1Division of Hematology-oncology, Department of Medicine, Medical University of South Carolina
Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.
Published March 7, 2013. Keywords: Medicine, Biomedical Engineering, Stem Cell Biology, Genetics, Immunology, Anatomy, Physiology, Cellular Biology, Surgery, Y Chromosome, Hematopoietic Stem Cells, HSC, stem cells, Bone Marrow Transplantation, Real-Time Polymerase Chain Reaction, rtPCR, PCR, Chimerism, Y chromosome specific gene, graft, engraftment, isolation, transplantation, cell culture, murine model, animal model
1Department of Anesthesia, Brigham and Woman's Hospital, 2Department of Radiology, Brigham and Woman's Hospital
We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.
Published September 21, 2009. Keywords: Developmental Biology, Mouse, skeletal muscle, in vivo, fluorescence imaging, cell therapy, longitudinal monitoring, quantification
JoVE Immunology and Infection
1Department of Pathology, Stanford University School of Medicine, 2Department of Microbiology & Immunology, Stanford University School of Medicine
We describe a method for the generation of in vitro derived mast cells, their engraftment into mast cell-deficient mice, and the analysis of the phenotype, numbers and distribution of engrafted mast cells at different anatomical sites. This protocol can be used to assess the functions of mast cells in vivo.
Published May 27, 2015. Keywords: Immunology, c-kit, stem cell factor, FcεRI, immunoglobulin E, mouse model, adoptive transfer, immunology, allergy
1Department of Neurology, Vanderbilt University Medical Center, 2Vanderbilt Ingram Cancer Center, Vanderbilt University Medical Center, 3Neurology Service, Veteran Affairs TVHS
Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models.
Published January 14, 2014. Keywords: Medicine, Glioma, Malignant glioma, primary orthotopic xenograft, isocitrate dehydrogenase
JoVE Immunology and Infection
1Molecular Pathology, Cancer Center and Center for Regenerative Medicine, Massachusetts General Hospital, 2Harvard Stem Cell Institute, 3GABBA - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto
Here, we present a protocol for cell transplantation of zebrafish skeletal muscle and embryonal rhabdomyosarcoma (ERMS) into adult immune compromised rag2E450fs homozygous mutant zebrafish. This protocol allows for the efficient analysis of regeneration and malignant transformation of muscle cells.
Published December 26, 2014. Keywords: Immunology, zebrafish, immune compromised, transplantation, muscle, rhabdomyosarcoma, rag2E450fs, rag2fb101, fluorescent, transgenic
1Department of Cell and Developmental Biology, University College London, 2Division of Regenerative Medicine, Stem Cells and Gene Therapy, San Raffaele Hospital
Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.…
Published January 20, 2014. Keywords: Bioengineering, Skeletal Muscle, Muscle Cells, Muscle Fibers, Skeletal, Pericytes, Stem Cells, Induced Pluripotent Stem Cells (iPSCs), Muscular Dystrophies, Cell Differentiation, animal models, muscle stem/progenitor cells, mesoangioblasts, muscle regeneration, iPSC-derived mesoangioblasts (IDEMs)
1Toronto General Research Institute, University Health Network, 2Department of Pathology, University Health Network, 3Division of General Surgery, University Health Network
Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.…
Published September 25, 2013. Keywords: Medicine, Liver Neoplasms, Hepatectomy, animal models, hepatocellular carcinoma, xenograft, cancer, liver, subcutaneous, intrahepatic, orthotopic, mouse, human, immunodeficient
JoVE Immunology and Infection
1Department of Developmental Biology and Cancer Research, Institute for Medical Research Israel-Canada, Hebrew University Medical School, 2Institute of Pulmonary Medicine, Hadassah-Hebrew University Medical Center
Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.
Published June 19, 2015. Keywords: Immunology, Neutrophil isolation, tumor-entrained neutrophils, high-density neutrophils, low-density neutrophils, anti-tumor cytotoxicity, BrdU labeling, CFSE labeling, luciferase assay, neutrophil depletion, anti-metastatic activity, lung metastatic seeding assay, neutrophil adoptive transfer.
