluripotent state to specific terminally-differentiated neural cells such as neurons, astrocytes, and oligodendrocytes. Expression levels of 26 genes consisting of marker genes for pluripotency, neural progenitors, and specific neuronal, astroglial, and oligodendrocytic cells are tracked using real time q-PCR. The time-course gene expression analysis of differentiating mESCs is combined with the hierarchal clustering and functional principal component analysis (FPCA) to elucidate the evolution of specific neural cells from mESCs at a molecular level. These statistical analyses identify three major gene clusters representing distinct phases of transition of stem cells from a pluripotent state to a terminally-differentiated neuronal or glial state. Temporal protein expression studies using immunohistochemistry demonstrate the generation of neural stem/progenitor cells and specific neural lineages and show a close agreement with the gene expression profiles of selected markers. Importantly, parallel gene and protein expression analysis elucidates long-term stability of certain proteins compared to those with a quick turnover. Describing the molecular regulation of neural cells commitment of mESCs due to stromal signaling will help identify major promoters of differentiation into specific cell types for use in cell replacement therapy applications.

  2. Gene function in early mouse embryonic stem cell differentiation

    Directory of Open Access Journals (Sweden)

    Campbell Pearl A

    2007-03-01

    Full Text Available Abstract Background Little is known about the genes that drive embryonic stem cell differentiation. However, such knowledge is necessary if we are to exploit the therapeutic potential of stem cells. To uncover the genetic determinants of mouse embryonic stem cell (mESC differentiation, we have generated and analyzed 11-point time-series of DNA microarray data for three biologically equivalent but genetically distinct mESC lines (R1, J1, and V6.5 undergoing undirected differentiation into embryoid bodies (EBs over a period of two weeks. Results We identified the initial 12 hour period as reflecting the early stages of mESC differentiation and studied probe sets showing consistent changes of gene expression in that period. Gene function analysis indicated significant up-regulation of genes related to regulation of transcription and mRNA splicing, and down-regulation of genes related to intracellular signaling. Phylogenetic analysis indicated that the genes showing the largest expression changes were more likely to have originated in metazoans. The probe sets with the most consistent gene changes in the three cell lines represented 24 down-regulated and 12 up-regulated genes, all with closely related human homologues. Whereas some of these genes are known to be involved in embryonic developmental processes (e.g. Klf4, Otx2, Smn1, Socs3, Tagln, Tdgf1, our analysis points to others (such as transcription factor Phf21a, extracellular matrix related Lama1 and Cyr61, or endoplasmic reticulum related Sc4mol and Scd2 that have not been previously related to mESC function. The majority of identified functions were related to transcriptional regulation, intracellular signaling, and cytoskeleton. Genes involved in other cellular functions important in ESC differentiation such as chromatin remodeling and transmembrane receptors were not observed in this set. Conclusion Our analysis profiles for the first time gene expression at a very early stage of m

  3. Differentiating osteomyelitis from bone infarction in sickle cell disease.

    Science.gov (United States)

    Wong, A L; Sakamoto, K M; Johnson, E E

    2001-02-01

    This brief review discusses one possible approach to evaluating the sickle cell patient with bone pain. The major differential diagnoses include osteomyelitis and bone infarction. Based on previous studies, we provide an approach to assessing and treating patients with the possible diagnosis of osteomyelitis. An algorithm has been provided, which emphasizes the importance of the initial history and physical examination. Specific radiographic studies are recommended to aid in making the initial assessment and to determine whether the patient has an infarct or osteomyelitis. Differentiating osteomyelitis from infarction in sickle cell patients remains a challenge for the pediatrician. This algorithm can be used as a guide for physicians who evaluate such patients in the acute care setting.

  4. Expression of the chitinase family glycoprotein YKL-40 in undifferentiated, differentiated and trans-differentiated mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Daniel J Hoover

    Full Text Available The glycoprotein YKL-40 (CHI3L1 is a secreted chitinase family protein that induces angiogenesis, cell survival, and cell proliferation, and plays roles in tissue remodeling and immune regulation. It is expressed primarily in cells of mesenchymal origin, is overexpressed in numerous aggressive carcinomas and sarcomas, but is rarely expressed in normal ectodermal tissues. Bone marrow-derived mesenchymal stem cells (MSCs can be induced to differentiate into various mesenchymal tissues and trans-differentiate into some non-mesenchymal cell types. Since YKL-40 has been used as a mesenchymal marker, we followed YKL-40 expression as undifferentiated MSCs were induced to differentiate into bone, cartilage, and neural phenotypes. Undifferentiated MSCs contain significant levels of YKL-40 mRNA but do not synthesize detectable levels of YKL-40 protein. MSCs induced to differentiate into chondrocytes and osteocytes soon began to express and secrete YKL-40 protein, as do ex vivo cultured chondrocytes and primary osteocytes. In contrast, MSCs induced to trans-differentiate into neurons did not synthesize YKL-40 protein, consistent with the general absence of YKL-40 protein in normal CNS parenchyma. However, these trans-differentiated neurons retained significant levels of YKL-40 mRNA, suggesting the mechanisms which prevented YKL-40 translation in undifferentiated MSCs remained in place, and that these trans-differentiated neurons differ in at least this way from neurons derived from neuronal stem cells. Utilization of a differentiation protocol containing β-mercaptoethanol resulted in cells that expressed significant amounts of intracellular YKL-40 protein that was not secreted, which is not seen in normal cells. Thus the synthesis of YKL-40 protein is a marker for MSC differentiation into mature mesenchymal phenotypes, and the presence of untranslated YKL-40 mRNA in non-mesenchymal cells derived from MSCs reflects differences between differentiated and

  5. The PLETHORA gene regulatory network guides growth and cell differentiation in Arabidopsis roots

    OpenAIRE

    Santuari, L.; Heidstra, R.

    2016-01-01

    Organ formation in animals and plants relies on precise control of cell state transitions to turn stem cell daughters into fully differentiated cells. In plants, cells cannot rearrange due to shared cell walls. Thus, differentiation progression and the accompanying cell expansion must be tightly coordinated across tissues. PLETHORA (PLT) transcription factor gradients are unique in their ability to guide the progression of cell differentiation at different positions in the growing Arabidopsis...

  6. Clonal, self-renewing and differentiating human and porcine urothelial cells, a novel stem cell population.

    Directory of Open Access Journals (Sweden)

    Hans M Larsson

    Full Text Available Although urothelial progenitor-like cells have been described in the human urinary tract, the existence of stem cells remains to be proven. Using a culture system that favors clonogenic epithelial cell growth, we evaluated and characterized clonal human urothelial cells. We isolated human urothelial cells that were clonogenic, capable of self-renewal and could develop into fully differentiated urothelium once re-implanted into the subcapsular space of nude mice. In addition to final urothelial cell differentiation, spontaneous formation of bladder-like microstructures was observed. By examining an epithelial stem cell signature marker, we found p63 to correlate with the self-renewal capacity of the isolated human urothelial clonal populations. Since a clinically relevant, long-term model for functional reconstitution of human cells does not exist, we sought to establish a culture method for porcine urothelial cells in a clinically relevant porcine model. We isolated cells from porcine ureter, urethra and bladder that were clonogenic and capable of self-renewal and differentiation into fully mature urothelium. In conclusion, we could isolate human and porcine cell populations, behaving as urothelial stem cells and showing clonogenicity, self-renewal and, once re-implanted, morphological differentiation.

  7. Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity (ACDC) assay

    Science.gov (United States)

    The Embryonic Stem Cell Test (EST) is an assay which evaluates xenobiotic-induced effects using three endpoints: mouse embryonic stem cell (mESC) differentiation, mESC viability, and 3T3-cell viability. Our research goal was to develop an improved high-throughput assay by establi...

  8. 3-Dimensional cell culture for on-chip differentiation of stem cells in embryoid body.

    Science.gov (United States)

    Kim, Choong; Lee, Kang Sun; Bang, Jae Hoon; Kim, Young Eyn; Kim, Min-Cheol; Oh, Kwang Wook; Lee, Soo Hyun; Kang, Ji Yoon

    2011-03-07

    This paper proposes a microfluidic device for the on-chip differentiation of an embryoid body (EB) formed in a microwell via 3-dimensional cultures of mouse embryonic carcinoma (EC) cells. The device adjusted the size of the EB by fluid volume, differentiated the EB by chemical treatment, and evaluated its effects in EC cells by on-chip immunostaining. A microfluidic resistance network was designed to control the size of the embryoid body. The duration time and flow rate into each microwell regulated the initial number of trapped cells in order to adjust the size of the EB. The docked cells were aggregated and formed a spherical EB on the non-adherent surface of the culture chip for 3 days. The EC cells in the EB were then differentiated into diverse cell lineages without attachment for an additional 4 days; meanwhile, retinoic acid (RA) was applied without serum to direct the cells into early neuronal lineage. On-chip immunostaining of the EB in the microwell with a neuronal marker was conducted to assess the differentiation-inducing ability of RA. The effect of RA on neuronal differentiation was analyzed with confocal microscopic images of the TuJ1 marker. The RA-treated cells expressed more neuronal markers and appeared as mature neuronal cells with long neurites. The fluorescence intensity of the TuJ1 in the RA-treated EB was twice that observed in the non-treated EB on day 5. It was demonstrated that the pre-screening of inducing chemicals on the early neuronal differentiation of EC cells in a single microfluidic chip was indeed feasible. This chip is expected to constitute a useful tool for assessing the early differentiation of ES cells without attachment, and is also expected to prove useful as an anti-cancer drug test platform for the cytotoxicity assay with cellular spheroids.

  9. Monocyte cell surface glycosaminoglycans positively modulate IL-4-induced differentiation toward dendritic cells.

    NARCIS (Netherlands)

    Dekker, E. den; Grefte, S.; Huijs, T.; Dam, G.B. ten; Versteeg, E.M.M.; Berk, L.C.J. van den; Bladergroen, B.A.; Kuppevelt, A.H.M.S.M. van; Figdor, C.G.; Torensma, R.

