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University of Hawai'i

6 ARTICLES PUBLISHED IN JoVE

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JoVE Journal

Osteoclast Derivation from Mouse Bone Marrow
Ruth Tevlin *1, Adrian McArdle *1,2, Charles K.F. Chan 2, John Pluvinage 2, Graham G. Walmsley 1,2, Taylor Wearda 1,2, Owen Marecic 1,2, Michael S. Hu 1, Kevin J. Paik 1, Kshemendra Senarath-Yapa 1, David A. Atashroo 1, Elizabeth R. Zielins 1, Derrick C. Wan 1, Irving L. Weissman 1,2, Michael T. Longaker 1,2
1Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

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Developmental Biology

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis
Elizabeth R. Zielins *1, Ruth Tevlin *1, Michael S. Hu 1, Michael T. Chung 1, Adrian McArdle 1, Kevin J. Paik 1, David Atashroo 1, Christopher R. Duldulao 1, Anna Luan 1, Kshemendra Senarath-Yapa 1, Graham G. Walmsley 1, Taylor Wearda 1, Michael T. Longaker 1,2, Derrick C. Wan 1
1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, 2Institute 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.

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Medicine

A Mouse Fetal Skin Model of Scarless Wound Repair
Graham G. Walmsley *1,2, Michael S. Hu *1,2,3, Wan Xing Hong 1,4, Zeshaan N. Maan 1, H. Peter Lorenz 1, Michael T. Longaker 1,2
1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 3Department of Surgery, John A. Burns School of Medicine, University of Hawai'i, 4University of Central Florida College of Medicine

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

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Developmental Biology

Murine Dermal Fibroblast Isolation by FACS
Graham G. Walmsley *1,2, Zeshaan N. Maan *1, Michael S. Hu *1,2,3, David A. Atashroo 1, Alexander J. Whittam 1, Dominik Duscher 1, Ruth Tevlin 1, Owen Marecic 1, H. Peter Lorenz 1, Geoffrey C. Gurtner 1, Michael T. Longaker 1,2
1Hagey Laboratory for Pediatric Regenerative Medicine, Department of Surgery, Division of Plastic and Reconstructive Surgery, Stanford University School of Medicine, 2Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 3Department of Surgery, John A. Burns School of Medicine, University of Hawai'i

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

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Medicine

Creation of Abdominal Adhesions in Mice
Clement D. Marshall 1, Michael S. Hu 1, Tripp Leavitt 1, Leandra A. Barnes 1, Alexander T.M. Cheung 1, Samir Malhotra 1, H. Peter Lorenz 1, Michael T. Longaker 1
1Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine

Abdominal adhesions that form after surgery are a major cause of pain, infertility, and hospitalization and reoperation for small bowel obstruction. Our surgical procedure for creating abdominal adhesions in mice is a reliable tool to study the mechanisms underlying the formation of adhesions.

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Developmental Biology

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model
Clement D. Marshall 1, Elizabeth R. Zielins 1, Elizabeth A. Brett 1, Charles P. Blackshear 1, Michael S. Hu 1, Tripp Leavitt 1, Leandra A. Barnes 1, H. Peter Lorenz 1, Michael T. Longaker 1, Derrick C. Wan 1
1Hagey Laboratory for Pediatric Regenerative Medicine, Division of Plastic and Reconstructive Surgery, Department of Surgery, Stanford University School of Medicine

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

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