1Department of Biomedical Physiology and Kinesiology, Simon Fraser University, 2The Biomedical Research Centre, University of British Columbia, 3Division of Neurology, Department of Medicine, Neuromuscular Disease Unit, VHHSC
We describe a protocol whereby busulfan conditioning permits the bone marrow of a recipient mouse to be replaced with bone marrow cells from donor mice ubiquitously expressing green fluorescent protein, in the absence of irradiation. This technique is useful to study bone marrow cell accumulation in the central nervous system.
Published April 1, 2015. Keywords: Medicine, busulfan, bone marrow transplantation, myelosuppressive conditioning, chimerism, hematopoietic stem cells, immunobiology, flow cytometry
JoVE Immunology and Infection
1Department of Microbiology and Immunology, University of North Carolina at Chapel Hill
This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.
Published May 13, 2015. Keywords: Immunology, Immunology, thymus, transplantation, tolerance, negative selection, T cell development
1Center for Regenerative Medicine, Massachusetts General Hospital
Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.
Published May 18, 2011. Keywords: Cellular Biology, zebrafish, kidney, regeneration, transplantation
1Department of Leukemia, MD Anderson Cancer Center, 2Wake Forest Baptist Medical Center, Institute for Regenerative Medicine
Complex tissue masses, from organs to tumors, are composed of various cellular elements. We elucidated the contribution of cellular phenotypes within a tissue utilizing multi-labeled fluorescent transgenic mice in combination with multiparameter immunofluorescent staining followed by spectral unmixing to decipher cell origin as well as cell characteristics based on protein expression.…
Published September 22, 2013. Keywords: Medicine, Immunology, Medicine, Cellular Biology, Molecular Biology, Genetics, Anatomy, Physiology, Biomedical Engineering, Immunohistochemistry (IHC), Microscopy, Fluorescence, Regeneration, Cellular Microenvironment, Tumor Microenvironment, Cell Biology, Investigative Techniques, Biological Phenomena, Mesenchymal stem cells (MSC), Tumor/Cancer associated fibroblasts (TAF/CAF), transgenic mouse model, regenerative medicine, wound healing, cancer
1Department of Surgery, University of California, 2Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, 3Biomedical Sciences Program, University of California
The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique
Published January 27, 2011. Keywords: Medicine, development, stem cells, transplantation, in utero
1Department of Medicine (Hematology, Oncology, and Transplant), University of Minnesota, Minneapolis, 2Stem Cell Institute, University of Minnesota, Minneapolis
This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.
Published April 23, 2013. Keywords: Stem Cell Biology, Bioengineering, Biomedical Engineering, Medicine, Physiology, Anatomy, Cellular Biology, Molecular Biology, Biochemistry, Hematology, Embryonic Stem Cells, ESCs, ES Cells, Hematopoietic Stem Cells, HSC, Pluripotent Stem Cells, Induced Pluripotent Stem Cells, iPSCs, Luciferases, Firefly, Immunotherapy, Immunotherapy, Adoptive, stem cells, differentiation, NK cells, in vivo imaging, fluorescent imaging, turboFP650, FACS, cell culture
1Light Microscopy Core Facility, NHLBI/NIH, 2Hematology Branch, NHLBI/NIH
Combinatorial 5 fluorescent proteins marking of hematopoietic stem and progenitor cells allows in vivo clonal tracking via confocal and two-photon microscopy, providing insights into bone marrow hematopoietic architecture during regeneration. This method allows non-invasive fate mapping of spectrally-coded HSPCs-derived cells in intact tissues for extensive periods of time following transplantation.
Published August 6, 2014. Keywords: Stem Cell Biology, LeGO imaging, clonal tracking, fluorescent proteins, confocal microscopy, multiphoton microscopy, hematopoiesis, lentiviral vectors, hematopoietic stem cells
1Department of Pediatrics, University of Colorado Anschutz Medical Campus, 2Department of Bone Marrow Transplantation, University Hospital of Essen
Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.