    2008-01-01

    IL-4 induces the differentiation of monocytes toward dendritic cells (DCs). The activity of many cytokines is modulated by glycosaminoglycans (GAGs). In this study, we explored the effect of GAGs on the IL-4-induced differentiation of monocytes toward DCs. IL-4 dose-dependently up-regulated the expr

  10. Vertebrate Ctr1 coordinates morphogenesis and progenitor cell fate and regulates embryonic stem cell differentiation

    OpenAIRE

    Haremaki, Tomomi; Fraser, Stuart T.; Kuo, Yien-Ming; Baron, Margaret H.; Weinstein, Daniel C.

    2007-01-01

    Embryogenesis involves two distinct processes. On the one hand, cells must specialize, acquiring fates appropriate to their positions (differentiation); on the other hand, they must physically construct the embryo through coordinated mechanical activity (morphogenesis). In early vertebrate development, fibroblast growth factor (FGF) regulates multiple embryonic events, including germ layer differentiation and morphogenesis; the cellular components that direct FGF signaling to evoke these diff...

  11. Differentially expressed genes in giant cell tumor of bone.

    Science.gov (United States)

    Babeto, Erica; Conceição, André Luis Giacometti; Valsechi, Marina Curado; Peitl Junior, Paulo; de Campos Zuccari, Débora Aparecida Pires; de Lima, Luiz Guilherme Cernaglia Aureliano; Bonilha, Jane Lopes; de Freitas Calmon, Marília; Cordeiro, José Antônio; Rahal, Paula

    2011-04-01

    Giant cells tumors of bone (GCTB) are benign in nature but cause osteolytic destruction with a number of particular characteristics. These tumors can have uncertain biological behavior often contain a significant proportion of highly multinucleated cells, and may show aggressive behavior. We have studied differential gene expression in GCTB that may give a better understanding of their physiopathology, and might be helpful in prognosis and treatment. Rapid subtractive hybridization (RaSH) was used to identify and measure novel genes that appear to be differentially expressed, including KTN1, NEB, ROCK1, and ZAK using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry in the samples of GCTBs compared to normal bone tissue. Normal bone was used in the methodology RaSH for comparison with the GCTB in identification of differentially expressed genes. Functional annotation indicated that these genes are involved in cellular processes related to their tumor phenotype. The differential expression of KTN1, ROCK1, and ZAK was independently confirmed by qRT-PCR and immunohistochemistry. The expression of the KTN1 and ROCK1 genes were increased in samples by qRT-PCR and immunohistochemistry, and ZAK had reduced expression. Since ZAK have CpG islands in their promoter region and low expression in tumor tissue, their methylation pattern was analyzed by MSP-PCR. The genes identified KTN1, ROCK1, and ZAK may be responsible for loss of cellular homeostasis in GCTB since they are responsible for various functions related to tumorigenesis such as cell migration, cytoskeletal organization, apoptosis, and cell cycle control and thus may contribute at some stage in the process of formation and development of GCTB.

  12. Cell culture plastics with immobilized interleukin-4 for monocyte differentiation

    DEFF Research Database (Denmark)

    Hansen, Morten; Hjortø, Gertrud Malene; Met, Ozcan

    2011-01-01

    at similar concentrations to the passive adsorption process, as measured by enzyme-linked immunosorbent assays, and the bound IL-4 did not leak into solution to any measurable extent during cell culture. However, covalently bound IL-4 was incapable of inducing monocyte differentiation. This may be caused...... by IL-4 denaturation or improper epitope presentation induced by the immobilization process, or by biological irresponsiveness of monocytes to IL-4 in immobilized formats....

  13. E-cadherin interactions are required for Langerhans cell differentiation

    OpenAIRE

    Mayumi, Nobuko; Watanabe, Eri; Norose, Yoshihiko; Watari, Eiji; Kawana, Seiji; Geijtenbeek, Teunis B H; Takahashi, Hidemi

    2012-01-01

    Human skin contains the following two distinct DC subsets: (i) Langerhans cells (LCs), expressing Langerin but not DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), are predominantly localized in the epidermis; and (ii) dermal DCs, expressing DC-SIGN but not Langerin, are observed mainly in the dermis. It is not known whether localization in the epidermis provides cues for LC differentiation. Here, we show that E-cadherin expressed by epidermal keratinocytes (KCs) ...

  14. Poorly Differentiated Squamous Cell Carcinoma Arising in Tattooed Skin

    OpenAIRE

    Deba P. Sarma; Dentlinger, Renee B.; Forystek, Amanda M.; Todd Stevens; Christopher Huerter

    2011-01-01

    Introduction. Tattoos have increasingly become accepted by mainstream Western society. As a result, the incidence of tattoo-associated dermatoses is on the rise. The presence of a poorly differentiated squamous cell carcinoma in an old tattooed skin is of interest as it has not been previously documented. Case Presentation. A 79-year-old white homeless man of European descent presented to the dermatology clinic with a painless raised nodule on his left forearm arising in a tattooed area. A...

  15. Cell-mediated remodeling of biomimetic encapsulating hydrogels triggered by adipogenic differentiation of adipose stem cells

    Science.gov (United States)

    Clevenger, Tracy N; Luna, Gabriel; Boctor, Daniel; Fisher, Steven K; Clegg, Dennis O

    2016-01-01

    One of the most common regenerative therapies is autologous fat grafting, which frequently suffers from unexpected volume loss. One approach is to deliver adipose stem cells encapsulated in the engineered hydrogels supportive of cell survival, differentiation, and integration after transplant. We describe an encapsulating, biomimetic poly(ethylene)-glycol hydrogel, with embedded peptides for attachment and biodegradation. Poly(ethylene)-glycol hydrogels containing an Arg–Gly–Asp attachment sequence and a matrix metalloprotease 3/10 cleavage site supported adipose stem cell survival and showed remodeling initiated by adipogenic differentiation. Arg–Gly–Asp–matrix metalloprotease 3/10 cleavage site hydrogels showed an increased number and area of lacunae or holes after adipose stem cell differentiation. Image analysis of adipose stem cells in Arg–Gly–Asp–matrix metalloprotease 3/10 cleavage site hydrogels showed larger Voronoi domains, while cell density remained unchanged. The differentiated adipocytes residing within these newly remodeled spaces express proteins and messenger RNAs indicative of adipocytic differentiation. These engineered scaffolds may provide niches for stem cell differentiation and could prove useful in soft tissue regeneration. PMID:27733898

  16. Quantitative single cell analysis of cell population dynamics during submandibular salivary gland development and differentiation

    Directory of Open Access Journals (Sweden)

    Deirdre A. Nelson

    2013-04-01

    Epithelial organ morphogenesis involves reciprocal interactions between epithelial and mesenchymal cell types to balance progenitor cell retention and expansion with cell differentiation for evolution of tissue architecture. Underlying submandibular salivary gland branching morphogenesis is the regulated proliferation and differentiation of perhaps several progenitor cell populations, which have not been characterized throughout development, and yet are critical for understanding organ development, regeneration, and disease. Here we applied a serial multiplexed fluorescent immunohistochemistry technology to map the progressive refinement of the epithelial and mesenchymal cell populations throughout development from embryonic day 14 through postnatal day 20. Using computational single cell analysis methods, we simultaneously mapped the evolving temporal and spatial location of epithelial cells expressing subsets of differentiation and progenitor markers throughout salivary gland development. We mapped epithelial cell differentiation markers, including aquaporin 5, PSP, SABPA, and mucin 10 (acinar cells; cytokeratin 7 (ductal cells; and smooth muscle α-actin (myoepithelial cells and epithelial progenitor cell markers, cytokeratin 5 and c-kit. We used pairwise correlation and visual mapping of the cells in multiplexed images to quantify the number of single- and double-positive cells expressing these differentiation and progenitor markers at each developmental stage. We identified smooth muscle α-actin as a putative early myoepithelial progenitor marker that is expressed in cytokeratin 5-negative cells. Additionally, our results reveal dynamic expansion and redistributions of c-kit- and K5-positive progenitor cell populations throughout development and in postnatal glands. The data suggest that there are temporally and spatially discreet progenitor populations that contribute to salivary gland development and homeostasis.

  17. High glucose suppresses embryonic stem cell differentiation into neural lineage cells

    Science.gov (United States)

    Yang, Penghua; Shen, Wei-bin; Reece, E. Albert; Chen, Xi; Yang, Peixin

    2017-01-01

    Abnormal neurogenesis occurs during embryonic development in human diabetic pregnancies and in animal models of diabetic embryopathy. Our previous studies in a mouse model of diabetic embryopathy have implicated that high glucose of maternal diabetes delays neurogenesis in the developing neuroepithelium leading to neural tube defects. However, the underlying process in high glucose-impaired neurogenesis is uncharacterized. Neurogenesis from embryonic stem (ES) cells provides a valuable model for understanding the abnormal neural lineage development under high glucose conditions. ES cells are commonly generated and maintained in high glucose (approximately 25 mM glucose). Here, the mouse ES cell line, E14, was gradually adapted to and maintained in low glucose (5 mM), and became a glucose responsive E14 (GR-E14) line. High glucose induced the endoplasmic reticulum stress marker, CHOP, in GR-E14 cells. Under low glucose conditions, the GR-E14 cells retained their pluripotency and capability to differentiate into neural lineage cells. GR-E14 cell differentiation into neural stem cells (Sox1 and nestin positive cells) was inhibited by high glucose. Neuron (Tuj1 positive cells) and glia (GFAP positive cells) differentiation from GR-E14 cells was also suppressed by high glucose. In addition, high glucose delayed GR-E14 differentiation into neural crest cells by decreasing neural crest markers, paired box 3 (Pax3) and paired box 7 (Pax7). Thus, high glucose impairs ES cell differentiation into neural lineage cells. The low glucose adapted and high glucose responsive GR-E14 cell line is a useful in vitro model for assessing the adverse effect of high glucose on the development of the central nervous system. PMID:26940741

  18. High glucose suppresses embryonic stem cell differentiation into neural lineage cells.

    Science.gov (United States)

    Yang, Penghua; Shen, Wei-bin; Reece, E Albert; Chen, Xi; Yang, Peixin

    2016-04-01

    Abnormal neurogenesis occurs during embryonic development in human diabetic pregnancies and in animal models of diabetic embryopathy. Our previous studies in a mouse model of diabetic embryopathy have implicated that high glucose of maternal diabetes delays neurogenesis in the developing neuroepithelium leading to neural tube defects. However, the underlying process in high glucose-impaired neurogenesis is uncharacterized. Neurogenesis from embryonic stem (ES) cells provides a valuable model for understanding the abnormal neural lineage development under high glucose conditions. ES cells are commonly generated and maintained in high glucose (approximately 25 mM glucose). Here, the mouse ES cell line, E14, was gradually adapted to and maintained in low glucose (5 mM), and became a glucose responsive E14 (GR-E14) line. High glucose induced the endoplasmic reticulum stress marker, CHOP, in GR-E14 cells. Under low glucose conditions, the GR-E14 cells retained their pluripotency and capability to differentiate into neural lineage cells. GR-E14 cell differentiation into neural stem cells (Sox1 and nestin positive cells) was inhibited by high glucose. Neuron (Tuj1 positive cells) and glia (GFAP positive cells) differentiation from GR-E14 cells was also suppressed by high glucose. In addition, high glucose delayed GR-E14 differentiation into neural crest cells by decreasing neural crest markers, paired box 3 (Pax3) and paired box 7 (Pax7). Thus, high glucose impairs ES cell differentiation into neural lineage cells. The low glucose adapted and high glucose responsive GR-E14 cell line is a useful in vitro model for assessing the adverse effect of high glucose on the development of the central nervous system.

