Practical Integration‐Free Episomal Methods for Generating Human Induced Pluripotent Stem Cells

Cody Kime1, Tim A. Rand2, Kathryn N. Ivey3, Deepak Srivastava4, Shinya Yamanaka5, Kiichiro Tomoda2

1 Present address: Laboratory for Retinal Regeneration, RIKEN Center for Developmental Biology, Kobe, 2 Roddenberry Center for Stem Cell Biology and Medicine at Gladstone, San Francisco, California, 3 Department of Pediatrics, University of California, San Francisco, California, 4 Department of Biochemistry and Biophysics, University of California, San Francisco, California, 5 Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 21.2
DOI:  10.1002/0471142905.hg2102s87
Online Posting Date:  October, 2015
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Abstract

The advent of induced pluripotent stem (iPS) cell technology has revolutionized biomedicine and basic research by yielding cells with embryonic stem (ES) cell‐like properties. The use of iPS‐derived cells for cell‐based therapies and modeling of human disease holds great potential. While the initial description of iPS cells involved overexpression of four transcription factors via viral vectors that integrated within genomic DNA, advances in recent years by our group and others have led to safer and higher quality iPS cells with greater efficiency. Here, we describe commonly practiced methods for non‐integrating induced pluripotent stem cell generation using nucleofection of episomal reprogramming plasmids. These methods are adapted from recent studies that demonstrate increased hiPS cell reprogramming efficacy with the application of three powerful episomal hiPS cell reprogramming factor vectors and the inclusion of an accessory vector expressing EBNA1. © 2015 by John Wiley & Sons, Inc.

Keywords: human; episomal; induced pluripotent stem cell; reprogramming

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Human Dermal Fibroblast Culture
  • Basic Protocol 2: Nucleofection of Fibroblasts with Plasmid DNA (Reprogramming Day 0)
  • Basic Protocol 3: Early HDF Reprogramming Cell Culture
  • Basic Protocol 4: Human iPS Cell Colony Growth, Picking, and Subculture
  • Alternate Protocol 1: Peripheral Blood Isolation and Culture
  • Alternate Protocol 2: Nucleofection of CD34+ PBMC with Plasmid DNA (Reprogramming Day 0)
  • Alternate Protocol 3: Early CD34+ PBMC Reprogramming Cell Culture
  • Support Protocol 1: Preparation of Plasmid DNA
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Human Dermal Fibroblast Culture

  Materials
  • Fibroblast culture medium (FCM; see recipe)
  • Gelatin substrate (see recipe)
  • DPBS (calcium‐ and magnesium‐free)
  • 0.05% Trypsin/EDTA solution (Life Technologies, cat. no. 25300‐054)
  • 6‐well or 100‐mm plates
  • 15‐ml conical vials
  • Centrifuge
  • 37°C incubator

Basic Protocol 2: Nucleofection of Fibroblasts with Plasmid DNA (Reprogramming Day 0)

  Materials
  • 3 μg total Episomal DNA per sample
  • Plasmids (prepare as described in the protocol 8Support Protocol):
    • pCXLE‐hOCT3/4‐shp53 (Addgene, cat. no. 27077)
    • pCXLE‐hSK (Addgene, cat. no. 27078)
    • pCXLE‐hUL (Addgene, cat. no. 27080)
    • pCXWB‐EBNA1 (Addgene, cat. no. 37624)
    • pCXLE‐EGFP (Addgene cat. no. 27082)
  • NHDF Nucleofector kit (Amaxa/Lonza, cat. no. VPD‐1001) containing nucleofection solution
  • HDF cultures (see protocol 1)
  • DPBS (calcium‐ and magnesium‐free)
  • 0.05% Trypsin/EDTA Solution (Life Technologies, cat. no. 25300‐054)
  • FCM (see recipe)
  • 6‐well gelatin‐coated plates (coat 6‐well plates for 15 min with 1 ml gelatin substrate per well; 3 wells for each nucleofection reprogramming sample; aspirate the gelatin substrate and add 2 ml of FCM per well, and then incubate at 37°C prior to plating nucleofected HDF)
  • 1.5‐ml tubes
  • Amaxa Nucleofector 2b
  • Program U‐023
  • 37°C incubator
  • 15‐ml conical vials
NOTE: Use at least 5 × 105 HDF per reprogramming sample, 70% to 90% confluent in FCM on a 100‐mm plate.

Basic Protocol 3: Early HDF Reprogramming Cell Culture

  Materials
  • 6‐well plates containing nucleofected HDF (see protocol 2)
  • FCM (see recipe)
  • Gelatin substrate (see recipe)
  • SNL Feeder Cells (mitotically inactivated; Applied Stem Cell, cat. no. ASF‐1318)
  • hES cell medium (see recipe)
  • ROCK inhibitor (Y‐27632; stock = 10 mM; SelleckChem, cat. no. S1049)
  • DPBS (calcium‐ and magnesium‐free)
  • 0.05% trypsin/EDTA solution (Life Technologies, cat. no. 25300‐054)
  • 100‐mm tissue culture grade plates
  • 37°C incubator
  • 15‐ml conical vials
  • Centrifuge
NOTE: SNL feeder cells that grow in cell culture can be purchased from Applied Stem Cell (cat. no. ASF‐1305). There are procedures for propagation and subsequent mitotic inactivation of SNLs for in‐house stock preparation (see Internet Resources; http://www.cira.kyoto‐u.ac.jp/e/research/images/protocol/pdf/hipsprotocolv2_090304.pdf).

