Reviewer
Hui Zhu
  • Scientist, Stanford University
Research fields
  • Cell Biology
Isolation of Primary Human Skeletal Muscle Cells
Authors:  Janelle M. Spinazzola and Emanuela Gussoni, date: 11/05/2017, view: 9240, Q&A: 0
Primary myoblast culture is a valuable tool in research of muscle disease, pathophysiology, and pharmacology. This protocol describes techniques for dissociation of cells from human skeletal muscle biopsies and enrichment for a highly myogenic population by fluorescence-activated cell sorting (FACS). We also describe methods for assessing myogenicity and population expansion for subsequent in vitro study.
Isolation of Keratan Sulfate Disaccharide-branched Chondroitin Sulfate E from Mactra chinensis
Authors:  Kyohei Higashi and Toshihiko Toida, date: 08/05/2017, view: 6517, Q&A: 0
Glycosaminoglycans (GAGs) including chondroitin sulfate (CS), dermatan sulfate (DS), heparin (HP), heparan sulfate (HS) and keratan sulfate (KS) are linear, sulfated repeating disaccharide sequences containing hexosamine and uronic acid (or galactose in the case of KS). Recently, a keratan sulfate (KS) disaccharide [GlcNAc6S(β1-3)Galactose(β1-]-branched CS-E was identified from the clam species M. chinensis. Here, we report the isolation protocol for KS-branched CS from M. chinensis.
Formaldehyde Fixation of Extracellular Matrix Protein Layers for Enhanced Primary Cell Growth
Authors:  Natalia V. Andreeva and Alexander V. Belyavsky, date: 07/05/2017, view: 8307, Q&A: 0
Coating tissue culture vessels with the components of the extracellular matrix such as fibronectin and collagens provides a more natural environment for primary cells in vitro and stimulates their proliferation. However, the effects of such protein layers are usually rather modest, which might be explained by the loss immobilized proteins due to their weak non-covalent association with the tissue culture plastic. Here we describe a simple protocol for a controlled fixation of fibronectin, vitronectin and collagen IV layers by formaldehyde, which substantially enhances the stimulation of primary cell proliferation by these extracellular proteins.
Screening for Novel Endogenous Inflammatory Stimuli Using the Secreted Embryonic Alkaline Phosphatase NF-κB Reporter Assay
Authors:  Lorena Zuliani-Alvarez, Anna M. Piccinini and Kim S Midwood, date: 04/05/2017, view: 9504, Q&A: 0
An immune response can be activated by pathogenic stimuli, as well as endogenous danger signals, triggering the activation of pattern recognition receptors and initiating signalling cascades that lead to inflammation. This method uses THP1-BlueTM cells, a human monocytic cell line which contains an embryonic alkaline phosphatase reporter gene allowing the detection of NF-κB-induced transcriptional activation. We validated this protocol by assessing NF-κB activation after stimulation of toll-like receptor 4 (TLR4) by two different agonists: lipopolysaccharide (LPS), derived from the cell wall of Gram negative bacteria, and tenascin-C, an extracellular matrix protein whose expression is induced upon tissue injury. We then used this protocol to screen for potential new endogenous TLR4 agonists, but this method can also be used as a quick, economical and reliable means to assay the activity of other inflammatory stimuli resulting in TLR-dependent NF-κB activation.
Ex vivo Culture of Fetal Mouse Gastric Epithelial Progenitors
Isolation and tridimensional culture of murine fetal progenitors from the digestive tract represents a new approach to study the nature and the biological characteristics of these epithelial cells that are present before the onset of the cytodifferentiation process during development. In 2013, Mustata et al. described the isolation of intestinal fetal progenitors growing as spheroids in the ex vivo culture system initially implemented by Sato et al. (2009) to grow adult intestinal stem cells. Noteworthy, fetal-derived spheroids have high self-renewal capacity making easy their indefinite maintenance in culture. Here, we report an adapted protocol for isolation and ex vivo culture and maintenance of fetal epithelial progenitors from distal pre-glandular stomach growing as gastric spheroids (Fernandez Vallone et al., 2016).