  19. Differentiation of human embryonic stem cells and induced pluripotent stem cells to cardiomyocytes: a methods overview.

    Science.gov (United States)

    Mummery, Christine L; Zhang, Jianhua; Ng, Elizabeth S; Elliott, David A; Elefanty, Andrew G; Kamp, Timothy J

    2012-07-20

    Since human embryonic stem cells were first differentiated to beating cardiomyocytes a decade ago, interest in their potential applications has increased exponentially. This has been further enhanced over recent years by the discovery of methods to induce pluripotency in somatic cells, including those derived from patients with hereditary cardiac diseases. Human pluripotent stem cells have been among the most challenging cell types to grow stably in culture, but advances in reagent development now mean that most laboratories can expand both embryonic and induced pluripotent stem cells robustly using commercially available products. However, differentiation protocols have lagged behind and in many cases only produce the cell types required with low efficiency. Cardiomyocyte differentiation techniques were also initially inefficient and not readily transferable across cell lines, but there are now a number of more robust protocols available. Here, we review the basic biology underlying the differentiation of pluripotent cells to cardiac lineages and describe current state-of-the-art protocols, as well as ongoing refinements. This should provide a useful entry for laboratories new to this area to start their research. Ultimately, efficient and reliable differentiation methodologies are essential to generate desired cardiac lineages to realize the full promise of human pluripotent stem cells for biomedical research, drug development, and clinical applications.

  20. Neuron-NG2 Cell Synapses: Novel Functions for Regulating NG2 Cell Proliferation and Differentiation

    Directory of Open Access Journals (Sweden)

    Qian-Kun Yang

    2013-01-01

    Full Text Available NG2 cells are a population of CNS cells that are distinct from neurons, mature oligodendrocytes, astrocytes, and microglia. These cells can be identified by their NG2 proteoglycan expression. NG2 cells have a highly branched morphology, with abundant processes radiating from the cell body, and express a complex set of voltage-gated channels, AMPA/kainate, and GABA receptors. Neurons notably form classical and nonclassical synapses with NG2 cells, which have varied characteristics and functions. Neuron-NG2 cell synapses could fine-tune NG2 cell activities, including the NG2 cell cycle, differentiation, migration, and myelination, and may be a novel potential therapeutic target for NG2 cell-related diseases, such as hypoxia-ischemia injury and periventricular leukomalacia. Furthermore, neuron-NG2 cell synapses may be correlated with the plasticity of CNS in adulthood with the synaptic contacts passing onto their progenies during proliferation, and synaptic contacts decrease rapidly upon NG2 cell differentiation. In this review, we highlight the characteristics of classical and nonclassical neuron-NG2 cell synapses, the potential functions, and the fate of synaptic contacts during proliferation and differentiation, with the emphasis on the regulation of the NG2 cell cycle by neuron-NG2 cell synapses and their potential underlying mechanisms.

  1. Transcription factor ABF-1 suppresses plasma cell differentiation but facilitates memory B cell formation.

    Science.gov (United States)

    Chiu, Yi-Kai; Lin, I-Ying; Su, Shin-Tang; Wang, Kuan-Hsiung; Yang, Shii-Yi; Tsai, Dong-Yan; Hsieh, Yi-Ting; Lin, Kuo-I

    2014-09-01

    Ag-primed B cells that result from an immune response can form either memory B cells or Ab-secreting plasma cells; however, the molecular machinery that controls this cellular fate is poorly understood. In this study, we show that activated B cell factor-1 (ABF-1), which encodes a basic helix-loop-helix transcriptional repressor, participates in this regulation. ABF-1 was prevalently expressed in purified memory B cells and induced by T follicular helper cell-mediated signals. ABF-1 expression declined by the direct repression of B lymphocyte-induced maturation protein-1 during differentiation. Ectopic expression of ABF-1 reduced the formation of Ab-secreting cells in an in vitro differentiation system of human memory B cells. Accordingly, knockdown of ABF-1 potentiates the formation of Ab-secreting cells. A transgenic mouse that expresses inducible ABF-1 in a B cell-specific manner was generated to demonstrate that the formation of germinal center and memory B cells was augmented by induced ABF-1 in an immune response, whereas the Ag-specific plasma cell response was dampened. This effect was associated with the ability of ABF-1 to limit cell proliferation. Together, our results demonstrate that ABF-1 facilitates formation of memory B cells but prevents plasma cell differentiation.

  2. Neural stem cells differentiated from iPS cells spontaneously regain pluripotency.

    Science.gov (United States)

    Choi, Hyun Woo; Kim, Jong Soo; Choi, Sol; Hong, Yean Ju; Kim, Min Jung; Seo, Han Geuk; Do, Jeong Tae

    2014-10-01

    Differentiated somatic cells can be reprogrammed into pluripotent stem cells by transduction of exogenous reprogramming factors. After induced pluripotent stem (iPS) cells are established, exogenous genes are silenced. In the pluripotent state, retroviral genes integrated in the host genome are kept inactive through epigenetic transcriptional regulation. In this study, we tried to determine whether exogenous genes remain silenced or are reactivated upon loss of pluripotency or on differentiation using an in vitro system. We induced differentiation of iPS cells into neural stem cells (NSCs) in vitro; the NSCs appeared morphologically indistinguishable from brain-derived NSCs and stained positive for the NSC markers Nestin and Sox2. These iPS cell-derived NSCs (iPS-NSCs) were also capable of differentiating into all three neural subtypes. Interestingly, iPS-NSCs spontaneously formed aggregates on long-term culture and showed reactivation of the Oct4-GFP marker, which was followed by the formation of embryonic stem cell-like colonies. The spontaneously reverted green fluorescent protein (GFP)-positive (iPS-NSC-GFP(+) ) cells expressed high levels of pluripotency markers (Oct4 and Nanog) and formed germline chimeras, indicating that iPS-NSC-GFP(+) cells had the same pluripotency as the original iPS cells. The reactivation of silenced exogenous genes was tightly correlated with the downregulation of DNA methyltransferases (Dnmts) during differentiation of iPS cells. This phenomenon was not observed in doxycycline-inducible iPS cells, where the reactivation of exogenous genes could be induced only by doxycycline treatment. These results indicate that pluripotency can be regained through reactivation of exogenous genes, which is associated with dynamic change of Dnmt levels during differentiation of iPS cells.

  3. The PLETHORA Gene Regulatory Network Guides Growth and Cell Differentiation in Arabidopsis Roots

    NARCIS (Netherlands)

    Santuari, Luca; Sanchez-Perez, Gabino F.; Luijten, Marijn; Rutjens, Bas; Terpstra, Inez; Berke, Lidija; Gorte, Maartje; Prasad, Kalika; Bao, Dongping; Timmermans-Hereijgers, Johanna L.P.M.; Maeo, Kenichiro; Nakamura, Kenzo; Shimotohno, Akie; Pencik, Ales; Novak, Ondrej; Ljung, Karin; Heesch, Van Sebastiaan; Bruijn, De Ewart; Cuppen, Edwin; Willemsen, Viola; Mähönen, Ari Pekka; Lukowitz, Wolfgang; Snel, Berend; Ridder, De Dick; Scheres, Ben; Heidstra, Renze

    2016-01-01

    Organ formation in animals and plants relies on precise control of cell state transitions to turn stem cell daughters into fully differentiated cells. In plants, cells cannot rearrange due to shared cell walls. Thus, differentiation progression and the accompanying cell expansion must be tightly

  4. The PLETHORA gene regulatory network guides growth and cell differentiation in Arabidopsis roots

    NARCIS (Netherlands)

    Santuari, L.; Heidstra, R.

    2016-01-01

    Organ formation in animals and plants relies on precise control of cell state transitions to turn stem cell daughters into fully differentiated cells. In plants, cells cannot rearrange due to shared cell walls. Thus, differentiation progression and the accompanying cell expansion must be tightly

  5. Survey of Differentially Methylated Promoters in Prostate Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Yipeng Wang

    2005-08-01

    Full Text Available DNA methylation, copy number in the genomes of three immortalized prostate epithelial, five cancer cell lines (LNCaP, PC3, PC3M, PC3M-Pro4, PC3MLN4 were compared using a microarray-based technique. Genomic DNA is cut with a methylation-sensitive enzyme Hpall, followed by linker ligation, polymerase chain reaction (PCR amplification, labeling, hybridization to an array of promoter sequences. Only those parts of the genomic DNA that have unmethylated restriction sites within a few hundred base pairs generate PCR products detectable on an array. Of 2732 promoter sequences on a test array, 504 (18.5% showed differential hybridization between immortalized prostate epithelial, cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, there were eight (CD44, CDKN1A, ESR1, PLAU, RARB, SFN, TNFRSF6, TSPY previously observed in prostate cancer, 13 previously known methylation targets in other cancers (ARHI, bcl-2, BRCA1, CDKN2C, GADD45A, MTAP, PGR, SLC26A4, SPARC, SYK, TJP2, UCHL1, WIT-1. The majority of genes that appear to be both differentially methylated, differentially regulated between prostate epithelial, cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, GG2-1, representing a rich new source of candidate genes used to study the role of DNA methylation in prostate tumors.