Published September 18, 2013. Keywords: Medicine, Leukemia, Receptor Protein-Tyrosine Kinases, Molecular Targeted Therapy, Therapeutics, novel small molecule inhibitor, receptor tyrosine kinase, leukemia
1Department of Ophthalmology, Saint Louis University
Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.
Published November 17, 2014. Keywords: Immunology, Transplantation, Allograft Responses, Immune Privilege, Cornea, Inflammatory cells, T cells, Macrophages
1Stem Cell Institute, Paul and Sheila Wellstone Muscular Dystrophy Center, Department of Neurology, University of Minnesota Medical School
The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.
Published April 8, 2014. Keywords: Cellular Biology, skeletal muscle, muscle stem cell, satellite cell, regeneration, myoblast transplantation, muscular dystrophy, self-renewal, differentiation, myogenesis
1Magdi Yacoub Institute, Imperial College London, 2National Heart and Lung Institute, Imperial College London, 3Australian Regenerative Medicine Institute, Monash University
Intramyocardial cell delivery in murine models of cardiovascular diseases, such as hypertension or myocardial infarction, is widely used to test the therapeutic potential of different cell types in regenerative studies. Therefore, a detailed description and a clear visualization of this surgical procedure will help to define the limits and advantages of cardiovascular cell therapeutic analyses in small rodents.…
Published January 24, 2014. Keywords: Medicine, intramyocardial cell injection, heart, grafting, cell therapy, stem cells, fibrotic tissue
1Program in Molecular Medicine and Department of Cancer Biology, University of Massachusetts Medical School, 2Departments of Surgery and Medicine, Weill Cornell Medical College, 3Departments of Surgery and Medicine, New York Presbyterian Hospital
A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.…
Published November 13, 2012. Keywords: Cancer Biology, Medicine, Genetics, Molecular Biology, Melanoma, zebrafish, Danio rerio, mitfa, melanocytes, tumor model, miniCoopR
1Department of Internal Medicine V, Innsbruck Medical University, 2Oncotyrol GmbH, 3Tyrolean Cancer Research Institute, 4Division of Vascular Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institute
Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.
Published May 1, 2015. Keywords: Medicine, CAM, angiogenesis, mesenchymal cells, multiple myeloma, GFP, 3- dimensional tissue culture, xenografts, tumor
JoVE Immunology and Infection
1Laboratory of Emerging Viruses, Heinrich Pette Institute, Leibniz Institute for Experimental Virology
There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.
Published June 25, 2015. Keywords: Immunology, Mouse models, vaccines, immunity, dendritic cells, influenza, T cells
1Department of Bioengineering, University of Illinois at Urbana-Champaign
This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.
Published June 14, 2015. Keywords: Medicine, Myocardial infarction, stem cell therapy, hydrogel, fibrin-based glue, cell-encapsulated patch, hydrogel adhesion
1Division of Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, 2Center for Regenerative Therapeutics, Brigham and Women's Hospital, 3Harvard Medical School, Harvard University, 4Harvard Stem Cell Institute, Harvard University, 5Harvard-MIT Division of Health Sciences and Technology, 6Department of Mechanical Engineering, Massachusetts Institute of Technology
This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.…
Published October 16, 2013. Keywords: Bioengineering, Microfluidics, Endothelial Cells, Leukocyte Rolling, HL-60 cells, TNF-α, P-selectin, E-selectin
1Emmy Noether-Group for Stem Cell Biology, Department of Molecular Embryology, Institute of Anatomy and Cell Biology, University of Freiburg, 2Spemann Graduate School of Biology and Medicine and Faculty of Biology, University of Freiburg, 3School of Life Sciences, Keele University, 4Center for Biological Signaling Studies (BIOSS), University of Freiburg
We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology.
