Human Induced Pluripotent Stem (hiPS) Cells from Urine Samples: A Non‐Integrative and Feeder‐Free Reprogramming Strategy

Clara Steichen1, Karim Si‐Tayeb2, Fanny Wulkan1, Thayane Crestani1, Graça Rosas3, Rafael Dariolli1, Alexandre C. Pereira1, Jose E. Krieger1

1 Heart Institute (InCor), University of São Paulo Medical School, São Paulo, 2 Université de Nantes, Nantes, 3 Emergency Medicine Department, University of São Paulo Medical School, São Paulo
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 21.7
DOI:  10.1002/cphg.26
Online Posting Date:  January, 2017
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Abstract

Human induced pluripotent stem (hiPS) cell technology has already revolutionized some aspects of fundamental and applied research such as study of disease mechanisms and pharmacology screening. The first clinical trial using hiPS cell‐derived cells began in Japan, only 10 years after the publication of the proof‐of concept article. In this exciting context, strategies to generate hiPS cells have evolved quickly, tending towards non‐invasive protocols to sample somatic cells combined with “safer” reprogramming strategies. In this unit, we describe a protocol combining both of these advantages to generate hiPS cells with episomal plasmid transfection from urine samples of individuals carrying the desired genotype. Based on previous published works, this simplified protocol requires minimal equipment and reagents, and is suitable both for scientists familiar with the hiPS cells technology and neophytes. HiPS cells displaying classical features of pluripotency and suitable for all desired downstream applications are generated rapidly (<10 weeks) and with high efficiency. © 2017 by John Wiley & Sons, Inc.

Keywords: human induced pluripotent stem (hiPS) cells; urine progenitor cells (UPCs); reprogramming; integration‐free; feeder‐free

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Urine Progenitor Cells (UPCs) from Human Urine Samples
  • Basic Protocol 2: Reprogramming of UPCs Into Induced Pluripotent Stem Cells
  • Support Protocol 1: Cell Passaging During Reprogramming
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Urine Progenitor Cells (UPCs) from Human Urine Samples

  Materials
  • Urine donor
  • 0.1% gelatin solution (see recipe)
  • 70% ethanol
  • Washing buffer (see recipe)
  • Urine Progenitor Cell mix (UPC mix; see recipe)
  • FGF2 (see recipe)
  • 1× TrypLE (Life Technologies, cat. no. 12563029)
  • DMSO (dimethylsulfoxide)
  • Liquid nitrogen
  • Sterile containers for urine
  • 24‐well and 12‐well plates (sterile, tissue‐culture treated)
  • 50‐ml and 15‐ml conical tubes (e.g., Corning Falcon; sterile)
  • Centrifuge
  • Inverted microscope
  • Cryopreservation vials
  • Freezing container: e.g., Mr Frosty (Nalgene)
  • Liquid nitrogen storage container and hardware
NOTE: All reagents and solutions should be prepared under sterile conditions and using sterile vessels.

Basic Protocol 2: Reprogramming of UPCs Into Induced Pluripotent Stem Cells

  Materials
  • Geltrex solution (see recipe)
  • Urine progenitor cells (UPCs; protocol 1) in 12‐well plates
  • DMEM/F12 medium (Life Technologies, cat. no. 11320033)
  • 1× TrypLE (Life Technologies, cat. no. 12563029)
  • Urine Progenitor Cell mix (UPC mix; see recipe)
  • FGF2 (see recipe)
  • OptiMEM (Life Technologies, cat. no. 31985062)
  • Lipofectamine 3000 transfection reagent (Life Technologies, cat. no. L3000008)
  • Epi5 iPS reprogramming kit (Life Technologies, cat. no. A15960)
  • E6 medium (Life Technologies, cat. no. A1516401)
  • E8 medium (Life Technologies, cat. no. A1517001)
  • ROCK Inhibitor Y27632 (see recipe)
  • 70% ethanol
  • Versene (Life Technologies, cat. no. 15040066)
  • 50‐ml and 15‐ml conical tubes (e.g., Corning Falcon; sterile)
  • 12‐well plates and 6‐well plates (sterile, tissue‐culture treated)
  • Inverted microscope
  • Digital microscope (optional): stereomicroscope type Lynx (Axiovision Lynx, Bench stand)
  • Syringe with 27‐G insulin syringe
  • 60 mm‐dishes (sterile, tissue‐culture treated)
  • Additional reagents and equipment for counting cells by trypan blue exclusion ( appendix 3G; Phelan, )
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Figures

Videos

Literature Cited

Literature Cited
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