Methods in molecular biology (Clifton, N.J.) (Meth Mol Biol)

Publications in this journal

• Article: Basic Protocols to Investigate hMSC Behavior onto Electrospun Fibers.

  Marco A Alvarez-Perez, Vincenzo Guarino, Valentina Cirillo, Luigi Ambrosio
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  ABSTRACT: Human mesenchymal stem cells (hMSC) currently represent a major cell resource in the research laboratory, to study differentiated-cell behavior in 3D scaffolds during the regeneration processes. Adhesion and differentiation of stem cells to a specific phenotype are achieved by culturing them in apposite culture media under precise conditions. Meanwhile, hydrolytic degradation of polymeric scaffolds allows implanted cells to synthesize their own extracellular matrix in situ after implantation so that the degeneration of the foreign scaffold is temporally matched by creation of the new innate one. In this context, structural properties and biochemical signals may concur to influence the cell response to the environmental stimuli during the culture. So, it becomes mandatory to introduce robust protocols to treat hMSC alone-before the culture-and in combination with the scaffolds for the next investigation by scanning electron microscopy. Here, we describe the protocols used to manage hMSC before and during the culture in order to obtain more detailed information on cell mechanisms mediated by polymeric scaffolds.
  Methods in molecular biology (Clifton, N.J.) 05/2013;
• Article: General Protocol for the Culture of Cells on Plasma-Coated Electrospun Scaffolds.

  A Géraldine Guex, Giuseppino Fortunato, Dirk Hegemann, Hendrik T Tevaearai, Marie-Noëlle Giraud
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  ABSTRACT: As opposed to culture on standard tissue-treated plastic, cell culture on three-dimensional scaffolds impedes additional challenges with respect to substrate preparation, cell seeding, culture maintenance, and analysis. We herewith present a general route for the culture of primary cells, differentiated cells, or stem cells on plasma-coated, electrospun scaffolds. We describe a method to prepare and fix the scaffolds in culture wells and discuss a convenient method for cell seeding and subsequent analysis by scanning electron microscopy or immunohistology.
  Methods in molecular biology (Clifton, N.J.) 05/2013;
• Article: Live Imaging of Early Mouse Embryos Using Fluorescently Labeled Transgenic Mice.

  Takaya Abe, Shinichi Aizawa, Toshihiko Fujimori
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  ABSTRACT: Live imaging is a powerful approach to understanding the dynamic processes that occur during development. Periimplantation mouse embryos are transparent, making them suitable for live imaging. In this chapter we describe the culturing and live imaging of mouse embryos, and also introduce the reporter mouse lines for organelle labeling we recently established.
  Methods in molecular biology (Clifton, N.J.) 05/2013;
• Article: Micro-CT Technique for Three-Dimensional Visualization of Human Stem Cells.

  Andrea Farini, Chiara Villa, Marzia Belicchi, Mirella Meregalli, Yvan Torrente
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  ABSTRACT: Micro-CT offers high spatial resolution of the distribution of stem cells and provides rapid reconstruction of 3D images and quantitative volumetric analysis. Together with real-time PCR analysis, micro-CT offers the possibility to obtain a quantification of the number of cells that are able to migrate from the bloodstream inside the muscular tissues. Here, we studied for the first time the kinetics of the human cells injected into the femoral artery of DMD animal model. It is fundamental to determine whether the cells disseminate and entrap only within the capillary system of downstream muscles and/or they are able to reach the non-injected muscles and other organs through blood flow. The efficient transplantation of stem cells to dystrophic-deficient muscle reinforced the utility of intra-arterial delivery of cells as a viable approach for cell-based clinical therapies of neuromuscular diseases.
  Methods in molecular biology (Clifton, N.J.) 05/2013;
• Article: Molecular Imaging and Tracking Stem Cells in Neurosciences.

  Toma Spiriev, Nora Sandu, Bernhard Schaller
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  ABSTRACT: Stem cell transplantation is a promising new therapeutic option in different neurological diseases. However, it is not yet possible to translate its potential from animal models to clinical application. One of the main problems of applying stem cell transplantation in clinical medium is the difficulty of detection, localization, and examination of the stem cells in vivo at both cellular and molecular levels. State-of-the-art molecular imaging techniques provide new and better means for noninvasive, repeated, and quantitative tracking of stem cell implant or transplant. From initial deposition to the survival, migration, and differentiation of the transplant/implanted stem cells, current molecular imaging methods allow monitoring of the infused cells in the same live recipient over time. The present review briefly summarizes and compares these molecular imaging methods for cell labeling and imaging in animal models as well as in clinical application and sheds light on consecutive new therapeutic options if appropriate.
  Methods in molecular biology (Clifton, N.J.) 05/2013;
• Article: Conversion of Primordial Germ Cells to Pluripotent Stem Cells: Methods for Cell Tracking and Culture Conditions.

