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UCLA

24 ARTICLES PUBLISHED IN JoVE

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JoVE Journal

Use of Human Perivascular Stem Cells for Bone Regeneration
Aaron W. James *1, Janette N. Zara *2, Mirko Corselli 2, Michael Chiang 1, Wei Yuan 2, Virginia Nguyen 1, Asal Askarinam 1, Raghav Goyal 1, Ronald K. Siu 3, Victoria Scott 1, Min Lee 3, Kang Ting 1, Bruno Péault 2,4, Chia Soo 2
1Dental and Craniofacial Research Institute and Section of Orthodontics, School of Dentistry, UCLA, 2UCLA and Orthopaedic Hospital, Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, UCLA, 3Department of Bioengineering, UCLA, 4Center for Cardiovascular Science, University of Edinburgh

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

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Medicine

Mouse Eye Enucleation for Remote High-throughput Phenotyping
Vinit B. Mahajan 1,2, Jessica M. Skeie 1,2, Amir H. Assefnia 2,3, MaryAnn Mahajan 1,2, Stephen H. Tsang 2,4
1Department of Ophthalmology and Visual Sciences, University of Iowa, 2Omics Laboratory, University of Iowa, 3School of Dentistry, UCLA, 4Bernard and Shirlee Brown Glaucoma Laboratory, Department of Ophthalmology, College of Physicians and Surgeons, Columbia University

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

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Medicine

Repair of a Critical-sized Calvarial Defect Model Using Adipose-derived Stromal Cells Harvested from Lipoaspirate
David D. Lo *1,2, Jeong S. Hyun *1,3, Michael T. Chung 1, Daniel T. Montoro 1, Andrew Zimmermann 1, Monica M. Grova 1,4, Min Lee 5, Derrick C. Wan 1, Michael T. Longaker 1
1Department of Surgery, Stanford University , 2Department of Surgery, Duke University , 3Department of Surgery, Saint Joseph Mercy Hospital, 4School of Medicine, University of California, San Francisco , 5School of Dentistry, University of California, Los Angeles

This protocol describes the isolation of adipose-derived stromal cells from lipoaspirate and the creation of a 4 mm critical-sized calvarial defect to evaluate skeletal regeneration.

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Biology

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue
Hassan Khalil 1, Wenxian Nie 1, Robert A Edwards 2, James Yoo 1
1Department of Surgery, UCLA, 2Department of Pathology, UC Irvine

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

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Biology

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle
William C.W. Chen 1, Arman Saparov 2,3, Mirko Corselli 4, Mihaela Crisan 5, Bo Zheng 6, Bruno Péault 7,8, Johnny Huard 9
1Stem Cell Research Center, Department of Bioengineering and Orthopedic Surgery, University of Pittsburgh, 2Department of Orthopedic Surgery, University of Pittsburgh, 3Nazarbayev University Research and Innovation System, Nazarbayev University, 4Department of Orthopaedic Surgery, UCLA Orthopaedic Hospital and the Orthopaedic Hospital Research Center, University of California at Los Angeles, 5Department of Cell Biology, Erasmus MC Stem Cell Institute, 6OHSU Center for Regenerative Medicine, Oregon Health & Science University, 7Centre for Cardiovascular Science and MRC Centre for Regenerative Medicine, Queen's Medical Research Institute and University of Edinburgh, 8David Geffen School of Medicine and the Orthopaedic Hospital Research Center, University of California at Los Angeles, 9Stem Cell Research Center, Department of Orthopedic Surgery and McGowan Institute for Regenerative Medicine, University of Pittsburgh

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

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Biology

Dissection and Mounting of Drosophila Pupal Eye Discs
Joy S. Tea 1, Albert Cespedes 1, Daniel Dawson 2, Utpal Banerjee 1,3, Gerald B. Call 2
1Department of Molecular, Cell and Developmental Biology (MCDB), University of California at Los Angeles (UCLA), 2Arizona College of Osteopathic Medicine (AZCOM), Midwestern University, 3MCDB, Broad Stem Cell Research Center, UCLA

