JoVE General
Transplantation of Cells Directly into the Kidney of Adult Zebrafish
Center 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.
JoVE Clinical and Translational Medicine
Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model
Division 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.
JoVE General
Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging
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.
JoVE Clinical and Translational Medicine
A Mouse Model of in Utero Transplantation
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
JoVE Clinical and Translational Medicine
In vivo Dual Substrate Bioluminescent Imaging
Case 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.
JoVE Clinical and Translational Medicine
Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model
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.
JoVE General
Homing of Hematopoietic Cells to the Bone Marrow
Center 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.
JoVE Bioengineering
Decellularization and Recellularization of Whole Livers
Perfusion decellularization is a novel technique to produce whole liver scaffolds that retains the organ's extracellular matrix composition and microarchitecture. Herein, the method of preparing whole organ scaffolds using perfusion decellularization and subsequent repopulation with hepatocytes is described. Functional and transplantable liver grafts can be generated using this technique.
JoVE General
Purification of Progenitors from Skeletal Muscle
The Biomedical Research Centre, University of British Columbia
Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.
JoVE Application Notes
CryoStor Cryopreservation Protocol - ADVERTISEMENT
BioLife Solutions, Inc.
CryoStor cryopreservation solutions are used to prepare and preserve cells in ultra low temperature environments, without the need for serum, proteins, or high levels of cytotoxic agents.
JoVE Clinical and Translational Medicine
Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle
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.
JoVE Bioengineering
Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and and Induced Pluripotent Stem Cells (iPSCs)
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.
JoVE Bioengineering
Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation
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.
JoVE Clinical and Translational Medicine
Molecular Imaging to Target Transplanted Muscle Progenitor Cells
1Imaging Program, Lawson Health Research Institute, 2Department of Anatomy and Cell Biology, Western University, 3Department of Medical Biophysics, Western University
A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.
JoVE Neuroscience
Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus
1Department of Molecular Biology and Biochemistry, University of California, Irvine, 2Sue and Bill Gross Stem Cell Center, University of California, Irvine, 3Institute for Immunology, University of California, Irvine
The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice.
JoVE General
In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells
Departments 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.
JoVE General
Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells
Childrens Hospital, Harvard Stem Cell Institute, Harvard Medical School
This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.
JoVE Clinical and Translational Medicine
A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice
1Monash Institute of Medical Research, 2MD Anderson Cancer Centre, University of Texas
In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.
JoVE Clinical and Translational Medicine
The Polyvinyl Alcohol Sponge Model Implantation
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.
JoVE Immunology and Infection
Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model
Department of Surgery, The Ohio State University Medical Center
Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.
JoVE Immunology and Infection
Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation
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.
JoVE General
Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney
1Department of Molecular Medicine, Maine Medical Center Research Institute, 2Molecular Medicine and Gene Therapy, Lund University Hospital
In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.
JoVE Bioengineering
Use of Human Perivascular Stem Cells for Bone Regeneration
1Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, UCLA, 2UCLA and Orthopaedic Hospital, Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, UCLA, 3Department of Bioengineering, UCLA, 4Center for Cardiovascular Science, University of Edinburgh
Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.
JoVE Clinical and Translational Medicine
Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice
1Department of Molecular Microbiology and Immunology, Bond Life Sciences Center, University of Missouri, 2Department of Biological Sciences, Columbia University, 3Department of Veterinary Pathobiology, Bond Life Sciences Center, University of Missouri
This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.
JoVE Clinical and Translational Medicine
High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors
Department of Pediatrics, Division of Cellular and Molecular Therapy, University of Florida
In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.
JoVE General
Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish
1Department of Molecular Pathology, Massachusetts General Hospital, Harvard Medical School, 2Department of Molecular Pathology, Massachusetts General Hospital Cancer Center, Harvard Stem Cell Institute
Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.
JoVE Neuroscience
Isolating Nasal Olfactory Stem Cells from Rodents or Humans
1NICN, Aix Marseille University, 2LNPM, Aix Marseille University, 3ENT Department, Aix Marseille University, 4Gene expression Laboratory, The Salk Institute for Biological Studies, 5Laboratory of Speech and Language, Aix Marseille University, 6Centre d'Investigations Cliniques en Biothérapie, Aix Marseille University
We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.
JoVE General
Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging
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.
JoVE General
Transplantation of Whole Kidney Marrow in Adult Zebrafish
Children's Hospital, Harvard Stem Cell Institute, Harvard Medical School
In this article, we demonstrate a method to perform HCT in adult zebrafish.
JoVE Clinical and Translational Medicine
Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model
1Department of Pathology, Vanderbilt University, 2Departments of Oncology, Surgery and Medical Biophysics, London Regional cancer program
Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.
JoVE Clinical and Translational Medicine
In vivo Measurement of the Mouse Pulmonary Endothelial Surface Layer
Division 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.
JoVE Clinical and Translational Medicine
In vitro Organoid Culture of Primary Mouse Colon Tumors
1Department of Molecular & Integrative Physiology, University of Michigan, 2Department of Internal Medicine, Division of Gastroenterology, University of Michigan
A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.
JoVE General
Dissection of the Adult Zebrafish Kidney
Department of Biological Sciences, University of Notre Dame
The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.
JoVE Clinical and Translational Medicine
Intraspinal Cell Transplantation for Targeting Cervical Ventral Horn in Amyotrophic Lateral Sclerosis and Traumatic Spinal Cord Injury
Department 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.
JoVE General
Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction
1San Diego Regenerative Medicine Institute, 2Xcelthera, 3Department of Neurosurgery, Harvard Medical School, 4Division of SCI Research, VA Boston Healthcare System, 5Program in Stem Cell & Regenerative Biology, Sanford-Burnham Medical Research Institute, 6La Jolla IVF
We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.
JoVE Neuroscience
Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction
1San Diego Regenerative Medicine Institute, 2Xcelthera, 3Department of Neurosurgery, Harvard Medical School, 4Division of SCI Research, VA Boston Healthcare System, 5Program in Stem Cell & Regenerative Biology, Sanford-Burnham Medical Research Institute, 6La Jolla IVF
We have established a protocol for induction of neuroblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human neuronal progenitors and neuronal cell types in the developing CNS for neural repair.
JoVE Neuroscience
The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells
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.
JoVE Clinical and Translational Medicine
Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction
Department of Physiology, Brody School of Medicine, East Carolina University
Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.
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