  Go Nagamatsu, Toshio Suda
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  ABSTRACT: Primordial germ cells (PGCs) are unipotent cells committed to germ lineage: PGCs can only differentiate into gametes in vivo. However, upon fertilization, germ cells acquire the capacity to differentiate into all cell types in the body, including germ cells. Therefore, germ cells are thought to have the potential for pluripotency. PGCs can convert to pluripotent stem cells in vitro when cultured under specific conditions that include bFGF, LIF, and the membrane-bound form of SCF (mSCF). Here, the culture conditions which efficiently convert PGCs to pluripotent embryonic germ (EG) cells are described, as well as methods used for identifying pluripotent candidate cells during culture.
  Methods in molecular biology (Clifton, N.J.) 05/2013;
• Article: High-Content Imaging and Analysis of Pluripotent Stem Cell-Derived Cardiomyocytes.

  Gábor Földes, Maxime Mioulane
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  ABSTRACT: Human pluripotent stem cells (hPSC) are investigated as a source of authentic human cardiac cells for drug discovery and toxicological tests. Cell-based assays using automated fluorescence imaging platform and high-content analysis characterize hypertrophic and toxicity profiles of compounds in hPSC-derived cardiomyocytes (hPSC-CM) at the cellular and subcellular levels. In purified population of hPSC-CM loaded with cell tracer probe and cell death markers, both hypertrophic and toxicity profiles can be assessed in live cardiomyocyte cultures. Alternatively, in non-purified cultures of hPSC-CM, hypertrophy, proliferation, and cell death assays can be performed specifically in the cardiomyocyte subpopulation using antibodies directed against cardiac proteins and a combination of cell death- and proliferation-specific fluorescent probes.
  Methods in molecular biology (Clifton, N.J.) 05/2013;
• Article: Noninvasive Imaging of Myocardial Blood Flow Recovery in Response to Stem Cell Intervention.

  Hualei Zhang, Rong Zhou
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  ABSTRACT: The recovery of myocardial blood flow is a major indicator of the effectiveness of cell-based therapies for ischemic heart diseases including myocardial infarction. Blood flow (also called perfusion) of the heart muscle can be noninvasively measured via imaging methods such as ultrasound, positron emission tomography (PET), or magnetic resonance imaging (MRI). Here, we describe an MRI technique, namely, spin labeling, to measure the volumetric blood flow (mL/min/g) in the heart. Specifically, we demonstrate how impaired blood flow in the infarcted region of the heart was recovered transiently (≥2 weeks) after the injection of endothelial progenitor cells.
  Methods in molecular biology (Clifton, N.J.) 05/2013;
• Article: Primary Culture and Live Imaging of Adult Neural Stem Cells and Their Progeny.

  Felipe Ortega, Benedikt Berninger, Marcos R Costa
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  ABSTRACT: Adult neural stem cells (NSC) generate neurons throughout life, but little is known about the sequence of events involved in the transition from NSC to neurons. Studying the intermediary steps involved in the specification of neuronal cells from NSCs requires observation of cells in real time. Here we describe a primary culture of the adult subependymal zone (SEZ) which allows for continuous live imaging to characterize the mode of cell division and lineage progression of adult NSCs and their progeny. To this end, cells are cultured at low density under adherent conditions and without growth factors. Under these conditions, NSCs display classical hallmarks of adult SEZ NSCs in vivo, such as astroglial marker expression and promoter activity, a slow cell cycle, and a predominantly neurogenic potential. Video time-lapse microscopy experiments using this cell preparation allow for studying the steps involved in the generation of fast-dividing precursors and neuroblasts from slow-dividing astroglia/NSCs.
  Methods in molecular biology (Clifton, N.J.) 05/2013;
• Article: Live Imaging, Identifying, and Tracking Single Cells in Complex Populations In Vivo and Ex Vivo.