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

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Developmental Biology

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue
James E. Baily 1, William C.W. Chen 2,3, Nusrat Khan 4, Iain R. Murray 4, Zaniah N. González Galofre 4, Johnny Huard 5,6, Bruno Péault 4,7
1Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, 2Department of Bioengineering and Orthopaedic Surgery, University of Pittsburgh, 3Research Laboratory of Electronics and Department of Biological Engineering, Massachusetts Institute of Technology, 4MRC Centre for Regenerative Medicine, University of Edinburgh, 5Stem Cell Research Center, Department of Orthopaedic Surgery, University of Pittsburgh, 6Department of Orthopaedic Surgery, University of Texas Health Science Center at Houston, 7Department of Orthopaedic Surgery, UCLA Orthopaedic Hospital, David Geffen School of Medicine, University of California at Los Angeles

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

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Medicine

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model
Aditya S. Shirali 1, Austin I. McDonald 2, Julia J. Mack 3, M. Luisa Iruela-Arispe 3
1Department of Surgery, University of California, Los Angeles, 2Molecular Biology Institute, University of California, Los Angeles, 3Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

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Neuroscience

Improving the Application of High Molecular Weight Biotinylated Dextran Amine for Thalamocortical Projection Tracing in the Rat
Dongsheng Xu *1, Jingjing Cui *1, Jia Wang 1, Zhiyun Zhang 1, Chen She 1, Wanzhu Bai 1
1Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences

Here, we present a refined protocol to effectively reveal biotinylated dextran amine (BDA) labeling with a fluorescent staining method through a reciprocal neural pathway. It is suitable for analyzing the fine structure of BDA labeling and distinguishing it from other neural elements under a confocal laser scanning microscope.

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Genetics

Determining Genome-wide Transcript Decay Rates in Proliferating and Quiescent Human Fibroblasts
Mithun Mitra 1,2, Ha Neul Lee 1,2,3, Hilary A. Coller 1,2,3
1Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, 2Department of Biological Chemistry, David Geffen School of Medicine, 3Molecular Biology Institute Interdepartmental Program, University of California, Los Angeles

We describe a protocol for generating proliferating and quiescent primary human dermal fibroblasts, monitoring transcript decay rates, and identifying differentially decaying genes.

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Biochemistry

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue
David Jelinek 1,2, Aimee Flores 1,3, Melanie Uebelhoer 1, Vincent Pasque 2, Kathrin Plath 2,3, M. Luisa Iruela-Arispe 1,3, Heather R. Christofk 2,3, William E. Lowry 1,3, Hilary A. Coller 1,2,3
1Department of Molecular, Cell and Developmental Biology, UCLA, 2Department of Biological Chemistry, David Geffen School of Medicine, 3Molecular Biology Institute Interdepartmental Program, UCLA

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

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Bioengineering

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart
Victoria Messerschmidt *1, Zachary Bailey *1, Kyung In Baek 2, Yichen Ding 2, Jeffrey J. Hsu 2, Richard Bryant 1, Rongsong Li 3, Tzung K. Hsiai 2, Juhyun Lee 1
1Department of Bioengineering, The University of Texas at Arlington, 2Department of Medicine (Cardiology) and Bioengineering, UCLA, 3College of Health Science and Environmental Engineering, Shenzhen Technology University

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

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Bioengineering

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart
Yichen Ding 1, Zachary Bailey 2, Victoria Messerschmidt 2, Jun Nie 3, Richard Bryant 2, Sandra Rugonyi 4, Peng Fei 3, Juhyun Lee 1,2, Tzung K. Hsiai 1
1Department of Bioengineering, University of California Los Angeles, 2Department of Bioengineering, University of Texas at Arlington, 3School of Optical and Electronic Information, Huazhong University of Science and Technology, 4Department of Biomedical Engineering, OSHU

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

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Chemistry

Automation of a Positron-emission Tomography (PET) Radiotracer Synthesis Protocol for Clinical Production
Eric Schopf *1, Christopher M. Waldmann *2,3, Jeffrey Collins 2,4, Christopher Drake 1, Roger Slavik 2,3, R. Michael van Dam 2,4
1SOFIE, 2Department of Molecular & Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles (UCLA), 3Ahmanson Translational Imaging Division, University of California, Los Angeles (UCLA), 4Crump Institute for Molecular Imaging, University of California, Los Angeles (UCLA)

Positron-emission tomography (PET) imaging sites that are involved in multiple early clinical research trials need robust and versatile radiotracer manufacturing capabilities. Using the radiotracer [18F]Clofarabine as an example, we illustrate how to automate the synthesis of a radiotracer using a flexible, cassette-based radiosynthesizer and validate the synthesis for clinical use.

