Visualization and Modeling of the In Vivo Distribution of Mesenchymal Stem Cells

Haolu Wang1, Camilla A. Thorling2, Zhi Ping Xu3, Darrell H. G. Crawford4, Xiaowen Liang2, Xin Liu2, Michael S. Roberts5

1 Department of Biliary‐Pancreatic Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 2 Therapeutics Research Centre, School of Medicine, The University of Queensland, Princess Alexandra Hospital, Woolloongabba, 3 Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, 4 School of Medicine, The University of Queensland, Greenslopes, 5 School of Pharmacy and Medical Science, University of South Australia, Adelaide
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2B.8
DOI:  10.1002/cpsc.39
Online Posting Date:  November, 2017
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This unit describes a protocol for elucidating the in vivo disposition of administered mesenchymal stem cells (MSCs). Specifically, direct visualization of donor cell spatiotemporal distribution and assessment of donor cell quantity in recipient organs are described. Protocols for data analysis are suggested, with the goal of developing a model to characterize and predict the physiological kinetics of administered MSCs. The use of this model is described, suggesting that it can be applied to abnormal conditions and has potential interspecies and inter‐route predictive capability. These universal methods can be employed, regardless of the type of stem cell or disease, to guide future experiments and design treatment protocols. © 2017 by John Wiley & Sons, Inc.

Keywords: Mesenchymal stem cells; in vivo imaging; modeling; distribution

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Table of Contents

  • Introduction
  • Basic Protocol 1: Short‐Term Visualization of MSC Spatiotemporal Disposition
  • Alternate Protocol 1: Long‐Term Visualization of MSC Spatiotemporal Disposition
  • Basic Protocol 2: Quantitation of Donor Cells in Recipient Organs
  • Basic Protocol 3: Modeling the In Vivo Distribution of MSCs
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Short‐Term Visualization of MSC Spatiotemporal Disposition

  • 70% (v/v) ethanol (POCD Scientific, cat. no. 02216122)
  • GFP‐MSCs
  • Phosphate‐buffered saline (PBS, sterile, Sigma, cat. no. P3813)
  • BALB/c nude mouse
  • Ketamine (Parnell Laboratories)
  • Xylazine (Bayer)
  • Opthalmic ointment (Puralube, cat. no. 9B‐76855)
  • Surgical drape (SurgiDry, cat. no. 00002)
  • Scissors (Elite Medical, cat. no. 360‐101)
  • Dissection scissors (Elite Medical, cat. no. 100‐130)
  • Forceps (Elite Medical, cat. no. 355‐092)
  • Cotton swabs (Livingstone, cat. no. Q03M)
  • Animal heating pad (Provet QLD, cat. no. 8556)
  • 1‐ml syringes (Terumo, cat. no. V00089)
  • 25‐ and 27‐G needles (BD PrecisionGlide, cat. no. 301629)
  • Sterile cotton gauze (Propax, cat. no. 5524)
  • Custom‐made metal plate attached to adjustable stand
  • 18‐mm coverslips (ProSciTech, cat. no. G405)
  • Two‐photon microscopy system: DermaInspect system (Jen‐Lab GmbH) or LaVision Biotec Nikon multiphoton system (LaVision BioTec)
  • 10× water‐immersion and/or 40× oil‐immersion objective
  • Filter sets for organ autofluorescence (740 nm ex., 350‐450 nm em.) and GFP (900 nm ex., 515‐620 nm em.)
  • Imaging software (e.g., ImageJ 1.44p, U.S. National Institutes of Health)

Alternate Protocol 1: Long‐Term Visualization of MSC Spatiotemporal Disposition

  • Mice carrying GFP‐MSCs (see protocol 1)
  • EDTA (Sigma Aldrich, cat. no. EDS)
  • Lysis buffer: 144 mM NH 4Cl (Sigma Aldrich, cat. no. A9434) in 17 mM Tris (Sigma Aldrich, cat. no. T1503), pH 7.2 (store up to 1 month at 4°C)
  • Phosphate‐buffered saline (PBS, Sigma, cat. no. P3813)
  • Fetal bovine serum (FBS, Gibco, cat. no. 10100139)
  • 2 mg/ml collagenase V (Sigma Aldrich, cat. no. C9263) in PBS
  • Surgical instruments: forceps, scissors, scalpels
  • 1‐ml tubes
  • Plastic dishes (Sarstedt, cat. no. 1505039)
  • 15‐ml Falcon tubes (Falcon, cat. no. 352096)
  • 10‐ml serological pipets (Corning, cat. no. 4488)
  • 70‐μm mesh filters (Falcon, cat. no. 08‐771‐2)
  • 5‐ml flow cytometry tubes (Falcon, cat. no. 14‐959‐40B)
  • Flow cytometer (Accuri C6 or FACSCalibur, BD Biosciences)
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Literature Cited

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