  Minjung Kang, Panagiotis Xenopoulos, Silvia Muñoz-Descalzo, Xinghua Lou, Anna-Katerina Hadjantonakis
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  ABSTRACT: Advances in optical imaging technologies combined with the use of genetically encoded fluorescent proteins have enabled the visualization of stem cells over extensive periods of time in vivo and ex vivo. The generation of genetically encoded fluorescent protein reporters that are fused with subcellularly localized proteins, such as human histone H2B, has made it possible to direct fluorescent protein reporters to specific subcellular structures and identify single cells in complex populations. This facilitates the visualization of cellular behaviors such as division, movement, and apoptosis at a single-cell resolution and, in principle, allows the prospective and retrospective tracking towards determining the lineage of each cell.
  Methods in molecular biology (Clifton, N.J.) 05/2013;
• Article: Covisualization of Methylcytosine, Global DNA, and Protein Biomarkers for In Situ 3D DNA Methylation Phenotyping of Stem Cells.

  Jian Tajbakhsh
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  ABSTRACT: DNA methylation and histone modifications are key regulatory mechanisms in cellular differentiation, and are skewed in complex diseases. Therefore, analyzing the higher nuclear organization of methylated DNA in conjunction with relevant cellular components, such as protein biomarkers, may well add cell-by-cell-specific spatial and temporal information to quantitative molecular data for the discovery of stem cell differentiation-related signaling networks and their exploitation in the therapeutic reprogramming of cells. The in situ fluorescent covisualization of methylated DNA (methylated CG dinucleotides = MeC), global DNA (gDNA), and proteins has been challenging, as the immunofluorescence detection of MeC sites requires thorough denaturing of double-stranded DNA for antigen (methylated carbon-5 of cytosine) retrieval. The protocol we present overcomes this obstacle through optimization of cell membrane permeabilization, acid treatment, and intermediate fixation steps to preserve immunostaining of biomarkers and delineate MeC and gDNA, while conserving the captured three-dimensional (3D) structure of the cells; making it suitable for high-resolution confocal microscopy, 3D visualization, and topological analyses of fixed cultured cells as well as fresh and frozen tissue sections.
  Methods in molecular biology (Clifton, N.J.) 04/2013;
• Article: Patterned Polymeric Surfaces to Study the Influence of Nanotopography on the Growth and Differentiation of Mesenchymal Stem Cells.

  Cristian Pablo Pennisi, Vladimir Zachar, Trine Fink, Leonid Gurevich, Peter Fojan
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  ABSTRACT: The implementation of micro- and nanotechnologies to biomaterials constitutes a unique platform to improve our understanding on microenvironmental regulation of stem cell functions. In the recent years, various methods have been developed for the fabrication of micro- and nanopatterned polymeric culture substrates, and many of these novel surfaces are opening possibilities for new applications. Here, we provide procedures for creating nanoscale topographic features on films of poly(lactic acid), a biodegradable polymer frequently used for the fabrication of tissue engineering scaffolds. In addition, we provide methods to assess the growth and differentiation of mesenchymal stem cells cultured on the substrates.
  Methods in molecular biology (Clifton, N.J.) 04/2013;
• Article: In Vitro Nanoparticle-Mediated Intracellular Delivery into Human Adipose-Derived Stem Cells.

  Rosario Sanchez-Martin, Victoria Cano-Cortés, Juan Antonio Marchal, Macarena Perán
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  ABSTRACT: Adipose-derived stem cells (ASCs) are multipotent cells that are emerging as an extremely promising therapeutic agent for tissue regeneration. The ability to manipulate ASC phenotypes by the delivery of biologically active cargoes is essential to understand their role and to design novel therapeutic strategies based on the use of ASCs. Here we describe a simple and efficient protocol for the conjugation and efficient delivery of biological materials into ASCs based on the use of polystyrene nanoparticles as carrier system.
  Methods in molecular biology (Clifton, N.J.) 04/2013;
• Article: Chondrogenic Differentiation of Menstrual Blood-Derived Stem Cells on Nanofibrous Scaffolds.

  Somaieh Kazemnejad, Amir-Hassan Zarnani, Manijeh Khanmohammadi, Sahba Mobini
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  ABSTRACT: Cartilage tissue engineering is a promising technology to restore and repair cartilage lesions in the body. In recent years, significant advances have been made using stem cells as a cell source for clinical goals of cartilage tissue engineering. Menstrual blood-derived stem cells (MenSCs) is a novel population of stem cells that demonstrate the potential and differentiate into a wide range of tissues including the chondrogenic lineage. Incorporation of cell culture with extracellular matrix (ECM) like substratum plays an important role in cartilage tissue regeneration by providing attachment sites as well as bioactive signals for cells to grow and differentiate into chondrogenic lineage. The electrospun nanofibers are a class of polymer-based biomaterials that have been extensively utilized in tissue engineering as ECM-like scaffold. This chapter discusses potential of electrospun nanofibers for cell-based cartilage tissue engineering and presents detailed protocols on immunophenotyping characterization and chondrogenic differentiation of MenSCs seeded in poly-ε-caprolactone (PCL) nanofibers. The isolated MenSCs are characterized using flow cytometry, seeded on the nanofibers, imaged using scanning electron microscopy, and subsequently differentiated into chondrogenic lineage in culture medium containing specific growth factors and cytokines. Immunofluorescence and alcian blue staining are used to evaluate the development of seeded MenSCs in PCL nanofibrous scaffold into chondrogenic lineage.
  Methods in molecular biology (Clifton, N.J.) 04/2013;
• Article: Transduction of Murine Embryonic Stem Cells by Magnetic Nanoparticle-Assisted Lentiviral Gene Transfer.