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JoVE Journal

Thermocapillary Convection Space Experiment on the SJ-10 Recoverable Satellite
Li Duan *1,2, Yongli Yin *3, Jia Wang *1, Qi Kang 1,2, Di Wu 1, Huan Jiang 1, Pu Zhang 1, Liang Hu 1
1National Microgravity Laboratory, Institute of Mechanics, Chinese Academy of Sciences, 2School of Engineering Sciences, University of Chinese Academy of Sciences, 3China Astronaut Research and Training Center

A protocol for the space payload design, the space experiment on thermocapillary convection, and analyses of experimental data and images are presented in this paper.

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Biology

Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization
Bianca Vezzani 1,2, Mario Gomez-Salazar 1, Joan Casamitjana 1, Carlo Tremolada 3, Bruno Péault 1,4
1MRC Center for Regenerative Medicine, University of Edinburgh, 2Dept. of Morphology, Surgery and Experimental Medicine, Section of General Pathology, University of Ferrara, 3Italian Image Institute, 4Orthopaedic Hospital Research Center and Broad Stem Cell Research Center, David Geffen School of Medicine, University of California

Here, we present human adipose tissue enzyme-free micro-fragmentation using a closed system device. This new method allows the obtainment of sub-millimeter clusters of adipose tissue suitable for in vivo transplantation, in vitro culture, and further cell isolation and characterization.

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Neuroscience

Visualizing the Calcitonin Gene-Related Peptide Immunoreactive Innervation of the Rat Cranial Dura Mater with Immunofluorescence and Neural Tracing
Jia Wang *1, Dongsheng Xu *1, Jingjing Cui 1, Chen She 1, Hui Wang 1, Shuang Wu 1, Ling Zou 1, Jianliang Zhang 1, Wanzhu Bai 1
1Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences

Here we present a protocol to visualize spatial correlation of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers and blood vessels in the cranial dura mater using immunofluorescence and fluorescent histochemistry with CGRP and phalloidin, respectively. In addition, the origin of these nerve fibers was retrograde traced with a fluorescent neural tracer.

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Cancer Research

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth
David Jelinek 1,2, Ellen Ran Zhang 1,2,3, Aaron Ambrus 1,2, Erin Haley 3, Emily Guinn 1,2, Austin Vo 1,2, Peter Le 1,2, Ayse Elif Kesaf 1,2, Jennifer Nguyen 1,2, Lily Guo 1,2, Destiny Frederick 1,2, Zhengyang Sun 1,2, Natalie Guo 3, Parker Sevier 1,2, Eric Bilotta 1,2, Kaiser Atai 1,2,4, Laurent Voisin 1,2, Hilary A. Coller 1,2,4
1Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, 2Department of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, 3Department of Molecular Biology, Princeton University, 4Molecular Biology Institute, University of California, Los Angeles

A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor growth.

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Chemistry

Optimization of Radiochemical Reactions using Droplet Arrays
Alejandra Rios 1,2, Travis S. Holloway 2,3, Jia Wang 2,4, R. Michael van Dam 1,2,3,4
1Physics and Biology in Medicine Interdepartmental Graduate Program, University of California Los Angeles (UCLA), 2Crump Institute of Molecular Imaging, UCLA, 3Department of Molecular & Medical Pharmacology, David Geffen School of Medicine, 4Department of Bioengineering, UCLA

This method describes the use of a novel high-throughput methodology, based on droplet chemical reactions, for the rapid and economical optimization of radiopharmaceuticals using nanomole amounts of reagents.