  6. Derivation, characterization and retinal differentiation of induced pluripotent stem cells

    Indian Academy of Sciences (India)

    Subba Rao Mekala; Vasundhara Vauhini; Usha Nagarajan; Savitri Maddileti; Subhash Gaddipati; Indumathi Mariappan

    2013-03-01

    Millions of people world over suffer visual disability due to retinal dystrophies which can be age-related or a genetic disorder resulting in gradual degeneration of the retinal pigmented epithelial (RPE) cells and photoreceptors. Therefore, cell replacement therapy offers a great promise in treating such diseases. Since the adult retina does not harbour any stem cells, alternative stem cell sources like the embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) offer a great promise for generating different cell types of the retina. Here, we report the derivation of four iPSC lines from mouse embryonic fibroblasts (MEFs) using a cocktail of recombinant retroviruses carrying the genes for Oct4, Sox2, Klf4 and cMyc. The iPS clone MEF-4F3 was further characterized for stemness marker expression and stable reprogramming by immunocytochemistry, FACS and RT-PCR analysis. Methylation analysis of the nanog promoter confirmed the reprogrammed epigenetic state. Pluripotency was confirmed by embryoid body (EB) formation and lineage-specific marker expression. Also, upon retinal differentiation, patches of pigmented cells with typical cobble-stone phenotype similar to RPE cells are generated within 6 weeks and they expressed ZO-1 (tight junction protein), RPE65 and bestrophin (mature RPE markers) and showed phagocytic activity by the uptake of fluorescent latex beads.

  7. Cellular interactions regulate stem cell differentiation in tri-culture.

    Science.gov (United States)

    Wang, I-Ning E; Bogdanowicz, Danielle R; Mitroo, Siddarth; Shan, Jing; Kala, Sonam; Lu, Helen H

    2016-11-01

    Currently, the mechanism governing the regeneration of the soft tissue-to-bone interface, such as the transition between the anterior cruciate ligament (ACL) and bone, is not known. Focusing on the ACL-to-bone insertion, this study tests the novel hypothesis that interactions between cells from the ligament (fibroblasts) and bone (osteoblasts) initiate interface regeneration. Specifically, these heterotypic cell interactions direct the fibrochondrogenic differentiation of interface-relevant cell populations, defined here as ligament fibroblasts and bone marrow stromal cells (BMSC). The objective of this study is to examine the effects of heterotypic cellular interactions on BMSC or fibroblast growth and biosynthesis, as well as expression of fibrocartilage-relevant markers in tri-culture. The effects of cell-cell physical contact and paracrine interactions between fibroblasts and osteoblasts were also determined. It was found that, in tri-culture with fibroblasts and osteoblasts, BMSC exhibited greater fibrochondrogenic potential than ligament fibroblasts. The growth of BMSC decreased while proteoglycan production and TGF-β3 expression increased. Moreover, tri-culture regulated BMSC response via paracrine factors, and interestingly, fibroblast-osteoblast contact further promoted proteoglycan and TGF-β1 synthesis as well as induced SOX9 expression in BMSC. Collectively, the findings of this study suggest that fibroblast-osteoblast interactions play an important role in regulating the stem cell niche for fibrocartilage regeneration, and the mechanisms of these interactions are directed by paracrine factors and augmented with direct cell-cell contact.

  8. Regulation of germinal center B-cell differentiation.

    Science.gov (United States)

    Zhang, Yang; Garcia-Ibanez, Laura; Toellner, Kai-Michael

    2016-03-01

    Germinal centers (GC) are the main sites where antigen-activated B-cell clones expand and undergo immunoglobulin gene hypermutation and selection. Iterations of this process will lead to affinity maturation, replicating Darwinian evolution on the cellular level. GC B-cell selection can lead to four different outcomes: further expansion and evolution, apoptosis (non-selection), or output from the GC with differentiation into memory B cells or plasma cells. T-helper cells in GC have been shown to have a central role in regulating B-cell selection by sensing the density of major histocompatibility complex (MHC):peptide antigen complexes. Antigen is provided on follicular dendritic cells in the form of immune complex. Antibody on these immune complexes regulates antigen accessibility by shielding antigen from B-cell receptor access. Replacement of antibody on immune complexes by antibody generated from GC-derived plasma cell output will gradually reduce the availability of antigen. This antibody feedback can lead to a situation where a slow rise in selection stringency caused by a changing environment leads to directional evolution toward higher affinity antibody.

  9. Differentiation mechanism and function of the cereal aleurone cells and hormone effects on them.

    Science.gov (United States)

    Zheng, Yankun; Wang, Zhong

    2014-11-01

    The cereal aleurone cells differentiate from the endosperm epidermis with the exception of endosperm transfer cells. Aleurone cells contain proteins, lipids, and minerals, and are important for digesting the endosperm storage products to nurse the embryo under effects of several hormones during the seed germination. The differentiation of aleurone cells is related to location effect and special gene expression. Moreover, the differentiation of aleurone cells is probably affected by the cues from maternal tissues. In the paper, differentiation mechanism and function of aleurone cells and hormone effects on them are reviewed. Some speculations about the differentiation mechanism of aleurone cells are given here.

  10. Sulfated polysaccharides and cell differentiation in the sea urchin embryo.

    Science.gov (United States)

    Løvtrup-Rein, H; Løvtrup, S

    1984-01-01

    The synthesis of sulfated polysaccharides during the embryonic development of Paracentrotus lividus has been investigated by incorporation of radioactive sulfate, glucose, glucosamine and fucose. The following substances become labelled: fucan sulfate (approximately 60%), heparan sulfate (approximately 20%) and dermatan sulfate (approximately 20%), and possibly a very slight amount of chondroitin sulfate. In animalized and vegetalized embryos, the rate of incorporation is significantly reduced, and furthermore dermatan sulfate is almost absent in animalized embryos. It is concluded that this substance is associated with the differentiation of vegetative cells, possibly the mesenchyme cells.

  11. Large chromatin domains in pluripotent and differentiated cells

    Institute of Scientific and Technical Information of China (English)

    Shibin Hu; Lu Cheng; Bo Wen

    2012-01-01

    Pluripotent stem cells are able to proliferate unlimitedly and to generate all somatic cell types,thus holding a great promise in medical applications.Epigenetic modifications are believed to play crucial roles in regulating pluripotency and differentiation.Recent genome-wide studies on mammalian systems have revealed several types of large chromatin domains which are associated with higherorder organization of the genome.The elucidation of genomic distribution and dynamics of these domains have shed light on the mechanisms underling pluripotency and lineage commitment.

  12. Natural Killer Cell Differentiation From Hematopoietic Stem Cells: A Comparative Analysis of Heparin and Stromal Cell Supported Methods

    OpenAIRE

    2011-01-01

    Natural killer (NK) cells differentiated from hematopietic stem cells (HSCs)may have significant clinical benefits over those from adult donors, including the ability to choose allo-reactive donors and potentially more robust in vivo expansion. Stromal-based methods have been used to study NK differentiation from HSCs. Stroma and cytokines support NK differentiation, but may have considerable regulatory hurdles. Recently, a clinical grade heparin-based method has been reported and could serve...

  13. Ghost cell differentiation and calcification in ameloblastic fibroma.

    Science.gov (United States)

    Luo, Hai Yan; Gao, Yan

    2013-01-01

    Ghost cell differentiation within an ameloblastic fibroma is extremely rare. The ghost cells found in an ameloblastic fibroma in previously reported cases were all associated with a typical calcifying odontogenic cyst. Here, we report a case of an ameloblastic fibroma with focal ghost cells and calcifications in some neoplastic epithelial islands, but without other histologic manifestations consistent with a calcifying odontogenic cyst. The patient was a 13-year-old Chinese boy who presented with a bony-hard swelling in the posterior mandibular region over a 6-month period. Radiographs showed a well-defined multilocular radiolucency associated with an unerupted tooth. The lesion was mostly cystic-solid and comprised of odontogenic epithelial strands, islands and myxoid ectomesenchymal component microscopically. Small groups of ghost cells and calcification were noted in the epithelial islands.

  14. Parameters Identification of Photovoltaic Cells Based on Differential Evolution Algorithm

    Directory of Open Access Journals (Sweden)

    Liao Hui

    2016-01-01

    Full Text Available For the complex nonlinear model of photovoltaic cells, traditional evolution strategy is easy to fall into the local optimal and its identification time is too long when taking parameters identification, then the difference algorithm is proposed in this study, which is to solve the problems of parameter identification in photovoltaic cell model, where it is very difficult to achieve with other identification algorithms. In this method, the random data is selected as the initial generation; the successful evolution to the next generation is done through a certain strategy of difference algorithm, which can achieve the effective identification of control parameters. It is proved that the method has a good global optimization and the fast convergence ability, and the simulation results are shown that the differential evolution has high identification ability and it is an effective method to identify the parameters of photovoltaic cells, where the photovoltaic cells can be widely used in other places with these parameters.

  15. In Vitro Differentiation Potential of Human Placenta Derived Cells into Skin Cells

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    Ruhma Mahmood

    2015-01-01

    Full Text Available Skin autografting is the most viable and aesthetic technique for treatment of extensive burns; however, this practice has potential limitations. Harvesting cells from neonatal sources (such as placental tissue is a simple, inexpensive, and noninvasive procedure. In the current study authors sought to evaluate in vitro potential of human placenta derived stem cells to develop into skin-like cells. After extensive washing, amniotic membrane and umbilical cord tissue were separated to harvest amniotic epithelial cells (AECs and umbilical cord mesenchymal stem cells (UC-MSCs, respectively. Both types of cells were characterized for the expression of embryonic lineage markers and their growth characteristics were determined. AECs and UC-MSCs were induced to differentiate into keratinocytes-like and dermal fibroblasts-like cells, respectively. After induction, morphological changes were detected by microscopy. The differentiation potential was further assessed using immunostaining and RT-PCR analyses. AECs were positive for cytokeratins and E-Cadherin while UC-MSCs were positive for fibroblast specific makers. AECs differentiated into keratinocytes-like cells showed positive expression of keratinocyte specific cytokeratins, involucrin, and loricrin. UC-MSCs differentiated into dermal fibroblast-like cells indicated expression of collagen type 3, desmin, FGF-7, fibroblast activation protein alpha, procollagen-1, and vimentin. In conclusion, placenta is a potential source of cells to develop into skin-like cells.

