January 2015: This Month in JoVE - Introducing JoVE Developmental Biology

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 Chevalier^1,3, 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. Agley^1,2, 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. Longaker^1,3, 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.