Published December 18, 2014. Keywords: Neuroscience, CD markers, surface antigens, intracellular antigens, flow cytometry, neurons, glial cells, neural stem cells, fluorescence-activated cell sorting (FACS), CD24, CD54, CFSE
1Case Comprehensive Cancer Center, Case Western Reserve University
Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.
Published October 11, 2011. Keywords: Medicine, firefly luciferase, Renilla Luciferase, breast cancer, metastasis, Smad
1Department of Radiology, Molecular Imaging Program at Stanford (MIPS), 2Stanford School of Medicine, Stanford University
We describe a technique for labeling and tracking stem cells with FDA-approved, superparamagnetic iron oxide (SPIO), ferumoxytol (Feraheme). This cellular imaging technique that utilizes magnetic resonance (MR) imaging for visualization, is readily accessible for long-term monitoring and diagnosis of successful or unsuccessful stem cell engraftments in patients.
Published November 4, 2011. Keywords: Medicine, USPIO, cell labeling, MR imaging, MRI, molecular imaging, iron oxides, ferumoxytol, cellular imaging, nanoparticles
1Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, 2The Department of Veterans Affairs Medical Center, 3Internal Medicine, Vanderbilt University School of Medicine
A useful tool to analyze the effects of drugs, growth factors, and/or manipulated cells in an animal model of wound repair is described. This technique utilizes the properties of a polyvinyl alcohol (PVA) sponge to deliver and contain the desired treatment and also provide a platform to be excised and analyzed.
Published April 18, 2012. Keywords: Medicine, Polyvinyl alcohol (PVA) sponge, engraftment, stem cells, granulation tissue, vascularization, tumorgenesis, drug delivery, wound model, physiology
1Human Oncology and Pathogenesis Program, Department of Medicine, Memorial Sloan-Kettering Cancer Center
This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.
Published July 5, 2014. Keywords: Medicine, Bone marrow, Leukemia, Hematopoiesis, Aspiration, Mouse Model, Hematopoietic Stem Cell
JoVE Immunology and Infection
1Center for Inflammatory Bowel Diseases, Division of Digestive Diseases, David Geffen School of Medicine, The University of California Los Angeles, Los Angeles
Bone marrow transplantation provides a way to change the genotype of the bone marrow derived cells. If the gene of interest is expressed in both bone marrow derived cells and non-bone marrow derived cells, bone marrow transplantation can change the bone marrow derived cells to a different genotype without changing the non-bone marrow derived cell genotype.
Published October 1, 2012. Keywords: Immunology, Genetics, Cellular Biology, Physiology, Bone marrow transplantation, colitis, mice, irradiation
JoVE Immunology and Infection
1Centre d'Immunologie et des Maladies Infectieuses (CIMI), INSERM, U1135, CNRS, ERL 8255, Sorbonne Universités, UPMC Univ Paris 06, CR7
Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.
Published February 27, 2015. Keywords: Immunology, Immunology, hematology, Intravital imaging, bone marrow, monocytes, cell trafficking.
1Department of Life Science and Facility for Imaging by Light Microscopy, Imperial College London, 2Department of Life Sciences, Imperial College London
The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.
Published September 4, 2014. Keywords: Cellular Biology, hematopoietic stem cell, multiphoton microscopy, cell tracking, bone marrow niche, calvarium, intra-vital confocal microscopy, time-lapse imaging, multi-modal microscopy
1Department of Hemostasis and Thrombosis, Leiden University Medical Center
Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.
Published February 8, 2015. Keywords: Medicine, Breast Cancer, Tumor growth, Orthotopic Injection, Metastasis
1Contrast Agent Research Group at the Center for Molecular and Functional Imaging, Department of Radiology, University of California San Francisco
For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.
Published March 31, 2008. Keywords: Cell Biology, cell labeling, stem cell, MR imaging, cell tracking, iron oxide, contrast agents, mesenchymal stem cells
1Reference and Translation Centre for Cardiac Stem Cell Therapy (RTC), Department of Cardiac Surgery, University Rostock, 2Institute for Experimental Surgery, University of Rostock
Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.