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Bioengineering

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC)
Xingjin Jian *1,2, Xiaojie Guo *1,2,3, Jia Wang 4, Zheng Lin Tan 1,2,6, Xin-hui Xing 1,2,5, Liyan Wang 3, Chong Zhang 1,2,5
1Department of Chemical Engineering, Institute of Biochemical Engineering, Tsinghua University, 2Key Laboratory of Industrial Biocatalysis, Ministry of Education, Tsinghua University, 3Luoyang TMAXTREE Biotechnology Co., Ltd., 4Biochemical Engineering Research Group, School of Chemical Engineering and Technology, Xi’an Jiaotong University, 5Center for Synthetic & Systems Biology, Tsinghua University, 6School of Life Science and Technology, Tokyo Institute of Technology

This protocol describes how to use the Microbial Microdroplet Culture system (MMC) to conduct automated microbial cultivation and adaptive evolution. MMC can cultivate and sub-cultivate microorganisms automatically and continuously and monitor online their growth with relatively high throughput and good parallelization, reducing labor and reagent consumption.

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Bioengineering

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart
Nicholas Kluge 1, Shyue-An Chan 1, Jeffrey L. Ardell 2,3, Corey Smith 1
1Department of Physiology and Biophysics, Case Western Reserve University, 2University of California Los Angeles (UCLA) Cardiac Arrhythmia Center, David Geffen School of Medicine, 3UCLA Neurocardiology Research Program of Excellence, UCLA

Established immunochemical methods to measure peptide transmitters in vivo rely on microdialysis or bulk fluid draw to obtain the sample for offline analysis. However, these suffer from spatiotemporal limitations. The present protocol describes the fabrication and application of a capacitive immunoprobe biosensor that overcomes the limitations of the existing techniques.

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Neuroscience

Visualizing the Morphological Characteristics of Neuromuscular Junction in Rat Medial Gastrocnemius Muscle
Jingjing Cui *1, Shuang Wu *1, Jia Wang 1, Yuqing Wang 1, Yuxin Su 1, Dongsheng Xu 1, Yihan Liu 1, Junhong Gao 1, Xianghong Jing 1, Wanzhu Bai 1
1Institute of Acupuncture and Moxibustion, China Academy of Chinese Medical Sciences

The protocol shows a method to examine spatial correlation among the pre-synaptic terminals, post-synaptic receptors, and peri-synaptic Schwann cells in the rat medial gastrocnemius muscle using fluorescent immunohistochemistry with different biomarkers, namely, neurofilament 200, vesicular acetylcholine transporter, alpha-bungarotoxin, and S100.

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Biochemistry

A Knowledge Graph Approach to Elucidate the Role of Organellar Pathways in Disease via Biomedical Reports
Alexander R. Pelletier 1,2,3, Dylan Steinecke 1,3,4, Dibakar Sigdel 1, Irsyad Adam 1, J. Harry Caufield 1, Vladimir Guevara-Gonzalez 1, Joseph Ramirez 1, Aarushi Verma 1, Kaitlyn Bali 1, Katherine Downs 1, Wei Wang 1,2,3, Alex Bui 3,4, Peipei Ping 1,2,3,4,5
1Department of Physiology, UCLA School of Medicine, 2Scalable Analytics Institute (ScAi) at Department of Computer Science, UCLA School of Engineering, 3NIH BRIDGE2AI Center at UCLA & NHLBI Integrated Cardiovascular Data Science Training Program, UCLA, 4Medical Informatics, University of California at Los Angeles (UCLA), 5Department of Medicine (Cardiology), UCLA School of Medicine

A computational protocol, CaseOLAP LIFT, and a use case are presented for investigating mitochondrial proteins and their associations with cardiovascular disease as described in biomedical reports. This protocol can be easily adapted to study user-selected cellular components and diseases.

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Biology

Pipeline for Multi-Scale Three-Dimensional Anatomic Study of the Human Heart
Peter Hanna *1,2, Shumpei Mori *1, Takanori Sato 1, Shili Xu 3,4,5
1University of California Los Angeles (UCLA) Cardiac Arrhythmia Center, UCLA Health System, David Geffen School of Medicine at UCLA, 2Neurocardiology Program of Excellence, UCLA, 3Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, 4Crump Institute for Molecular Imaging, David Geffen School of Medicine at UCLA, 5Jonsson Comprehensive Cancer Center, David Geffen School of Medicine at UCLA

This protocol presents a comprehensive pipeline to analyze samples obtained from human hearts that span the microscopic and macroscopic scales.

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