Rhesus Macaque iPSC Generation and Maintenance

Ravi Chandra Yada1, So Gun Hong1, Yongshun Lin2, Thomas Winkler1, Cynthia E. Dunbar1

1 Hematology Branch, National Heart, Lung and Blood Institute (NHLBI), National Institutes of Health, Bethesda, Maryland, 2 iPSC Core, Center for Molecular Medicine, NHLBI, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 4A.11
DOI:  10.1002/cpsc.25
Online Posting Date:  May, 2017
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Abstract

The rhesus macaque (Macaca mulatta) is physiologically and phylogenetically similar to humans, and therefore represents an invaluable model for the pre‐clinical assessment of the safety and feasibility of iPSC‐derived cell therapies. The use of an excisable polycistronic lentiviral STEMCCA vector to reprogram rhesus fibroblasts or bone marrow stromal cells (BMSCs) into RhiPSCs is described. After reprogramming, the pluripotency transgenes can be removed by transient expression of Cre, leaving a residual genetic tag that may be useful for identification of RhiPSC‐derived tissues in vivo. Finally, the steps to maintain pluripotency during passaging of RhiPSCs, required for successful utilization of RhiPSCs, is described. © 2017 by John Wiley & Sons, Inc.

Keywords: induced pluripotent stem cells (iPSCs); reprogramming; non‐human primate; rhesus macaque

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Rhesus‐Induced Pluripotent Stem Cell Generation
  • Support Protocol 1: MEF‐Conditioned Medium (CM) Preparation for Feeder‐Free Culture of iPSCs
  • Basic Protocol 2: Rhesus iPSC Culture and Maintenance
  • Basic Protocol 3: RhiPSC Characterization
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Rhesus‐Induced Pluripotent Stem Cell Generation

  Materials
  • Rhesus fibroblasts or BMSCs
  • Fibroblast culture medium or BMSC medium (see recipes)
  • Polybrene (see recipe)
  • STEMCCA virus
  • Ultrapure water with 0.1% gelatin (Millipore, cat. no. ES‐006‐B)
  • Mouse embryonic fibroblasts (MEF), 2 million cells/vial (GlobalStem, cat. no. GSC‐6201G)
  • MEF culture medium (see recipe)
  • 0.25% trypsin‐EDTA (1×) (Gibco, cat. no. 25200‐056)
  • RhiPSC medium (see recipe)
  • Valproic acid (VPA) (see recipe)
  • ROCK inhibitor (Y‐27632) (see recipe)
  • Opti‐MEM reduced serum medium (ThermoFisher, cat. no. 31985062)
  • EditPro Stem Transfection Reagent (MTI‐GlobalStem, cat. no. 31985062)
  • 6‐well plates
  • 37ºC, 5% CO 2, 20% O 2 incubator
  • 15‐ml conical tubes
  • Centrifuge
  • Aspirator
  • 10‐cm dishes, optional
  • Hypoxia chamber (BioSpherix, cat. no. C‐274)
  • O 2 controller (BioSpherix, cat. no. P110)
  • CO 2 controller (BioSpherix, cat. no. P120)
  • Cell scraper (Corning, cat. no. 3010)
  • Matrigel‐coated 12‐well plates (unit 1.2)

Support Protocol 1: MEF‐Conditioned Medium (CM) Preparation for Feeder‐Free Culture of iPSCs

  Materials
  • Ultrapure water with 0.1% gelatin (Millipore, cat. no. ES‐006‐B)
  • Mouse embryonic fibroblasts (MEF), 2 million cells/vial (GlobalStem, cat. no. GSC‐6201G)
  • MEF medium (see recipe)
  • RhiPSC medium without bFGF (see recipe)
  • PBS (Lonza, cat. no. 17‐516Q)
  • Puro‐T2A‐Cre‐GFP plasmid (Merling et al., )
  • Puromycin (see recipe)
  • 165‐cm2 tissue culture flasks
  • 37°C incubator
  • Aspirator
  • 0.22‐μm filters
  • Matrigel‐coated 12‐well plates (unit 1.2)

Basic Protocol 2: Rhesus iPSC Culture and Maintenance

  Materials
  • MEF plates (see protocol 1)
  • Thawing medium: RhiPSC medium supplemented with either ROCK inhibitor (Y‐27632) (Stemgent, cat. no. 04‐0012) or RevitalCell Supplement (100×) (optional; ThermoFisher Scientific, cat. no. A2644501)
  • RhiPSC medium (see recipe)
  • PBS
  • Matrigel (BD Bioscience, cat. no. 354277) plates
  • CM (see protocol 2Support Protocol)
  • Dispase (see recipe)
  • Freezing medium: ES‐defined FBS (Hyclone, cat. no. SH30070.02E)/10% DMSO or CryoStor CS10 (StemCell Technologies cat. no. 07930)
  • Liquid nitrogen
  • 15‐ml conical tubes
  • 37ºC water bath
  • Centrifuge
  • Stereo microscope (e.g., Leica KL 200 LED)
  • 37ºC, 5% CO 2, 5% O 2 incubator
  • Cell scraper
  • Cryovials
  • Freezing containers
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Figures

Videos

Literature Cited

Literature Cited
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