We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.
1Division of Neonatology, Department of Pediatrics, Leiden University Medical Center, 2Division of Fetal Therapy, Department of Obstetrics, Leiden University Medical Center, 3Department of Obstetrics, Leiden University Medical Center
Twin-to-twin transfusion syndrome and twin anemia polycythemia sequence are two potentially devastating problems in perinatal medicine. Both disorders occur only in monochorionic twins and result from unbalanced blood flow through placental vascular anastomoses. We provide a simple protocol to accurately evaluate the presence of vascular anastomoses using colored dye injection of placental vessels after birth.
In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.
Guide Wire Assisted Catheterization and Colored Dye Injection for Vascular Mapping of Monochorionic Twin Placentas
1Division of Pediatric and Fetal Surgery, Department of Surgery, University of California, San Francisco, 2Department of Pathology, University of Alberta, 3Department of Obstretics and Gynecology, University of California, San Francisco, 4Department of Radiology, University of California, San Francisco
Vascular mapping of monochorionic (MC) twin placentas after birth provides a means for detailed demonstration of vascular connections between the twins’ circulations. Imbalance of these connections is thought to play a pivotal role in the development of complications of MC twinning including twin-to-twin transfusion syndrome.
Here are some highlights from the September 2011 Issue of Journal of Visualized Experiments (JoVE).
1Division of Developmental Neuroscience, United Centers for Advanced Research and Translational Medicine (ART), Tohoku University Graduate School of Medicine, 2The Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation (JST)
Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.
1Department of Microbiology and Immunology, Tulane University Medical School, 2Physician/Scientist Program, Tulane University Medical School, 3Department of Molecular and Cellular Biology, Baylor College of Medicine
Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.
1Department of Hematology and Oncology, Brigham and Women's Hospital and Harvard Medical School, 2Department of Cell Biology and Genetics, Erasmus University Medical Center, 3Department of Medicine, Howard Hughes Medical Institute, Brigham and Women's Hospital and Harvard Medical School
This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.
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.
A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.
The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.
1Molecular Biology of Neural Development, Institut de Recherches Cliniques de Montréal, 2Division of Experimental Medicine and Program in Neuroengineering, McGill University, 3Program in Neuroengineering, McGill University, 4Montreal Neurological Institute, 5Department of Anatomy and Cell Biology, McGill University, 6Department of Biology, McGill University, 7Department of Medicine, Universite de Montreal - University of Montreal
This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.
The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.
A Cell Free Assay System Estimating the Neutralizing Capacity of GM-CSF Antibody using Recombinant Soluble GM-CSF Receptor
1Bioscience Medical Research Center, Niigata University Medical and Dental Hospital, 2First department of Internal Medicine, School of Medicine, Kyorin University, 3Neosilk Laboratory, Immuno Biological Laboratories Co., Ltd.
We designed a cell-free receptor binding assay in order to estimate the binding of granulocyte-macrophage colony-stimulating factor (GM-CSF) to the receptors. It enables us to evaluate competitive inhibition of biotinylated GM-CSF binding to soluble GM-CSF receptor alpha by GM-CSF autoantibody with excellent reproducibility.
DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord
The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.
Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells
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.
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.
This article describes a method for labeling embryonic skin and thymus blood vessels.
Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)
This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.
Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions
This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.
Visualization and Genetic Manipulation of Dendrites and Spines in the Mouse Cerebral Cortex and Hippocampus using In utero Electroporation
1Division of Molecular Neurobiology, MRC National Institute for Medical Research, 2Confocal and Image Analysis Laboratory, National Institute for Medical Research, 3Physiopathologie de la plasticité neuronale, Neurocentre Magendie, Université de Bordeaux
This article describes in detail a protocol to electroporate in utero the cerebral cortex and the hippocampus at E14.5 in mice. We also show that this is a valuable method to study dendrites and spines in these two cerebral regions.
Expansion of Embryonic and Adult Neural Stem Cells by In Utero Electroporation or Viral Stereotaxic Injection
Controlling the expansion of somatic stem cells is a major factor hampering their study and use in therapy. Here we describe a system to temporally control neural stem cells expansion during development and adulthood, which can be used to increase the number of neurons generated in the mouse brain.
1Department of Obstretrics & Gynaecology, Schulich School of Medicine and Dentistry, University of Western Ontario, 2Department of Biochemistry, Schulich School of Medicine and Dentistry, University of Western Ontario, 3Children's Health Research Institute
Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.
1Department of Medical Oncology, Dana-Farber Cancer Institute, 2Harvard Medical School, Boston, MA, 3Sheba Cancer Research Center, Chaim Sheba Medical Center, 4Department of Pathology, Brigham and Women's Hospital
The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.
This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.
In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.
1Institute for Cell Engineering Neuroregeneration and Stem Cell Programs, Johns Hopkins University School of Medicine, 2Departments of Neurology, Neuroscience, and Oncology, Johns Hopkins University School of Medicine
In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.
Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.
This study describes the development of an in vitro electroporation technique that allows for the manipulation of gene expression in the lower rhombic lip of midgestation embryos.
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.
A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting
1Malawi-Liverpool-Wellcome Trust Clinical Research Programme, 2Liverpool School of Tropical Medicine, 3Department of Microbiology, Division of Medical Parasitology, New York University School of Medicine
This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.
Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.
1Departments of Pathology and Cell Biology, and Neuroscience, Columbia University College of Physicians and Surgeons, 2Department of Ophthalmology, Columbia University College of Physicians and Surgeons
Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.
The dissection and growth of cells from an individual brain area facilitates investigation of cellular and physiological parameters. We describe a method for primary cell culturing that produces neuron-enriched cultures in a serum-free environment.