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Harvard Stem Cell Institute

13 ARTICLES PUBLISHED IN JoVE

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Biology

Homing of Hematopoietic Cells to the Bone Marrow
Rushdia Z. Yusuf 1, David T. Scadden 1
1Center for Regenerative Medicine, MGH - Massachusetts General Hospital

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

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Biology

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish
Jessica S. Blackburn 1, Sali Liu 1, David M. Langenau 2
1Department of Molecular Pathology, Massachusetts General Hospital, Harvard Medical School, 2Department of Molecular Pathology, Massachusetts General Hospital Cancer Center, Harvard Stem Cell Institute

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

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Bioengineering

Generation of Aligned Functional Myocardial Tissue Through Microcontact Printing
Ayhan Atmanli 1, Ibrahim J. Domian 1,2
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.

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Medicine

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair
Dongsu Park 1, Joel A. Spencer 2, Charles P. Lin 2, David T. Scadden 1
1Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Stem Cell Institute, 2Wellman Center for Photomedicine and Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

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Immunology and Infection

Normal and Malignant Muscle Cell Transplantation into Immune Compromised Adult Zebrafish
Inês M. Tenente *1,2,3, Qin Tang *1,2, John C. Moore 1,2, David M. Langenau 1,2
1Molecular Pathology, Cancer Center and Center for Regenerative Medicine, Massachusetts General Hospital, 2Harvard Stem Cell Institute, 3GABBA - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto

Here, we present a protocol for cell transplantation of zebrafish skeletal muscle and embryonal rhabdomyosarcoma (ERMS) into adult immune compromised rag2E450fs homozygous mutant zebrafish. This protocol allows for the efficient analysis of regeneration and malignant transformation of muscle cells.

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Developmental Biology

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae
Elliott J. Hagedorn 1,2, Jennifer L. Cillis 3, Caitlyn R. Curley 3, Taylor C. Patch 3, Brian Li 1,2, Bradley W. Blaser 1,2,7, Raquel Riquelme 1,2, Leonard I. Zon 1,2,4,5,6, Dhvanit I. Shah 1,2,3,4,5
1Division of Hematology/Oncology, Boston Children’s Hospital, 2Harvard Medical School, 3Division of Hematology, Department of Medicine, Brigham and Women’s Hospital, 4Harvard Stem Cell Institute, 5Broad Institute of Massachusetts Institute of Technology, 6Howard Hughes Medical Institute, 7Division of Hematologic Malignancies, Dana-Farber Cancer Institute

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

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JoVE Journal

Analysis of Hematopoietic Stem Progenitor Cell Metabolism
Giorgia Scapin 1,2,3, Marie C. Goulard 1,2,3, Priyanka R. Dharampuriya 1,2,3, Jennifer L. Cillis 1,2,3, Dhvanit I. Shah 1,2,3
1Nationwide Children's Hospital, 2The Ohio State University College of Medicine, 3The Ohio State University Comprehensive Cancer Center

Hematopoietic stem progenitor cells (HSPCs) transition from a quiescent state to a differentiation state due to their metabolic plasticity during blood formation. Here, we present an optimized method for measuring mitochondrial respiration and glycolysis of HSPCs.

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Developmental Biology

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice
Laura Ortinau 1,2, Kevin Lei 1, Youngjae Jeong 1, Dongsu Park 1,2,3
1Department of Molecular & Human Genetics, Baylor College of Medicine, 2Center for Skeletal Biology, Baylor College of Medicine, 3Department of Pathology & Immunology, Baylor College of Medicine

This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy.

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Neuroscience

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells
Sovannarath Pong 1,2,3, Paulo Lizano 1,2,3,4, Rakesh Karmacharya 1,3,4,5
1Center for Genomic Medicine, Massachusetts General Hospital, 2Department of Psychiatry, Beth Israel Deaconess Medical Center, 3Chemical Biology and Therapeutic Science Program, Broad Institute of MIT and Harvard, 4Department of Psychiatry, Harvard Medical School, 5Schizophrenia and Bipolar Disorder Program, McLean Hospital

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

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Neuroscience

Co-Culturing Microglia and Cortical Neurons Differentiated from Human Induced Pluripotent Stem Cells
Kara Lopez-Lengowski 1,2, Annie Kathuria 1,2,3, Kaia Gerlovin 1,2, Rakesh Karmacharya 1,2,3,4,5,6,7
1Center for Genomic Medicine, Massachusetts General Hospital, 2Chemical Biology Program, Broad Institute of MIT & Harvard, 3Department of Psychiatry, Harvard Medical School, 4Schizophrenia and Bipolar Disorder Program, McLean Hospital, 5Program in Neuroscience, Harvard University, 6Program in Chemical Biology, Harvard University, 7Harvard Stem Cell Institute

This protocol describes a methodology to differentiate microglia from human iPSCs and maintain them in co-culture with iPSC-derived cortical neurons in order to study mechanistic underpinnings of neuroimmune interactions using human neurons and microglia.

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Developmental Biology

Spatial High-resolution Analysis of Gene Expression Levels in Tendons
Steffany Villaseñor 1, Mor Grinstein 1,2,3
1Center for Regenerative Medicine, Department of Orthopedic Surgery, Massachusetts General Hospital, Harvard Medical School, 2Harvard Stem Cell Institute, 3Center Of Regeneration and Longevity (CORAL), Tel-Aviv Sourasky Medical Center

This article describes how to perform an optimized in situ protocol for tendons. This method discusses tissue preparation, section permeabilization, probe design, and signal amplification methods.

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Developmental Biology

Two-Photon Microscopy for the Study of Tendons
Steffany Villaseñor 1, Mor Grinstein 1,2,3
1Center for Regenerative Medicine, Department of Orthopedic Surgery, Massachusetts General Hospital, Harvard Medical School, 2Harvard Stem Cell Institute, 3Center Of Regeneration and Longevity (CORAL), Tel-Aviv Sourasky Medical Center

This article outlines the process of preparing, setting up, and imaging tendons using multiphoton microscopy. Additionally, it covers the application of SHG for analyzing collagen fibril alignment and the creation of a 3D representation of tendons. This methodology proves highly valuable in characterizing tendon cells and their ECM during injury and development.

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Immunology and Infection

Stromal Cell Isolation From Hematopoietic Organs
Trine Kristiansen *1,2,3, Christina Mayerhofer *1,2,3, Karin Gustafsson 1,2,3, David T. Scadden 1,2,3
1Center for Regenerative Medicine, Massachusetts General Hospital, 2Harvard Stem Cell Institute, 3Department of Stem Cell and Regenerative Biology, Harvard University

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

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