Ex vivo Culture of Adult Mouse Antral Glands
The tri-dimensional culture, initially described by Sato et al. (2009) in order to isolate and characterize epithelial stem cells of the adult small intestine, has been subsequently adapted to many different organs. One of the first examples was the isolation and culture of antral stem cells by Barker et al. (2010), who efficiently generated organoids that recapitulate the mature pyloric epithelium in vitro. This ex vivo approach is suitable and promising to study gastric function in homeostasis as well as in disease. We have adapted Barker’s protocol to compare homeostatic and regenerating tissues and here, we meticulously describe, step by step, the isolation and culture of antral glands as well as the isolation of single cells from antral glands that might be useful for culture after cell sorting as an example (Fernandez Vallone et al., 2016).
Retinal Differentiation of Mouse Embryonic Stem Cells
Author:  Anna La Torre, date: 07/05/2016, view: 14182, Q&A: 5
Groundbreaking studies from Dr. Yoshiki Sasai’s laboratory have recently introduced novel methods to differentiate mouse and human Embryonic Stem Cells (mESCs and hESCs) into organ-like 3D structures aimed to recapitulate developmental organogenesis programs (Eiraku et al., 2011; Eiraku and Sasai, 2012; Nakano et al., 2012; Kamiya et al., 2011). We took advantage of this method to optimize a 3D protocol to efficiently generate retinal progenitor cells and subsequently retinal neurons in vitro. This culture system provides an invaluable platform both to study early developmental processes and to obtain retinal neurons for transplantation approaches. The protocol described here has been successfully applied to several mouse ESC (including the R1, WD44 and G4 cell lines) and mouse induced-Pluripotent Stem Cell (iPSCs) lines.
Preparation of Synovial Mesenchymal Stem Cells from a Rat Knee Joint
Authors:  Nobutake Ozeki, Takeshi Muneta, Mitsuru Mizuno and Ichiro Sekiya, date: 05/05/2016, view: 9308, Q&A: 2
Mesenchymal stem cells (MSCs), first described in human bone marrow, are emerging as promising cell-based therapeutics for a wide range of diseases (Caplan and Correa, 2011). MSCs have been isolated from various organs in the body, and synovial MSCs were first reported by De Bari et al. (2001). We previously reported that synovial MSCs have superior proliferation and chondrogenic potentials as compared to bone marrow-, muscle-, and adipose- derived MSCs in humans (Sakaguchi et al., 2005) and rats (Yoshimura et al., 2007). In addition, administration of synovial MSCs for osteochondral defect promoted cartilage regeneration in a rabbit (Koga et al., 2008) and a pig model (Nakamura et al., 2012). In 2008, we started a clinical trial in human and obtained satisfactory results of symptoms and regenerated cartilage by Magnetic Resonance Imaging (Sekiya et al., 2015). We have also engaged in multiple research lines using synovial MSCs for meniscus regeneration in rats (Horie et al., 2009; Horie et al., 2012; Katagiri et al., 2013; Okuno et al., 2014; Ozeki et al., 2015). In this article, we demonstrated how to harvest the synovium including infrapatellar fat pad from a rat knee joint, and to describe the technique of isolation and culture of rat synovial MSCs.
Glioma Associated Stem Cells (GASCs) Isolation and Culture
Glioma Associated Stem Cells (GASCs) represent a population of non-tumorigenic multipotent stem cells hosted in the microenvironment of human gliomas. In vitro, these cells are able, through the release of exosomes, to increase the biological aggressiveness of glioma-initiating cells. The clinical importance of this finding is supported by the strong prognostic value associated with the GASCs surface immunophenotype thus suggesting that this patient-based approach can provide a groundbreaking method to predict prognosis and to exploit novel strategies that target the tumor stroma (Bourkoula et al., 2014).
Monocyte-MSC Co-cultures
Authors:  Sara M. Melief, C. L. M. Schrama and Helene Roelofs, date: 01/20/2015, view: 11574, Q&A: 0
To assess the effect of multipotent stromal cells (MSC) on monocytes, 3-day cultures were performed of freshly isolated monocytes in MSC-conditioned medium (CM). As a control condition, monocytes were stimulated with low dose macrophage colony-stimulating factor (M-CSF). Monocytes were isolated from peripheral blood mononuclear cell (PBMC) populations by magnetic activated cell sorting (MACS) using CD14 microbeads.
We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.