  Sarah Rieck, Katrin Zimmermann, Daniela Wenzel
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  ABSTRACT: Genetic modification of embryonic stem (ES) cells is a valuable technique when combined with cell replacement strategies. Obtaining stable transgene expression and low-cytotoxicity lentiviral transduction of ES cells is advantageous. It has been shown that the efficiency of transfection and transduction approaches can be increased by magnetic nanoparticles (MNPs). Here, we present a protocol for MNP-assisted lentiviral transduction of adherent mouse ES cells. The application of MNPs increased transduction efficiency and provided the opportunity of cell positioning by a magnetic field.
  Methods in molecular biology (Clifton, N.J.) 04/2013;
• Article: Engineering of a 3D Nanostructured Scaffold Made of Functionalized Self-Assembling Peptides and Encapsulated Neural Stem Cells.

  Carla Cunha, Silvia Panseri, Fabrizio Gelain
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  ABSTRACT: Three-dimensional (3D) in vitro models of cell culture aim to fill the gap between the standard 2D cell studies and the in vivo environment. Especially for neural tissue regeneration approaches where there is little regenerative capacity, such models must rely on scaffolds that mimic the extracellular matrix in providing support; allowing the natural flow of oxygen, nutrients, and growth factors; and possibly favoring neural cell regrowth. Their combined use with stem cells has many potentialities for tissue engineering applications. Here, we describe a new 3D model of stem cell culture, using a nanostructured biomaterial, made of self-assembling peptides, where adult neural stem cells are completely embedded. This new 3D cell culture system takes advantage of the nano- and microfiber assembling process of these biomaterials under physiological conditions. The assembled scaffold forms an intricate and biologically active matrix able to display specifically designed functional motifs such as RGD, BMHP1, and BMHP2. Such model has the potential to be tailored to develop ad hoc designed peptides for specific cell lines.
  Methods in molecular biology (Clifton, N.J.) 03/2013;
• Article: Peptide Nanofiber Scaffolds for Multipotent Stromal Cell Culturing.

  Seher Ustun, Samet Kocabey, Mustafa O Guler, Ayse B Tekinay
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  ABSTRACT: Self-assembled peptide nanofibers are versatile materials providing suitable platforms for regenerative medicine applications. This chapter describes the use of peptide nanofibers as extracellular matrix mimetic scaffolds for two-dimensional (2D) and three-dimensional (3D) multipotent stromal cell culture systems and procedures for in vitro experiments using these scaffolds. Preparation of 2D and 3D peptide nanofiber scaffolds and cell culturing procedures are presented as part of in vitro experiments including cell adhesion, viability, and spreading analysis. Analysis of cellular differentiation on peptide nanofiber scaffolds is described through immunocytochemistry, qRT-PCR, and other biochemical experiments towards osteogenic and chondrogenic lineage.
  Methods in molecular biology (Clifton, N.J.) 03/2013;
• Article: Lanthanide-Based Upconversion Nanoparticles for Connexin-Targeted Imaging in Co-cultures.

  Sounderya Nagarajan, Yong Zhang
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  ABSTRACT: From the perspective of deep tissue imaging, it is required that the excitation light can penetrate deep enough to excite the sample of interest and the fluorescence emission is strong enough to be detected. The longer wavelengths like near infrared are absorbed less by the tissue and are scattered less implying deeper penetration. This has drawn interest to the class of nanoparticles called upconversion nanoparticles (UCNs) which has an excitation in the near-infrared wavelength and the emission is in the visible/near-infrared wavelength (depending on the doped ions). Here, we discuss surface modification of the UCNs to make them hydrophilic allowing dispersion in physiological buffers and enabling conjugation of antibody to their surface. It was of interest to use connexin 43 gap junction protein-specific antibody on UCNs to target cardiac cell such as H9c2 and co-culture of bone marrow stem cells and H9c2.
  Methods in molecular biology (Clifton, N.J.) 03/2013;