Basic Protocol 4: Human iPS Cell Colony Growth, Picking, and Subculture

  Materials
  • Reprogramming cells (see protocol 3 or protocol 7)
  • hES cell medium (see recipe)
  • Gelatin substrate (see recipe)
  • SNL feeder cells (mitotically inactivated; Applied Stem Cell, cat. no. ASF‐1318
  • FCM (see recipe)
  • DPBS (calcium‐ and magnesium‐free)
  • ROCK inhibitor (Y‐27632; stock = 10 mM; SelleckChem, cat. no. S1049)
  • Accutase (Millipore, cat. no. SCR005)
  • 24‐well tissue culture grade plates
  • 37°C incubator
  • 96‐well plates (for manual colony dispersion)
  • Picking microscope (e.g., EVOS XL)
  • 200‐μl barrier tip on a 200‐μl pipet
  • 1‐ml pipet tips
  • 1000‐μl pipets

Alternate Protocol 1: Peripheral Blood Isolation and Culture

  Materials
  • Sodium citrate or sodium heparin vacutainer with 10 ml of patient blood
  • 70% ethanol
  • MACS buffer (see recipe)
  • Ice
  • Ficoll‐Paque
  • Miltenyi MACS System with CD34+ Microbeads Kit for Human (Miltenyi, cat. no. 130‐046‐702) containing FcR Blocking Reagent
  • Stem Pro 34 SFM Kit (Life Technologies, cat.no. 10639‐011)
  • Centrifuge
  • Pasteur pipets or plastic serological pipets
  • Transfer pipets
  • Magnetic separator
  • 6‐well ultra‐low attachment (ULA) 37°C incubator plates (make sure your plates are ULA)
  • 50‐ and 15‐ml conical vials
  • 37°C incubator

Alternate Protocol 2: Nucleofection of CD34+ PBMC with Plasmid DNA (Reprogramming Day 0)

  Materials
  • Plasmids (prepared as described in the protocol 8Support Protocol)
  • pCXLE‐hOCT3/4‐shp53 (Addgene, cat. no. 27077)
  • pCXLE‐hSK (Addgene, cat. no. 27078)
  • pCXLE‐hUL (Addgene, cat. co. 27080)
  • pCXLE‐EGFP (Addgene, cat. no. 27082)
  • pCXWB‐EBNA1 (Addgene, cat. no. 37624)
  • 16 μg total Episomal DNA per sample.
  • Amaxa Nucleofector 2b Program U‐008
  • Human CD34 Cell Nucleofector Kit (Amaxa/Lonza, cat. no. VPA‐1003) containing complete nucleofection solution
  • CD34+ PBMC (at least 4‐10 × 105 for each nucleofection; see protocol 5)
  • MACS buffer (see recipe)
  • Stem Pro 34 SFM Kit (Life Technologies, cat. no. 10639‐011)
  • 37°C incubator

Alternate Protocol 3: Early CD34+ PBMC Reprogramming Cell Culture

  Materials
  • 6‐well plates containing nucleofected CD34+ PBMC (see protocol 6)
  • Gelatin substrate (see recipe)
  • SNL feeder cells (mitotically inactivated; Applied Stem Cell, cat. no. ASF‐1318)
  • FCM (see recipe)
  • Stem Pro 34 SFM Kit (Life Technologies, cat. no. 10639‐011) containing Stem Pro 34 medium
  • hES cell medium (see recipe)
  • ROCK inhibitor [Y‐27632; stock = (10 mM); SelleckChem, cat. no. S1049]
  • DPBS (calcium‐ and magnesium‐free)
  • 100‐mm tissue culture grade plates
  • 37°C incubator
  • Centrifuge
  • 15‐ml conical vials

Support Protocol 1: Preparation of Plasmid DNA

  Materials
  • DH5α (Life Technologies or New England Biolabs)
  • pCXLE‐hOCT3/4‐shp53 (Addgene, cat. no. 27077)
  • pCXLE‐hSK (Addgene, cat. no. 27078)
  • pCXLE‐hUL (Addgene, cat. no. 27080)
  • pCXLE‐EGFP (Addgene, cat. no. 27082)
  • pCXWB‐EBNA1 (Addgene, cat. no. 37624)
  • LB medium
  • Ampicillin
  • QIAGEN EndoFree Maxiprep Kit (QIAGEN, cat. no. 12362)
  • Nuclease‐free water or TE buffer
  • Shaking incubator
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Figures

Videos

Literature Cited

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