Published November 5, 2013. Keywords: Stem Cell Biology, migration, intravital microscopy, cremaster muscle, bone marrow, endothelium, microsurgery
1Department of Biomedical Engineering, Soft Tissue Biomechanics and Engineering, Eindhoven University of Technology, The Netherlands
Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.
Published March 19, 2013. Keywords: Bioengineering, Biomedical Engineering, Biophysics, Biomechanics, Anatomy, Physiology, Stem Cell Biology, Medicine, Cellular Biology, Molecular Biology, Genetics, Tissue Engineering, skeletal muscle, muscle progenitor cells, biophysical stimulation, iPS cells, myoblasts, muscle tissue, soft tissue, stem cells, cell culture, collagen, Matrigel, animal model
1Departments of Radiology and Medicine (Cardiology), Stanford University School of Medicine
With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.
Published May 2, 2008. Keywords: Cell Biology, molecular imaging, firefly luciferase, bioluminescence, reporter gene, human embryonic stem cells, teratoma, stem cell transplantation.
1Center for Regenerative Medicine, MGH - Massachusetts General Hospital
This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.
Published March 18, 2009. Keywords: Immunology, HSC, homing, engraftment, transplantation
1Division of Pediatric Pathology, Department of Pathology and Laboratory Medicine, Medical College of Wisconsin, 2Department of Physiology and Cell Biology, The Ohio State University, 3Department of Human Nutrition, Foods and Exercise, Virginia Tech, 4Division of Biomedical Informatics, Department of Biostatistics, Department of Computer Science, University of Kentucky, 5Division of Genetics and Genomics, The Manton Center for Orphan Disease Research, Boston Children's Hospital, Harvard Medical School, 6Cure Congenital Muscular Dystrophy, 7Joshua Frase Foundation, 8Department of Rehabilitation Medicine, University of Washington, 9Department of Physiology, University of Arizona
The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.
Published July 15, 2014. Keywords: Basic Protocol, Tissue, Freezing, Muscle, Isopentane, Pathology, Functional Testing, Cell Culture
1Department of Pediatric General and Thoracic Surgery, Cincinnati Children's Hospital Medical Center, 2Department of Medicine, Section of Gastroenterology and Hepatology, Baylor College of Medicine
We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.
Published March 6, 2015. Keywords: Medicine, Intestinal stem cells, 3-dimensional cell culture, human, small intestine, colon, biopsy, enteroids, minigut, epithelial organoids, in-vitro, colonoids, enterospheres
1Comprehensive Transplant Center, Feinberg School of Medicine, Northwestern University, 2Department of Surgery, Feinberg School of Medicine, Northwestern University, 3Department of Biomedical Engineering, Northwestern University, 4Simpson Querrey Institute for BioNanotechnology in Medicine, Northwestern University, 5Department of Internal Medicine, University of New Mexico HSC, 6Department of Pathology, University of New Mexico HSC, 7Department of Chemical and Biological Engineering, Northwestern University, 8Chemistry of Life Processes Institute, Northwestern University, 9Department of Surgery, Jesse Brown VA Medical Center
This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.…
Published August 10, 2015. Keywords: Bioengineering, Bioreactor, cell seeding, decellularization, extracellular matrix, epithelial, kidney, perfusion, recellularization, renal, resazurin, scaffold, tissue engineering
JoVE Developmental Biology
1Department of Molecular Pharmacology & Experimental Therapeutics, Mayo Clinic College of Medicine, Center for Individualized Medicine, 2Tufts University School of Medicine, 3Department of Biochemistry and Molecular Biology, Mayo Clinic
The goal of this study is to demonstrate how the mosaic transgenesis strategy can be used in zebrafish to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation and progression in vivo.