  16. Directed differentiation of airway epithelial cells of human bone marrow mesenchymal stem cells.

    Science.gov (United States)

    Li, Jian-Dong

    2016-11-01

    The ability to generate lung and airway epithelial cells from human bone marrow mesenchymal stem cells (hBMSCs) would have applications in regenerative medicine, modeling of lung disease, drug screening, and studies of human lung development. In this research, hBMSCs were cultured in specialized airway epithelial cell growth media for differentiation of airway epithelial cells, including keratinocyte growth factor transferrin, bovine pituitary extract, epinephrine, triiodothyronine and retinoic acid. The surfactant protein C, a specific marker of type II pneumocytes, and its corresponding protein were demonstrated by immunofluorescence and western blotting after differentiation of airway epithelial cells, respectively. These cells were then transferred into an induced acute lung injury model. The results showed that the hBMSCs could induce differentiation in airway epithelial cells under the special conditions of the medium, the result for surfactant protein C was positive in differentiated airway epithelial cells using immunofluorescence and western blotting, and these cells were successfully colonized in the injured lung airway. In conclusion, our research shows that a population of airway epithelial cells can be specifically generated from hBMSCs and that induced cells may be allowed to participate in tissue repair.

  17. Differentiation within autologous fibrin scaffolds of porcine dermal cells with the mesenchymal stem cell phenotype

    Energy Technology Data Exchange (ETDEWEB)

    Puente, Pilar de la, E-mail: pilardelapuentegarcia@gmail.com [Tissue Bank, San Francisco Clinic Foundation, Av./Facultad 51, 5°, 24004 León (Spain); Ludeña, Dolores [Pathology Service, University Hospital of Salamanca, P/San Vicente 58-182, 37007 Salamanca (Spain); López, Marta; Ramos, Jennifer; Iglesias, Javier [Tissue Bank, San Francisco Clinic Foundation, Av./Facultad 51, 5°, 24004 León (Spain)

    2013-02-01

    Porcine mesenchymal stem cells (pMSCs) are an attractive source of cells for tissue engineering because their properties are similar to those of human stem cells. pMSCs can be found in different tissues but their dermal origin has not been studied in depth. Additionally, MSCs differentiation in monolayer cultures requires subcultured cells, and these cells are at risk of dedifferentiation when implanting them into living tissue. Following this, we attempted to characterize the MSCs phenotype of porcine dermal cells and to evaluate their cellular proliferation and differentiation in autologous fibrin scaffolds (AFSs). Dermal biopsies and blood samples were obtained from 12 pigs. Dermal cells were characterized by flow cytometry. Frozen autologous plasma was used to prepare AFSs. pMSC differentiation was studied in standard structures (monolayers and pellets) and in AFSs. The pMSCs expressed the CD90 and CD29 markers of the mesenchymal lineage. AFSs afforded adipogenic, osteogenic and chondrogenic differentiation. The porcine dermis can be proposed to be a good source of MSCs with adequate proliferative capacity and a suitable expression of markers. The pMSCs also showed optimal proliferation and differentiation in AFSs, such that these might serve as a promising autologous and implantable material for use in tissue engineering. -- Highlights: ► Low fibrinogen concentration provides a suitable matrix for cell migration and differentiation. ► Autologous fibrin scaffolds is a promising technique in tissue engineering. ► Dermal cells are an easily accessible mesenchymal stem cell source. ► Fibrin scaffolds afforded adipogenic, osteogenic and chondrogenic differentiation.

  18. bFGF promotes adipocyte differentiation in human mesenchymal stem cells derived from embryonic stem cells

    OpenAIRE

    Xinghui Song; Yanwei Li; Xiao Chen; Guoli Yin; Qiong Huang; Yingying Chen; Guowei Xu; Linlin Wang

    2014-01-01

    In this work we describe the establishment of mesenchymal stem cells (MSCs) derived from embryonic stem cells (ESCs) and the role of bFGF in adipocyte differentiation. The totipotency of ESCs and MSCs was assessed by immunofluorescence staining and RT-PCR of totipotency factors. MSCs were successfully used to induce osteoblasts, chondrocytes and adipocytes. MSCs that differentiated into adipocytes were stimulated with and without bFGF. The OD/DNA (optical density/content of total DNA) and exp...

  19. Prion protein expression regulates embryonic stem cell pluripotency and differentiation.

    Directory of Open Access Journals (Sweden)

    Alberto Miranda

    Full Text Available Cellular prion protein (PRNP is a glycoprotein involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs. Although the physiological function of PRNP is largely unknown, its key role in prion infection has been extensively documented. This study examines the functionality of PRNP during the course of embryoid body (EB differentiation in mouse Prnp-null (KO and WT embryonic stem cell (ESC lines. The first feature observed was a new population of EBs that only appeared in the KO line after 5 days of differentiation. These EBs were characterized by their expression of several primordial germ cell (PGC markers until Day 13. In a comparative mRNA expression analysis of genes playing an important developmental role during ESC differentiation to EBs, Prnp was found to participate in the transcription of a key pluripotency marker such as Nanog. A clear switching off of this gene on Day 5 was observed in the KO line as opposed to the WT line, in which maximum Prnp and Nanog mRNA levels appeared at this time. Using a specific antibody against PRNP to block PRNP pathways, reduced Nanog expression was confirmed in the WT line. In addition, antibody-mediated inhibition of ITGB5 (integrin αvβ5 in the KO line rescued the low expression of Nanog on Day 5, suggesting the regulation of Nanog transcription by Prnp via this Itgb5. mRNA expression analysis of the PRNP-related proteins PRND (Doppel and SPRN (Shadoo, whose PRNP function is known to be redundant, revealed their incapacity to compensate for the absence of PRNP during early ESC differentiation. Our findings provide strong evidence for a relationship between Prnp and several key pluripotency genes and attribute Prnp a crucial role in regulating self-renewal/differentiation status of ESC, confirming the participation of PRNP during early embryogenesis.

  20. Differentiation of Human Dental Pulp Stem Cells into Dopaminergic Neuron-like Cells in Vitro.

    Science.gov (United States)

    Chun, So Young; Soker, Shay; Jang, Yu-Jin; Kwon, Tae Gyun; Yoo, Eun Sang

    2016-02-01

    We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.

  1. Matrices secreted during simultaneous osteogenesis and adipogenesis of mesenchymal stem cells affect stem cells differentiation.

    Science.gov (United States)

    Cai, Rong; Nakamoto, Tomoko; Hoshiba, Takashi; Kawazoe, Naoki; Chen, Guoping

    2016-04-15

    The extracellular matrix (ECM) plays a pivotal role in regulating stem cell functions. The ECM dynamically changes during tissue development. It remains a great challenge to mimic the dynamically changing ECM. In this study, we prepared novel types of extracellular matrices that could mimic the dynamic variation of extracellular matrices, which were derived from simultaneous osteogenesis and adipogenesis of human bone marrow-derived mesenchymal stem cells (MSCs). Four ECMs simultaneously mimicking early osteogenesis and early adipogenesis (EOEA), early osteogenesis and late adipogenesis (EOLA), late osteogenesis and early adipogenesis (LOEA), late osteogenesis and late adipogenesis (LOLA) were prepared. The stepwise osteogenesis-co-adipogenesis-mimicking matrices had different compositions and different effects on the osteogenic and adipogenic differentiation of MSCs. The matrices could provide very useful tools to investigate the interaction between ECM and stem cells and the role of ECM on stem cell differentiation. Extracellular matrices (ECMs) are dynamically remodeled to regulate stem cell functions during tissue development. Until now, mimicking the ECM variation during stem cell differentiation to single cell type has been reported. However, there is no report on simultaneous mimicking of stem cell differentiation to two types of cells. In this study, we prepared the mixture ECMs derived from simultaneous osteogenesis and adipogenesis of MSCs at different stages and found that they could regulate stem cell differentiation. The concept is new and the ECMs are novel. No such ECMs have been reported previously. The matrices will provide very useful tools to investigate the interaction between ECM and stem cells and the role of ECM on stem cell differentiation. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  2. DNA double-strand break induction in Ku80-deficient CHO cells following Boron Neutron Capture Reaction

    Directory of Open Access Journals (Sweden)

    Masunaga Shinichiro

    2011-09-01

    Full Text Available Abstract Background Boron neutron capture reaction (BNCR is based on irradiation of tumors after accumulation of boron compound. 10B captures neutrons and produces an alpha (4He particle and a recoiled lithium nucleus (7Li. These particles have the characteristics of high linear energy transfer (LET radiation and have marked biological effects. The purpose of this study is to verify that BNCR will increase cell killing and slow disappearance of repair protein-related foci to a greater extent in DNA repair-deficient cells than in wild-type cells. Methods Chinese hamster ovary (CHO-K1 cells and a DNA double-strand break (DSB repair deficient mutant derivative, xrs-5 (Ku80 deficient CHO mutant cells, were irradiated by thermal neutrons. The quantity of DNA-DSBs following BNCR was evaluated by measuring the phosphorylation of histone protein H2AX (gamma-H2AX and 53BP1 foci using immunofluorescence intensity. Results Two hours after neutron irradiation, the number of gamma-H2AX and 53BP1 foci in the CHO-K1 cells was decreased to 36.5-42.8% of the levels seen 30 min after irradiation. In contrast, two hours after irradiation, foci levels in the xrs-5 cells were 58.4-69.5% of those observed 30 min after irradiation. The number of gamma-H2AX foci in xrs-5 cells at 60-120 min after BNCT correlated with the cell killing effect of BNCR. However, in CHO-K1 cells, the RBE (relative biological effectiveness estimated by the number of foci following BNCR was increased depending on the repair time and was not always correlated with the RBE of cytotoxicity. Conclusion Mutant xrs-5 cells show extreme sensitivity to ionizing radiation, because xrs-5 cells lack functional Ku-protein. Our results suggest that the DNA-DSBs induced by BNCR were not well repaired in the Ku80 deficient cells. The RBE following BNCR of radio-sensitive mutant cells was not increased but was lower than that of radio-resistant cells. These results suggest that gamma-ray resistant cells have

  3. Erythropoietin and the effect of oxygen during proliferation and differentiation of human neural progenitor cells

    Directory of Open Access Journals (Sweden)

    Frech Moritz J

    2010-12-01

    Full Text Available Abstract Background Hypoxia plays a critical role in various cellular mechanisms, including proliferation and differentiation of neural stem and progenitor cells. In the present study, we explored the impact of lowered oxygen on the differentiation potential of human neural progenitor cells, and the role of erythropoietin in the differentiation process. Results In this study we demonstrate that differentiation of human fetal neural progenitor cells under hypoxic conditions results in an increased neurogenesis. In addition, expansion and proliferation under lowered oxygen conditions also increased neuronal differentiation, although proliferation rates were not altered compared to normoxic conditions. Erythropoietin partially mimicked these hypoxic effects, as shown by an increase of the metabolic activity during differentiation and protection of differentiated cells from apoptosis. Conclusion These results provide evidence that hypoxia promotes the differentiation of human fetal neural progenitor cells, and identifies the involvement of erythropoietin during differentiation as well as different cellular mechanisms underlying the induction of differentiation mediated by lowered oxygen levels.

