JoVE Bioengineering
Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells
1Institute of Biomaterials and Biomedical Engineering / Department of Chemical Engineering and Applied Chemistry, University of Toronto, 2Institute of Biomaterials and Biomedical Engineering, University of Toronto
Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.
JoVE Bioengineering
Engineering a Bilayered Hydrogel to Control ASC Differentiation
1Department of Extremity Trauma Research and Regenerative Medicine, United States Army Institute of Surgical Research, 2Department of Biomedical Engineering, The University of Texas at Austin
This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.
JoVE Clinical and Translational Medicine
Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate
1Department of Surgery, Stanford University, 2Department of Surgery, Duke University, 3Department of Surgery, Saint Joseph Mercy Hospital, 4School of Medicine, University of California, San Francisco, 5School of Dentistry, University of California, Los Angeles
This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.
JoVE Bioengineering
Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres
Department of Regenerative Medicine, United States Army Institute of Surgical Research
A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.
JoVE General
Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures
Stem Cell Research Unit, Medical University of Graz, Austria
Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.
JoVE General
Isolating Stem Cells from Soft Musculoskeletal Tissues
1Stem Cell Research Center, Childrens Hospital of Pittsburgh of UPMC, 2Department of Bioengineering, University of Pittsburgh, 3Department of Orthopedic Surgery, University of Pittsburgh, 4Department of Pathology, University of Pittsburgh, 5Department of Molecular Genetics & Biochemistry, University of Pittsburgh
Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to flask.
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 General
Xenotransplantation of Human Stem Cells into the Chicken Embryo
1Department of Physiology, University of Oslo, 2Norwegian Center for Stem Cell Research, University of Oslo
In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.
JoVE General
Bioengineering Human Microvascular Networks in Immunodeficient Mice
Department of Cardiac Surgery, Children's Hospital Boston, Harvard Medical School
Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.
JoVE Bioengineering
Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth
1Department of Biological Systems Engineering, University of Nebraska-Lincoln, 2Department of Engineering Mechanics, University of Nebraska-Lincoln
The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (μMRE).
JoVE General
Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis
1Department of Biomedical Engineering, Tulane University, 2Department of Biomedical Engineering, University of Virginia, 3Center for Stem Cell Research and Regenerative Medicine, Tulane University
This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.
JoVE Bioengineering
Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)
1Transplant and Stem Cell Immunobiology Lab (TSI) and CVRC, University Hospital Hamburg, University Heart Center Hamburg, 2Department of Experimental and Clinical Pharmacology and Toxicology, University Heart Center Hamburg, 3CT Surgery, Stanford University School of Medicine
This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.
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 Clinical and Translational Medicine
Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma
1Institute of Pathology, Laboratory of Molecular Tumor Pathology, Charité - Universitätsmedizin Berlin, 2Institute for Chemistry and Biochemistry, Free University Berlin, 3Laboratory for Functional Genomics Charité (LFGC), Charité - Universitätsmedizin Berlin, 4Comprehensive Cancer Center Charité, Charité - Universitätsmedizin Berlin
This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).
JoVE Neuroscience
Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells
1Department of Neurosurgery, University of Florida, 2Department of Anatomical Sciences, Shiraz University of Medical Sciences, 3STEMCELL Technologies, Inc.
This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.
JoVE Clinical and Translational Medicine
Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods
1Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran, 2Department of Endocrinology & Female Infertility, Reproductive Biomedicine Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran
The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.
JoVE General
Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells
School of Biosciences, University of Birmingham
The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.
JoVE Neuroscience
Isolation, Enrichment, and Maintenance of Medulloblastoma Stem Cells
Department of Cell and Developmental Biology, Vanderbilt University
This protocol describes the isolation, enrichment, and maintenance of medulloblastoma tumor stem cells derived from mutant mice with ectopic Sonic hedgehog pathway activity.
JoVE Clinical and Translational Medicine
Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model
Institute of Human Physiology and Clinical Experimental Research, Semmelweis University
We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.
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 Clinical and Translational Medicine
Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting
Stem Cell and Cancer Research Institute, McMaster University
The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.
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 General
Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells
1UCSF Diabetes Center and Department of Cell and Tissue Biology, University of California, San Francisco, 2Department of Biology, University of Copenhagen, Denmark, 3National Institute of Nutrition and Seafood Research, Bergen, Norway
Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.
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
Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs
Department of Chemical Engineering and Materials Science, City of Hope Cancer Center
Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.
JoVE General
Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells
Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.
JoVE General
Isolation of Stem Cells from Human Pancreatic Cancer Xenografts
Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine
Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.
JoVE General
Isolation and Culture of Adult Epithelial Stem Cells from Human Skin
Department of Cancer Biology, University of Massachusetts Medical School
A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.