Published March 31, 2015. Keywords: Developmental Biology, zebrafish, animal model, mosaic transgenesis, coinjection, functional genomics, tumor initiation
1Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, 2The Ritchie Centre, Monash Institute of Medical Research, Monash University
We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.
Published December 21, 2014. Keywords: Medicine, Amnion Membrane, Amniotic, Stem Cells, Epithelial, Cell Therapy, Perinatal, Placenta
1Department of Neuroscience, The University of Connecticut Health Center, 2Department of Genetics and Developmental Biology, The University of Connecticut Health Center, 3Stem Cell Institute, The University of Connecticut Health Center
This procedure yields telencephalic neurons by going through checkpoints which are similar to those observed during human development. The cells are allowed to spontaneously differentiate, are exposed to factors which push them towards the neural lineage, are isolated, and are plated onto coverslips to allow for terminal differentiation and maturation.
Published April 14, 2013. Keywords: Stem Cell Biology, Neuroscience, Neurobiology, Developmental Biology, Cellular Biology, Molecular Biology, Stem Cells, Embryonic Stem Cells, ESCs, Pluripotent Stem Cells, Induced Pluripotent Stem Cells, iPSC, neural differentiation, forebrain, glutamatergic neuron, neural patterning, development, neurons
JoVE Developmental Biology
1Department of Orthodontics and Craniofacial Biology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, 2Department of Biological Structure, University of Washington School of Medicine, 3Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center
This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.
Published July 20, 2015. Keywords: Developmental Biology, Head muscles, levator veli palatini muscle, digastric muscle, masseter muscle, satellite cells, isolation primary cells, cleft palate, regenerative medicine, tissue engineering, stem cells, differentiation, myofibers
1Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado School of Medicine
The endothelial glycocalyx/endothelial surface layer is ideally studied using intravital microscopy. Intravital microscopy is technically challenging in a moving organ such as the lung. We demonstrate how simultaneous brightfield and fluorescent microscopy may be used to estimate endothelial surface layer thickness in a freely-moving in vivo mouse lung.
Published February 22, 2013. Keywords: Medicine, Cellular Biology, Anatomy, Physiology, Biomedical Engineering, Biophysics, Surgery, Endothelium, Vascular, Inflammation, Pulmonary Circulation, Intravital Microscopy, endothelial surface layer, endothelial, glycocalyx, pulmonary microvasculature, catheter, tracheostomy, venous, catheterization, lung injury, mouse, animal model
1Department of Pediatrics, University of Colorado School of Medicine
The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.
Published September 24, 2014. Keywords: Medicine, intracranial, glioblastoma, mouse, orthotopic, brain tumor, stereotaxic, micropump, brain injection
1Harper Cancer Research Institute, 2Microbiology and Immunology, Indiana University School of Medicine, 3Department of Biological Sciences, University of Notre Dame
Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.
Published October 20, 2014. Keywords: Cellular Biology, Embryonic stem cell, Embryoid body, Hematopoietic Progenitor Cells, Retrovirus, Gene Expression, Temporal Gene Expression
1Department of Obstetrics, Gynecology and Reproductive Sciences, Reproductive Immunology Unit, Yale University School of Medicine, 2NatureMost Laboratories, 3Bruker Preclinical Imaging
We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.
Published November 2, 2014. Keywords: Cancer Biology, ovarian cancer, recurrence, in vivo imaging, tumor burden, cancer stem cells, chemotherapy
1Department of Neuroscience, Thomas Jefferson University Medical College
Neural precursor transplantation is a promising strategy for protecting and/or replacing lost/dysfunctional cervical phrenic motor neurons in spinal cord injury (SCI) and the motor neuron disorder, amyotrophic laterals sclerosis (ALS). We provide a protocol for cell delivery to cervical spinal cord ventral horn in rodent models of ALS and SCI.
Published September 18, 2011. Keywords: Medicine, cell transplantation, engraftment, graft, spinal cord, stem cells, precursors, ALS, amyotrophic lateral sclerosis, motor neuron, SCI, spinal cord injury