  4. Differentiation of Equine Mesenchymal Stromal Cells into Cells of Neural Lineage: Potential for Clinical Applications

    Directory of Open Access Journals (Sweden)

    Claudia Cruz Villagrán

    2014-01-01

    Full Text Available Mesenchymal stromal cells (MSCs are able to differentiate into extramesodermal lineages, including neurons. Positive outcomes were obtained after transplantation of neurally induced MSCs in laboratory animals after nerve injury, but this is unknown in horses. Our objectives were to test the ability of equine MSCs to differentiate into cells of neural lineage in vitro, to assess differences in morphology and lineage-specific protein expression, and to investigate if horse age and cell passage number affected the ability to achieve differentiation. Bone marrow-derived MSCs were obtained from young and adult horses. Following demonstration of stemness, MSCs were neurally induced and microscopically assessed at different time points. Results showed that commercially available nitrogen-coated tissue culture plates supported proliferation and differentiation. Morphological changes were immediate and all the cells displayed a neural crest-like cell phenotype. Expression of neural progenitor proteins, was assessed via western blot or immunofluorescence. In our study, MSCs generated from young and middle-aged horses did not show differences in their ability to undergo differentiation. The effect of cell passage number, however, is inconsistent and further experiments are needed. Ongoing work is aimed at transdifferentiating these cells into Schwann cells for transplantation into a peripheral nerve injury model in horses.

  5. Spheroid culture for enhanced differentiation of human embryonic stem cells to hepatocyte-like cells.

    Science.gov (United States)

    Subramanian, Kartik; Owens, Derek Jason; Raju, Ravali; Firpo, Meri; O'Brien, Timothy D; Verfaillie, Catherine M; Hu, Wei-Shou

    2014-01-15

    Stem cell-derived hepatocyte-like cells hold great potential for the treatment of liver disease and for drug toxicity screening. The success of these applications hinges on the generation of differentiated cells with high liver specific activities. Many protocols have been developed to guide human embryonic stem cells (hESCs) to differentiate to the hepatic lineage. Here we report cultivation of hESCs as three-dimensional aggregates that enhances their differentiation to hepatocyte-like cells. Differentiation was first carried out in monolayer culture for 20 days. Subsequently cells were allowed to self-aggregate into spheroids. Significantly higher expression of liver-specific transcripts and proteins, including Albumin, phosphoenolpyruvate carboxykinase, and asialoglycoprotein receptor 1 was observed. The differentiated phenotype was sustained for more than 2 weeks in the three-dimensional spheroid culture system, significantly longer than in monolayer culture. Cells in spheroids exhibit morphological and ultrastructural characteristics of primary hepatocytes by scanning and transmission electron microscopy in addition to mature functions, such as biliary excretion of metabolic products and cytochrome P450 activities. This three-dimensional spheroid culture system may be appropriate for generating high quality, functional hepatocyte-like cells from ESCs.

  6. Differentiation of mouse bone marrow derived stem cells toward microglia-like cells

    Directory of Open Access Journals (Sweden)

    Stolzing Alexandra

    2011-08-01

    Full Text Available Abstract Background Microglia, the macrophages of the brain, have been implicated in the causes of neurodegenerative diseases and display a loss of function during aging. Throughout life, microglia are replenished by limited proliferation of resident microglial cells. Replenishment by bone marrow-derived progenitor cells is still under debate. In this context, we investigated the differentiation of mouse microglia from bone marrow (BM stem cells. Furthermore, we looked at the effects of FMS-like tyrosine kinase 3 ligand (Flt3L, astrocyte-conditioned medium (ACM and GM-CSF on the differentiation to microglia-like cells. Methods We assessed in vitro-derived microglia differentiation by marker expression (CD11b/CD45, F4/80, but also for the first time for functional performance (phagocytosis, oxidative burst and in situ migration into living brain tissue. Integration, survival and migration were assessed in organotypic brain slices. Results The cells differentiated from mouse BM show function, markers and morphology of primary microglia and migrate into living brain tissue. Flt3L displays a negative effect on differentiation while GM-CSF enhances differentiation. Conclusion We conclude that in vitro-derived microglia are the phenotypic and functional equivalents to primary microglia and could be used in cell therapy.

  7. Differentiated cell behavior: a multiscale approach using measure theory.

    Science.gov (United States)

    Colombi, Annachiara; Scianna, Marco; Tosin, Andrea

    2015-11-01

    This paper deals with the derivation of a collective model of cell populations out of an individual-based description of the underlying physical particle system. By looking at the spatial distribution of cells in terms of time-evolving measures, rather than at individual cell paths, we obtain an ensemble representation stemming from the phenomenological behavior of the single component cells. In particular, as a key advantage of our approach, the scale of representation of the system, i.e., microscopic/discrete vs. macroscopic/continuous, can be chosen a posteriori according only to the spatial structure given to the aforesaid measures. The paper focuses in particular on the use of different scales based on the specific functions performed by cells. A two-population hybrid system is considered, where cells with a specialized/differentiated phenotype are treated as a discrete population of point masses while unspecialized/undifferentiated cell aggregates are represented by a continuous approximation. Numerical simulations and analytical investigations emphasize the role of some biologically relevant parameters in determining the specific evolution of such a hybrid cell system.

  8. [Progress in early pancreas development and reprogramming of terminally differentiated cells into β cells].

    Science.gov (United States)

    Mingjun, Cao; Huansheng, Dong; Qingjie, Pan; Hongjun, Wang; Xiao, Dong

    2014-06-01

    Type 1 diabetes mellitus (T1DM) is an autoimmune disease in which the immune system attacks insulin-secreting β cells, thus leading to an absolute deficiency of insulin. Patients must rely on exogenous insulin, which cannot effectively prevent diabetes complications. Generation of insulin-secreting cells by reprogramming of pluripotent stem cells or somatic cells is a potential approach for the treatment of T1DM. These cells can be used for cell therapy and drug screening, and may eventually provide a cure for the disease. Significant progress has been made in generating insulin-secreting cells through the expression of β cell specific transcription factors in stem cells or somatic cells. In this review, we summarize recent research progress in early pancreas development, β cell specific transcription factors and reprogramming of terminally differentiated cells into β cells.

  9. Role of p53 mutation in the effect of boron neutron capture therapy on oral squamous cell carcinoma

    Directory of Open Access Journals (Sweden)

    Ohnishi Ken

    2009-12-01

    Full Text Available Abstract Background Boron neutron capture therapy (BNCT is a selective radiotherapy, being effective for the treatment of even advanced malignancies in head and neck regions as well as brain tumors and skin melanomas. To clarify the role of p53 gene, the effect of BNCT on oral squamous cell carcinoma (SCC cells showing either wild- (SAS/neo or mutant-type (SAS/mp53 p53 was examined. Methods Cells were exposed to neutron beams in the presence of boronophenylalanine (BPA at Kyoto University Research Reactor. Treated cells were monitored for modulations in colony formation, proliferation, cell cycle, and expression of cell cycle-associated proteins. Results When SAS/neo and SAS/mp53 cells were subjected to BNCT, more suppressive effects on colony formation and cell viability were observed in SAS/neo compared with SAS/mp53 cells. Cell cycle arrest at the G1 checkpoint was observed in SAS/neo, but not in SAS/mp53. Apoptotic cells increased from 6 h after BNCT in SAS/neo and 48 h in SAS/mp53 cells. The expression of p21 was induced in SAS/neo only, but G2 arrest-associated proteins including Wee1, cdc2, and cyclin B1 were altered in both cell lines. Conclusion These results indicate that oral SCC cells with mutant-type are more resistant to BNCT than those with wild-type p53, and that the lack of G1 arrest and related apoptosis may contribute to the resistance. At a physical dose affecting the cell cycle, BNCT inhibits oral SCC cells in p53-dependent and -independent manners.

  10. Structural basis for hemoglobin capture by Staphylococcus aureus cell-surface protein, IsdH.

    Science.gov (United States)

    Krishna Kumar, Kaavya; Jacques, David A; Pishchany, Gleb; Caradoc-Davies, Tom; Spirig, Thomas; Malmirchegini, G Reza; Langley, David B; Dickson, Claire F; Mackay, Joel P; Clubb, Robert T; Skaar, Eric P; Guss, J Mitchell; Gell, David A

    2011-11-01

    Pathogens must steal iron from their hosts to establish infection. In mammals, hemoglobin (Hb) represents the largest reservoir of iron, and pathogens express Hb-binding proteins to access this source. Here, we show how one of the commonest and most significant human pathogens, Staphylococcus aureus, captures Hb as the first step of an iron-scavenging pathway. The x-ray crystal structure of Hb bound to a domain from the Isd (iron-regulated surface determinant) protein, IsdH, is the first structure of a Hb capture complex to be determined. Surface mutations in Hb that reduce binding to the Hb-receptor limit the capacity of S. aureus to utilize Hb as an iron source, suggesting that Hb sequence is a factor in host susceptibility to infection. The demonstration that pathogens make highly specific recognition complexes with Hb raises the possibility of developing inhibitors of Hb binding as antibacterial agents.

