Protocol
Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells
Ieng Lam Lei1, Lei Bu2, Zhong Wang1
1Cardiac Surgery, University of Michigan, 2Leon H Charney Division of Cardiology, New York University School of Medicine
In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.
Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes
Estelle Hirsinger1, João Emanuel Carvalho2, Christine Chevalier1,3, Georges Lutfalla5, Jean-François Nicolas1, Nadine Peyriéras4, Michael Schubert2
1Département de Biologie du Développement et Cellules Souches, Institut Pasteur, 2Laboratoire de Biologie du Développement de Villefranche-sur-Mer (UMR7009 CNRS/UPMC Univ Paris 06), Sorbonne Universités, 3Equipe Epigenetic Control of Normal and Pathological Hematopoiesis, Centre de Recherche en Cancérologie de Marseille, 4Plateforme BioEmergences IBiSA FBI, CNRS-NED, Institut de Neurobiologie Alfred Fessard, 5Unité de Dynamique des Interactions Membranaires Normales et Pathologiques, CNRS UMR5235/DAA/cc107/Université Montpellier II
We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.
Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle
Chibeza C. Agley1,2, Anthea M. Rowlerson1, Cristiana P. Velloso1, Norman L. Lazarus1, Stephen D. R. Harridge1
1Centre of Human and Aerospace Physiological Sciences, King‘s College London, 2Wellcome Trust-Medical Research Council, Cambridge Stem Cell Institute
The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.
Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis
Elizabeth R. Zielins*1, Ruth Tevlin*1, Michael S. Hu1, Michael T. Chung1, Adrian McArdle1, Kevin J. Paik1, David Atashroo1, Christopher R. Duldulao1, Anna Luan1, Kshemendra Senarath-Yapa1, Graham G. Walmsley1, Taylor Wearda1, Michael T. Longaker1,3, Derrick C. Wan1
1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, 2Stem Cell and Biomaterials Engineering Laboratory, Department of Bioengineering, Stanford University School of Medicine, 3Institute for Stem Cell Biology and Regenerative Medicine, Stanford University
The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.