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 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
Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration
Department of Biological Sciences, University of Notre Dame
Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.
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 General
Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells
1The Sprott Center for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, 2Department of Cellular and Molecular Medicine, University of Ottawa
An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.
JoVE Neuroscience
The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry
1Department of Neurosurgery, The University of Florida, 2Laboratory for Stem Cell Research, Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran
This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.
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 General
Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors
1Institute for Research in Biomedicine, Bellinzona (Switzerland), 2Dipartimento di Biologia e Genetica per le Scienze Mediche, Universitá degli Studi di Milano
A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.
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 General
A Quantitative Assay for Insulin-expressing Colony-forming Progenitors
1Department of Biotechnology & Bioinformatics, California State University Channel Islands, 2Department of Diabetes, Endocrinology and Metabolism, Beckman Research Institute of City of Hope, 3The Irell & Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope
A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.
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 General
Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium
1Department of Dermatology, Brigham and Women's Hospital, 2Department of Dermatology, Brigham and Women's Hospital and Harvard Medical School
This report provides a visual depiction of parallel-plate flow chamber analysis for studying leukocyte endothelial interactions under physiologic shear stress. This method is particularly useful for investigating the role of endothelial (E)-selectin and leukocyte E-selectin ligands that trigger leukocyte rolling on endothelial cell surfaces.
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
Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood
Herman B Wells Center for Pediatric Research, Indiana University School of Medicine
Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.
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
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 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
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 Neuroscience
Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay
1 Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran, 2Department of Neurosurgery, University of Florida
This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.
JoVE General
Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death
1Department of Biological Sciences, University of Alberta, 2Department of Agriculture, Food and Nutrition Sciences, University of Alberta
An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.
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.
JoVE General
Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue
We have derived a strategy to detect sequential incorporation of thymidine analogues (CldU and IdU) into tissues of adult mice to quantify two successive rounds of cell division. This strategy is useful to detect cell turnover of long-lived tissues, oncogenic transformation, or transit-amplifying cells.
JoVE Neuroscience
An Optimized Procedure for Fluorescence-activated Cell Sorting (FACS) Isolation of Autonomic Neural Progenitors from Visceral Organs of Fetal Mice
Division of Genetic Medicine, Department of Medicine, Vanderbilt University School of Medicine
An optimized procedure to purify neural crest-derived neuronal progenitors from fetal mouse tissues is described. This method takes advantage of expression from fluorescent reporter alleles to isolate discrete populations by fluorescence-activated cell sorting (FACS). The technique can be applied to isolate neuronal subpopulations throughout development or from adult tissues.
JoVE Bioengineering
Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing
1Cardiovascular Research Center, Massachusetts General Hospital and Harvard Medical School, 2Harvard Stem Cell Institute
The generation of aligned myocardial tissue is a key requirement for adapting the recent advances in stem cell biology to clinically useful purposes. Herein we describe a microcontact printing approach for the precise control of cell shape and function. Using highly purified populations of embryonic stem cell derived cardiac progenitors, we then generate anisotropic functional myocardial tissue.
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 General
Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons
1Department of Genetics and Development, Columbia University, 2Department of Pathology and Cell Biology, Columbia University, 3Department of Neuroscience, Columbia University, 4Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School
Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.
JoVE General
Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells
1Charles C. Gates Regenerative Medicine and Stem Cell Biology Program, University of Colorado Denver, 2Department of Medicine, University of Colorado Denver, 3Cancer Center, University of Colorado Denver, 4Webb Waring Institute, University of Colorado Denver
In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.
JoVE General
Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines
Department of Surgery, Weill Cornell Medical College
A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.
JoVE General
Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures
1Institute of Human Genetics and Northeast England Stem Cell Institute, Newcastle University, 2Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin
Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.
JoVE Neuroscience
Isolating LacZ-expressing Cells from Mouse Inner Ear Tissues using Flow Cytometry
Department of Otolaryngology-Head and Neck Surgery, Stanford University School of Medicine
Flow cytometry is a powerful tool allowing for the isolation and study of specific cell populations. This protocol describes steps for isolating LacZ-expressing cells from cochlear tissues from neonatal transgenic mice. Dissociated cochlear cells were labeled using fluorescent-conjugated substrates of β-galactosidase prior to separation via flow cytometry.
JoVE General
Isolation of Basal Cells and Submucosal Gland Duct Cells from Mouse Trachea
Department of Pediatrics, David Geffen School of Medicine at UCLA
Here we demonstrate our protocol for isolation of basal and submucosal gland duct cells from mouse tracheas. We also demonstrate the method of injecting stem cells into the dorsal mouse fat pad to create an in vivo model of submucosal gland regeneration.
JoVE General
Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)
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 Neuroscience
High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium
1Neural Development Group, Division of Cell and Developmental Biology, College of Life Sciences, University of Dundee, Dundee, UK, 2Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee, UK
Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.
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 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
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
Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells
Stem Cell Research Unit, Medical University of Graz, Austria
Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.
JoVE Neuroscience
Derivation of Glial Restricted Precursors from E13 mice
1Hugo W. Moser Research Institute at Kennedy Krieger, Johns Hopkins University, 2Department of Neurology, Johns Hopkins School of Medicine, 3University of Maryland, 4Experimental Neurology, Biogen Idec, 5The Brain Science Institute, Johns Hopkins School of Medicine, 6Department of Pediatrics, Johns Hopkins School of Medicine
This protocol outlines the derivation of Glial Restricted Precursors from fetal spinal cords and maintained in vitro either for transplantation or for the study of oligodendrocytic lineage.
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 Neuroscience
Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay
1Department of Anatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran, 2Department of Neurosurgery, The University of Florida
This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.
JoVE General
Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration
Program in Epithelial Biology, Stanford University School of Medicine
Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.
JoVE Immunology and Infection
Depletion and Reconstitution of Macrophages in Mice
1Department of Graduate Studies, University of British Columbia, 2Department of Molecular Biology, Vrije Universiteit Amsterdam, 3Department of Pediatrics, University of British Columbia
Macrophages play a central role in homeostasis and pathology in many tissues. The protocol presented here describes methods for depleting macrophages in vivo, deriving polarized macrophages from bone marrow aspirates, and adoptively transferring macrophages into mice. These techniques allow determination of the role that polarized macrophages play in health and disease.
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
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
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 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
Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology
1Neural Regeneration Laboratory and Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 2Carleton Immersive Media Studio, Azrieli School of Architecture and Urbanism, Carleton University
Here, we describe how to produce, expand, and immunolabel postnatal hippocampal neural progenitor cells (NPCs) in three-dimensional (3D) culture. Next, using hybrid visualization technologies, we demonstrate how digital images of immunolabelled cryosections can be used to reconstruct and map the spatial position of immunopositive cells throughout the entire 3D neurosphere.
JoVE General
Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay
Genes and Environment Laboratory, University of California, Berkeley
A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.
JoVE General
Isolation of Valvular Endothelial Cells
Department of Biomedical Engineering, Cornell University
We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.
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