  11. Inhibition of FGF signaling accelerates neural crest cell differentiation of human pluripotent stem cells.

    Science.gov (United States)

    Jaroonwitchawan, Thiranut; Muangchan, Pattamon; Noisa, Parinya

    2016-12-02

    Neural crest (NC) is a transient population, arising during embryonic development and capable of differentiating into various somatic cells. The defects of neural crest development leads to neurocristopathy. Several signaling pathways were revealed their significance in NC cell specification. Fibroblast growth factor (FGF) is recognized as an important signaling during NC development, for instance Xenopus and avian; however, its contributions in human species are remained elusive. Here we used human pluripotent stem cells (hPSCs) to investigate the consequences of FGF inhibition during NC cell differentiation. The specific-FGF receptor inhibitor, SU5402, was used in this investigation. The inhibition of FGF did not found to affect the proliferation or death of hPSC-derived NC cells, but promoted hPSCs to commit NC cell fate. NC-specific genes, including PAX3, SLUG, and TWIST1, were highly upregulated, while hPSC genes, such as OCT4, and E-CAD, rapidly reduced upon FGF signaling blockage. Noteworthy, TFAP-2α, a marker of migratory NC cells, abundantly presented in SU5402-induced cells. This accelerated NC cell differentiation could be due to the activation of Notch signaling upon the blockage of ERK1/2 phosphorylation, since NICD was increased by SU5402. Altogether, this study proposed the contributions of FGF signaling in controlling human NC cell differentiation from hPSCs, the crosstalk between FGF and Notch, and might imply to the influences of FGF signaling in neurocristophatic diseases.

  12. Human dental pulp stem cells express many pluripotency regulators and differentiate into neuronal cells

    Institute of Scientific and Technical Information of China (English)

    Behnam Ebrahimi; Mohammad Mehdi Yaghoobi; Ali Mohammadi Kamal-abadi; Maryam Raoof

    2011-01-01

    Stem cells were isolated from human dental pulp using an optimized method, in which pulp pieces were digested by enzymes and immobilized to enhance cell outgrowth. Stem cell marker expression was detected by reverse transcription-PCR (RT-PCR), and differentiation markers were detected by real-time quantitative RT-PCR and immunocytochemistry. Results showed that dental pulp stem cells actively expressed nanog, oct4, nucleostemin slain-1, jmjd1a, jmjd2c, and cyclin D1. When stem cells were induced to differentiate into neurons, nucleostemin, nanog, and cyclin D1 expres-sion significantly decreased, whereas expression of neuronal markers, such as microtubule asso-ciated protein-2 and neurofilament-heavy, significantly increased. These results suggested that stem cells exited a pluripotent state and entered a neuronal differentiation pathway. In addition, results demonstrated that human dental pulp serves as a reservoir of stem cells that express defined stem cell markers; these cells were easily isolated and were induced to differentiate towards a desired cell lineage.

  13. Direct Differentiation of Human Pluripotent Stem Cells into Haploid Spermatogenic Cells

    Science.gov (United States)

    Easley, Charles A.; Phillips, Bart T.; McGuire, Megan M.; Barringer, Jennifer M.; Valli, Hanna; Hermann, Brian P.; Simerly, Calvin R.; Rajkovic, Aleksander; Miki, Toshio; Orwig, Kyle E.; Schatten, Gerald P.

    2013-01-01

    Summary Human embryonic (hESCs) and induced pluripotent stem cells (hiPSCs) have been shown to differentiate into primordial germ cells (PGCs) but not into spermatogonia nor haploid spermatocytes or spermatids. Here we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages including post-meiotic, spermatid-like cells in vitro without genetic manipulation. Furthermore, our procedure mirrors spermatogenesis in vivo by differentiating pluripotent stem cells into UTF1, PLZF and CDH1-positive spermatogonia-like cells, HIWI and HILI-positive spermatocyte-like cells, and haploid cells expressing acrosin, transition protein 1 and protamine 1, proteins found uniquely in either spermatids and/or sperm. These spermatids show uniparental genomic imprints similar to human sperm on two loci: H19 and IGF2. These results demonstrate that male pluripotent stem cells have the capability to directly differentiate into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro. PMID:22921399

  14. Differentiation of adult human bone marrow mesenchymal stem cells into Schwann-like cells in vitro

    Institute of Scientific and Technical Information of China (English)

    YANG Li-ye; ZHENG Jia-kun; WANG Chao-yang; LI Wen-yu

    2005-01-01

    Objective: To investigate the differentiative capability of adult human bone marrow mesenchymal stem cells (BMSCs) into Schwann-like cells. Methods: Bone marrows were aspirated from healthy donors and mononuclear cells were separated by Percoll lymphocytes separation liquid (1.073 g/ml) with centrifugation, cells were cultured in DMEM/F12 (1:1) medium containing 10% fetal bovine serum (FBS), 20 ng/ml epidermal growth factor (EGF) and 20 ng/ml basic fibroblast growth factor (bFGF). Cells of passage 1 were identified with immunocytochemistry. Conclusions: Bone marrow contains the stem cells with the ability of differentiating into Schwann-like cells, which may represent an alternative stem cell sources for neural transplantation.

  15. Generation of patient-specific pluripotent stem cells and directed differentiation of embryonic stem cells for regenerative medicine

    Institute of Scientific and Technical Information of China (English)

    Minyue Ma; Jiahao Sha; Zuomin Zhou; Qi Zhou; Qingzhang Li

    2008-01-01

    Embryonic stem(ES) cells are pluripotent cells that can give rise to derivatives of all three embryonic germ layers. Due to its characteristics, the patient-specific ES cells are of great potential for transplantation therapies. Several strategies can reprogramme somatic cells back to pluripotent stem cells: nuclear transfer, fusion with ES cells, treatment with cell extract and induction by specific factors. Considering the future clinical use, the differentiation from ES to neurons, cardiomyocytes and many other types of cell scurrently provide basic cognition and experience to regenerative medicine. This article will review two courses, the reprogramming of differentiated cells and the differentiation of ES cells to specific cell types.

  16. Fibrin-fiber architecture influences cell spreading and differentiation.

    Science.gov (United States)

    Bruekers, Stéphanie M C; Jaspers, Maarten; Hendriks, José M A; Kurniawan, Nicholas A; Koenderink, Gijsje H; Kouwer, Paul H J; Rowan, Alan E; T S Huck, Wilhelm

    2016-09-02

    The mechanical and structural properties of the extracellular matrix (ECM) play an important role in regulating cell fate. The natural ECM has a complex fibrillar structure and shows nonlinear mechanical properties, which are both difficult to mimic synthetically. Therefore, systematically testing the influence of ECM properties on cellular behavior is very challenging. In this work we show two different approaches to tune the fibrillar structure and mechanical properties of fibrin hydrogels. Addition of extra thrombin before gelation increases the protein density within the fibrin fibers without significantly altering the mechanical properties of the resulting hydrogel. On the other hand, by forming a composite hydrogel with a synthetic biomimetic polyisocyanide network the protein density within the fibrin fibers decreases, and the mechanics of the composite material can be tuned by the PIC/fibrin mass ratio. The effect of the changes in gel structure and mechanics on cellular behavior are investigated, by studying human mesenchymal stem cell (hMSC) spreading and differentiation on these gels. We find that the trends observed in cell spreading and differentiation cannot be explained by the bulk mechanics of the gels, but correlate to the density of the fibrin fibers the gels are composed of. These findings strongly suggest that the microscopic properties of individual fibers in fibrous networks play an essential role in determining cell behavior.

  17. Mesenchymal stem cells from rat olfactory bulbs can differentiate into cells with cardiomyocyte characteristics.

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    Huang, Yuahn-Sieh; Li, I-Hsun; Chueh, Sheau-Huei; Hueng, Dueng-Yuan; Tai, Ming-Cheng; Liang, Chang-Min; Lien, Shiu-Bii; Sytwu, Huey-Kang; Ma, Kuo-Hsing

    2015-12-01

    Mesenchymal stromal/stem cells (MSCs) are widely distributed in different tissues such as bone marrow, adipose tissues, peripheral blood, umbilical cord and amnionic fluid. Recently, MSC-like cells were also found to exist in rat olfactory bulb and are capable of inducing differentiation into mesenchymal lineages - osteocytes, chondrocytes and adipocytes. However, whether these cells can differentiate into myocardial cells is not known. In this study, we examined whether olfactory bulb-derived MSCs could differentiate into myocardial cells in vitro. Fibroblast-like cells isolated from the olfactory bulb of neonatal rats were grown under four conditions: no treatment; in the presence of growth factors (neuregulin-1, bFGF and forskolin); co-cultured with cardiomyocytes; and co-cultured with cardiomyocytes plus neuregulin-1, bFGF and forskolin. Cell differentiation into myocardial cells was monitored by RT-PCR, light microscopy immunofluorescence, western blot analysis and contractile response to pharmacological treatments. The isolated olfactory bulb-derived fibroblast-like cells expressed CD29, CD44, CD90, CD105, CD166 but not CD34 and CD45, consistent with the characteristics of MSCs. Long cylindical cells that spontaneously contracted were only observed following 7 days of co-culture of MSCs with rat cardiomyocytes plus neuregulin-1, bFGF and forskolin. RT-PCR and western blot analysis indicated that the cylindrical cells expressed myocardial markers, such as Nkx2.5, GATA4, sarcomeric α-actinin, cardiac troponin I, cardiac myosin heavy chain, atrial natriuretic peptide and connexin 43. They also contained sarcomeres and gap junction and were sensitive to pharmacological treatments (adrenal and cholinergic agonists and antagonists). These findings indicate that rat olfactory bulb-derived fibroblast-like cells with MSC characteristics can differentiate into myocardial-like cells.