JoVE General
Isolation of CD133+ Liver Stem Cells for Clonal Expansion
1Department of Pediatrics and Pharmacology, Pennsylvania State College of Medicine, 2Department of Pharmacology, Pennsylvania State College of Medicine, 3Department of Pediatrics, University of California Los Angeles, School of Medicine
Here we describe the isolation of CD133 expressing liver stem cells and cancer stem cells from whole murine liver, a process that requires tissue digestion, cell enrichment, and flow cytometry isolation. We include methods for advanced single cell isolation and clonal expansion.
JoVE Immunology and Infection
Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging
1Experimental and Clinical Research Center, A joint cooperation between the Charité Medical Faculty and the Max Delbrück Center for Molecular Medicine, 2Berlin Ultrahigh Field Facility (B.U.F.F.), Max Delbrück Center for Molecular Medicine
Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.
JoVE General
Propagation of Human Embryonic Stem (ES) Cells
Center for Regenerative Medicine, MGH - Massachusetts General Hospital
JoVE General
ES Cell-derived Neuroepithelial Cell Cultures
McLean Hospital, Harvard Medical School
Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).
JoVE Immunology and Infection
Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT
1 , Whitehead Institute for Biomedical Research, 2Departments of Microbiology and Biological Sciences, National University of Singapore, 3Department of Biology, Massachusetts Institute of Technology
We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.
JoVE General
Isolation of Retinal Stem Cells from the Mouse Eye
Molecular Genetics, University of Toronto
In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.
JoVE Immunology and Infection
Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells
Department of Microbiology & Immunology, Pennsylvania State University College of Medicine
We provide a reproducible method to induce type 1 diabetes (T1D) in mice within two weeks by the adoptive transfer of islet antigen-specific, primary CD4+ T cells.
JoVE Immunology and Infection
Isolation of Precursor B-cell Subsets from Umbilical Cord Blood
1Department of Pathology and Anatomical Sciences, University of Missouri-Columbia, 2Laboratory for Infectious Disease Research, University of Missouri-Columbia
Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.
JoVE Bioengineering
A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo
1Department of Biomedical Engineering, University of Wisconsin-Madison, 2Department of Biomedical Engineering, Materials Science Program, Laboratory for Optical and Computational Instrumentation, University of Wisconsin-Madison
A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of ˜1,000 cells in peripheral tissues.
JoVE Clinical and Translational Medicine
Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer
1Department of Oncology, Georgetown University, 2Lombardi Comprehensive Cancer Center, Georgetown University, 3Stem Cell Dynamics, Helmholtz Zentrum München - German Research Center for Environmental Health, 4Department of Medicine, Georgetown University, 5Department of Nanobiomedical Science and WCU Research Center of Nanobiomedical Science, Dankook University
Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.
JoVE Immunology and Infection
Flow Cytometry Analysis of Immune Cells Within Murine Aortas
1Deptartment of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, 2Division of Inflammation Biology, LaJolla Institute for Allergy and Immunology
This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.
JoVE Clinical and Translational Medicine
Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging
1Department of Biomedical Engineering, University of Wisconsin-Madison, 2Materials Science Program, University of Wisconsin-Madison, 3Department of Neurological Surgery, University of Wisconsin-Madison, 4Carbone Comprehensive Cancer Center and Center for Stem Cell and Regenerative Medicine, University of Wisconsin-Madison
A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.
JoVE General
Isolation of Immune Cells from Primary Tumors
1Tumor Immunity and Tolerance Section, Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, National Cancer Institute - Frederick, 2KEWB Productions
In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.
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 Neuroscience
Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue
1Department of Neurology, Beth Israel Deaconess Medical Center, 2Department of Obstetrics and Gynecology, Brigham and Women's Hospital, 3Department of Pathology, Beth Israel Deaconess Medical Center, 4Department of Pathology, Division of Neuropathology, Brigham and Women's Hospital
A simple and reliable method on isolation and culture of neural stem cells from discarded human fetal cortical tissue is described. Cultures derived from known human neurological disorders can be used for characterization of pathological cellular and molecular processes, as well as provide a platform to assess pharmacological efficacy.
JoVE General
Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP
Yale Stem Cell Center, Department of Genetics, Yale School of Medicine
A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.
JoVE General
Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector
1Center for Regenerative Medicine (CReM), Boston University School of Medicine, 2Department of Hematology, Children's Hospital of Philadelphia, 3Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia
Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.
JoVE General
Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging
This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.
JoVE General
Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors
We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.
JoVE Neuroscience
Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells
1Alaska Basic Neuroscience Program, Institute of Arctic Biology, University of Alaska at Fairbanks, 2Department Biochemistry, Hood College, 3Department of Cell Biology, Neuronascent, Inc., 4Research and Development, Neuronascent, Inc.
Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.
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 General
Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny
1Department of Medicine, Hematology-Oncology, University of California, Los Angeles, 2Department of Biological Chemistry, University of California, Los Angeles
mStrawberry OP9 cells allow for complete evaluation of all ES-derived progeny from co-culture.
More Results...
