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
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 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 General
Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation
Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.
JoVE General
Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells
Department of Vertebrate Genomics, Max Planck Institute for Molecular Genetics
The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgfβ1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.
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
In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging
Division of Cardiovascular Medicine, Stanford University
In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.
JoVE General
Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures
Department of Engineering, University of California, Merced
Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.
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 Clinical and Translational Medicine
Teratoma Generation in the Testis Capsule
1Department of Chemical Physiology, Scripps Research Institute, 2Department of Chemical Physiology, Scripps Research Institute, 3Department of Reproductive Medicine, University of California
Human pluripotent stem cells (hPSCs) have the potential to treat a myriad of different diseases. The utility of these cells lies in the fact that they can differentiate into any cell type in the body. Here we describe the teratoma assay, which is used to demonstrate the pluripotence of hPSCs.
JoVE General
Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format
1Max Planck Institute for Molecular Biomedicine, 2Medical Faculty, University of Münster
Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.
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 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 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
Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping
Institute of Biomedical Sciences, Federal University of Rio De Janeiro-UFRJ
Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.
JoVE Bioengineering
Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages
1Stem Cell Lab, School of Psychiatry, Faculty of Medicine, The University of New South Wales, 2Siriraj Center of Excellence for Stem cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, 3Neuropsychiatric Institute, Prince of Wales Hospital
We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.
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
Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts
1Department of Molecular Carcinogenesis and Center for Cancer Epigenetics, University of Texas M.D. Anderson Cancer Center, 2Department of Cell Biology, Poznan University of Medical Sciences, 3Department of Molecular Biology, The Scripps Research Institute
We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.
JoVE General
Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach
1Department of Bioengineering, University of Pittsburgh, 2Department of Chemical Engineering, University of Pittsburgh
The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing cells.
JoVE General
Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies
Pluripotent stem cells growing in suspension differentiate into embryoid bodies (EBs). Here we demonstrate how to obtain high quality EB cryosections useful for studying cellular and molecular aspects of embryogenesis, while preserving their organization as aggregates.
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 Editorial
JoVE 7th Issue
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 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 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 General
Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations
Medical Research Council Centre for Regenerative Medicine, University of Edinburgh
This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.
JoVE General
Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set
Research and Development, Stemgent
We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.
JoVE Editorial
October 2011: This Month in JoVE
Here are some highlights from the October 2011 Issue of Journal of Visualized Experiments (JoVE).
JoVE General
Derivation of Human Embryonic Stem Cells by Immunosurgery
Department of Molecular and Cell Biology, Harvard
The ability of human embryonic stem cells to self-renew and differentiate into all cell types of the body suggests that they hold great promise for both medical applications and as a research tool for addressing fundamental questions in development and disease. Here, we provide a concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos by immunosurgical isolation of the inner cell mass.
JoVE General
Naïve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures
1Department of Dermatology and Institute for Medical Engineering, Technische Universität München, 2Department of Dermatology and Allergology, Technische Universität München
We report a method to isolate naïve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.
JoVE General
Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia
1Division of Cardiovascular Medicine, Stanford University, 2Department of Radiology, Stanford University
The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by 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
From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs
David Geffen School of Medicine, University of California, Los Angeles
This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. Part 1 of 3.
JoVE Neuroscience
Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain
1Laboratory of Molecular Medicine and Neuroscience, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 2Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health
Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.
JoVE Bioengineering
Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications
1Department of Biology, University of Victoria, 2Department of Mechanical Engineering, Division of Medical Sciences, University of Victoria
This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.
JoVE General
Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes
1Buck Institute for Research on Aging, 2Department of Physiology & Biophysics, University of Washington
Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.
JoVE General
Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells
We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.
JoVE General
Selective Capture of 5-hydroxymethylcytosine from Genomic DNA
1Department of Human Genetics, Emory University School of Medicine, 2Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago
Described is a two-step labeling process using β-glucosyltransferase (β-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.
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 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 General
Passaging HuES Human Embryonic Stem Cell-lines with Trypsin
Department of Molecular and Cell Biology, Harvard
In this video we demonstrate how our lab routinely passages HuES human embryonic stem cell lines with trypsin.
JoVE General
Engineering Cell-permeable Protein
Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.
JoVE General
Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium
The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.
JoVE General
Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium
This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.
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 General
From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel
David Geffen School of Medicine, University of California, Los Angeles
This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3.
JoVE General
From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel
David Geffen School of Medicine, University of California, Los Angeles
This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 3 of 3.
JoVE Immunology and Infection
Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)
Blood Research Institute, BloodCenter of Wisconsin
For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.
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.
More Results...