  18. Differentiation of Adipose-derived Stem Cells into Schwann Cell Phenotype in Comparison with Bone Marrow Stem Cells

    Directory of Open Access Journals (Sweden)

    Zolikha Golipoor

    2010-06-01

    Full Text Available Objective(sBone marrow is the traditional source of human multipotent mesenchymal stem cells (MSCs, but adipose tissue appears to be an alternative and more readily available source. In this study, rat adipose-derived stem cells (ADSCs were induced to differentiate into Schwann-like cells and compared with rat bone marrow stem cells (BMSCs for their Schwann-like cells differentiation potential. Materials and MethodsBMSCs and ADSCs were characterized for expression of MSCs-specific markers, osteogenic and adipogenic differentiation. They were induced to differentiate into Schwann-like cells and analyzed for expression of the Schwann specific markers. The immunocytochemical differentiation markers were S-100 and real time quantitative Real-time polymerase chain reaction (RT-PCR markers were S100, P75 and glial fibrillary acidic protein (GFAP. 3-(4, 5-Dimethylthiazol- 2-yl-2, 5-diphenyltetrazolium bromide (MTT assay and Annexin V-Fluorescein isothiocyanate (FITC/ Propidium iodide (PI double labeling method were employed to detect early stage cell apoptosis.ResultsBMSCs and ADSCs showed similarities in expression of the MSC-specific markers, osteogenic and adipogenic differentiation. Both quantitative RT-PCR and immunocytochemical analysis demonstrated that BMSCs and ADSCs had equal expression of the Schwann-specific markers following Schwann-like cells differentiation. However, gene expression of P75 was higher in BMSCs compared with ADSCs. MTT assay and flow cytometry found that of the total BMSCs and ADSCs in the culture medium, 20% to 30% of the cells died, but the remaining cell population remained strongly attached to the substrate and differentiated.ConclusionComparative analysis showed that Schwann-like cell differentiation potential of ADSCs was slightly decreased in comparison with BMSCs. Therefore, BMSCs are more favorable choice than ADSCs for tissue engineering.

  19. Bone marrow mesenchymal stem cells stimulate proliferation and neuronal differentiation of retinal progenitor cells.

    Directory of Open Access Journals (Sweden)

    Jing Xia

    Full Text Available During retina development, retinal progenitor cell (RPC proliferation and differentiation are regulated by complex inter- and intracellular interactions. Bone marrow mesenchymal stem cells (BMSCs are reported to express a variety of cytokines and neurotrophic factors, which have powerful trophic and protective functions for neural tissue-derived cells. Here, we show that the expanded RPC cultures treated with BMSC-derived conditioned medium (CM which was substantially enriched for bFGF and CNTF, expressed clearly increased levels of nuclear receptor TLX, an essential regulator of neural stem cell (NSC self-renewal, as well as betacellulin (BTC, an EGF-like protein described as supporting NSC expansion. The BMSC CM- or bFGF-treated RPCs also displayed an obviously enhanced proliferation capability, while BMSC CM-derived bFGF knocked down by anti-bFGF, the effect of BMSC CM on enhancing RPC proliferation was partly reversed. Under differentiation conditions, treatment with BMSC CM or CNTF markedly favoured RPC differentiation towards retinal neurons, including Brn3a-positive retinal ganglion cells (RGCs and rhodopsin-positive photoreceptors, and clearly diminished retinal glial cell differentiation. These findings demonstrate that BMSCs supported RPC proliferation and neuronal differentiation which may be partly mediated by BMSC CM-derived bFGF and CNTF, reveal potential limitations of RPC culture systems, and suggest a means for optimizing RPC cell fate determination in vitro.

  20. An improved protocol that induces human embryonic stem cells to differentiate into neural cells in vitro.

    Science.gov (United States)

    Zhou, Jun-Mei; Chu, Jian-Xin; Chen, Xue-Jin

    2008-01-01

    Human embryonic stem (ES) cells have the capacity for self-renewal and are able to differentiate into any cell type. However, obtaining high-efficient neural differentiation from human ES cells remains a challenge. This study describes an improved 4-stage protocol to induce a human ES cell line derived from a Chinese population to differentiate into neural cells. At the first stage, embryonic bodies (EBs) were formed in a chemically-defined neural inducing medium rather than in traditional serum or serum-replacement medium. At the second stage, rosette-like structures were formed. At the third stage, the rosette-like structures were manually selected rather than enzymatically digested to form floating neurospheres. At the fourth stage, the neurospheres were further differentiated into neurons. The results show that, at the second stage, the rate of the formation of rosette-like structures from EBs induced by noggin was 88+/-6.32%, higher than that of retinoic acid 55+/-5.27%. Immunocytochemistry staining was used to confirm the neural identity of the cells. These results show a major improvement in obtaining efficient neural differentiation of human ES cells.

  1. Capacity of Human Dental Follicle Cells to Differentiate into Neural Cells In Vitro

    Directory of Open Access Journals (Sweden)

    Shingo Kanao

    2017-01-01

    Full Text Available The dental follicle is an ectomesenchymal tissue surrounding the developing tooth germ. Human dental follicle cells (hDFCs have the capacity to commit to differentiation into multiple cell types. Here we investigated the capacity of hDFCs to differentiate into neural cells and the efficiency of a two-step strategy involving floating neurosphere-like bodies for neural differentiation. Undifferentiated hDFCs showed a spindle-like morphology and were positive for neural markers such as nestin, β-III-tubulin, and S100β. The cellular morphology of several cells was neuronal-like including branched dendrite-like processes and neurites. Next, hDFCs were used for neurosphere formation in serum-free medium containing basic fibroblast growth factor, epidermal growth factor, and B27 supplement. The number of cells with neuronal-like morphology and that were strongly positive for neural markers increased with sphere formation. Gene expression of neural markers also increased in hDFCs with sphere formation. Next, gene expression of neural markers was examined in hDFCs during neuronal differentiation after sphere formation. Expression of Musashi-1 and Musashi-2, MAP2, GFAP, MBP, and SOX10 was upregulated in hDFCs undergoing neuronal differentiation via neurospheres, whereas expression of nestin and β-III-tubulin was downregulated. In conclusion, hDFCs may be another optimal source of neural/glial cells for cell-based therapies to treat neurological diseases.

  2. β-Cell neogenesis: experimental considerations in adult stem cell differentiation.

    Science.gov (United States)

    Iskovich, Svetlana; Goldenberg-Cohen, Nitza; Stein, Jerry; Yaniv, Isaac; Farkas, Daniel L; Askenasy, Nadir

    2011-04-01

    The contribution of stem cells derived from adult tissues to the recovery of pancreatic islets from chemical injury is controversial. Analysis of nonhematopoietic differentiation of bone marrow-derived cells has yielded positive and negative results under different experimental conditions. Using the smallest subset of bone marrow cells lacking immuno-hematopoietic lineage markers, we have detected incorporation and conversion into insulin-producing cells. Donor cells identified by genomic markers silence green fluorescent protein (GFP) expression as a feature of differentiation, in parallel to expressing PDX-1 and proinsulin. Here we elaborate potential experimental difficulties that might result in false-negative results. The use of GFP as a reporter protein is suboptimal for differentiation experiments: (a) the bone marrow of GFP donors partially expresses the reporter protein, (b) differentiating bone marrow cells silence GFP expression, and (c) the endocrine pancreas is constitutively negative for GFP. In addition, design of the experiments, data analysis, and interpretation encounter numerous objective and subjective difficulties. Rigorous evaluation under optimized experimental conditions confirms the capacity of adult bone marrow-derived stem cells to adopt endocrine developmental traits, and demonstrates that GFP downregulation and silencing is a feature of differentiation.

  3. Acrylamide inhibits cellular differentiation of human neuroblastoma and glioblastoma cells.

    Science.gov (United States)

    Chen, Jong-Hang; Chou, Chin-Cheng

    2015-08-01

    This study explores human neuroblastoma (SH-SY5Y) and human glioblastoma (U-1240 MG) cellular differentiation changes under exposure to acrylamide (ACR). Differentiation of SH-SY5Y and U-1240 MG cells were induced by retinoic acid (RA) and butyric acid (BA), respectively. Morphological observations and MTT assay showed that the induced cellular differentiation and cell proliferation were inhibited by ACR in a time- and dose-dependent manner. ACR co-treatment with RA attenuated SH-SY5Y expressions of neurofilament protein-L (NF-L), microtubule-associated protein 1b (MAP1b; 1.2 to 0.7, p < 0.001), MAP2c (2.2 to 0.8, p < 0.05), and Janus kinase1 (JAK1; 1.9 to 0.6, p < 0.001), while ACR co-treatment with BA attenuated U-1240 MG expressions of glial fibrillary acidic protein (GFAP), MAP1b (1.2 to 0.6, p < 0.001), MAP2c (1.5 to 0.7, p < 0.01), and JAK1 (2.1 to 0.5, p < 0.001), respectively. ACR also decreased the phosphorylation of extracellular-signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) in U-1240 MG cells, while caffeine reversed this suppression of ERK and JNK phosphorylation caused by ACR treatment. These results showed that RA-induced neurogenesis of SH-SY5Y and BA-induced astrogliogenesis of U-1240 MG cells were attenuated by ACR and were associated with down-regulation of MAPs expression and JAK-STAT signaling.

  4. Differentiation of Mesenchymal Stem Cells Into Dopaminergic Neuron-like Cells in vitro

    Institute of Scientific and Technical Information of China (English)

    LI GUO; HONG-XUE FAN; FEI YIN; HONG-QI MENG; LING LING; TA-NA HU-HE; PENG LI; CHUN-XIA ZHANG; SHUN YU; DE-SHENG DUAN

    2005-01-01

    Objective To explore the way to induce mesenchymal stem cells (MSCs) to differentiate into dopaminergic neurons in vitro. Methods MSCs were obtained from rat bone marrow, cultured and passaged. MSCs used in this experiment had multipotency, which was indirectly proved by being induced to differentiate into chondrocytes and adipocytes. MSCs were cultured in medium containing 0.5 mmol/L IBMX for 2 days. Then the medium was replaced with induction medium, which contained GDNF, IL-1β, mesencephalic glial-cell-conditioned medium and flash-frozen mesencephalic membrane fragments. The surface markers of the differentiated neurons, such as NSE, nestin, MAP-2a, b and TH were detected by immunocytochemistry and Western blot after MSCs were cultured in induction medium for 7 days and 15 days. Results MSCs differentiated into neural progenitors and expressed nestin after MSCs were incubated with medium containing IBMX for 2 d. After the medium was replaced with induction medium containing many inducing agents, MSCs differentiated into neuron-like cells and dopaminergic neuron-like cells and expressed NSE, MAP-2a, b and TH. The percentage of NSE-positive cells, MAP-2a, b-positive cells and TH-positive cells was 30.032±2.489%, 41.580±5.101% and 34.958±5.534%, respectively after MSCs were induced in medium containing GDNF, IL-1β, mesencephalic glial-cell-conditioned medium and flash-frozen mesencephalic membrane fragments for 15 days. Conclusion MSCs can differentiate into dopaminergic neuron-like cells and are a new cell source for the treatment of neurodegeneration diseases and have a great potential for wide application.

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