cGMP Generation of Human Induced Pluripotent Stem Cells with Messenger RNA

Yuhui Ni1, Yuanyuan Zhao1, Luigi Warren2, Jennifer Higginbotham3, Jiwu Wang3

1 Allele Biotechnology and Pharmaceuticals, Inc, San Diego, California, 2 Cellular Programming Inc, La Jolla, California, 3 Scintillon Institute, San Diego, California
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 4A.6
DOI:  10.1002/cpsc.18
Online Posting Date:  November, 2016
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Reprogramming somatic cells to generate induced pluripotent stem cells (iPSCs) has presented the biomedical community with a powerful platform to develop new models for human disease. To fully realize the promise of this technology in cell therapy and regenerative medicine, creating iPSCs under current Good Manufacture Practice (cGMP) conditions is paramount. Some reports have described efforts in this regard, resulting in iPSC lines that are cGMP compliant. The technology developed at Allele Biotechnology for footprint‐free, feeder‐free, and xeno‐free reprogramming using only mRNA is very suitable for creating iPSC lines through an established cGMP process. This technology has resulted in a licensing agreement between Allele Biotechnology and Ocata (formerly ACT, now a wholly owned division of Astellas) for clinical applications. All reagents and vessels are certified as cGMP‐produced, all equipment and software are certifiable, and all procedures are carried out in Industry ISO 7 or Class 10,000‐grade cleanrooms. In this revised version of the unit, we describe the core improvements to implement steps toward cGMP‐compliant generation of iPSCs. Recreating a process close to cGMP production in academic research will make these findings more applicable to translational research. © 2016 by John Wiley & Sons, Inc.

Keywords: induced pluripotent stem cells; iPSC; mRNA reprogramming; feeder‐free; xeno‐free; cGMP

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Table of Contents

  • Introduction
  • Basic Protocol 1: cGMP Reprogramming of Human Fibroblasts to Pluripotency
  • Support Protocol 1: Live Staining of iPSC Colonies
  • Support Protocol 2: Generation of in Vitro Transcription Template Constructs
  • Support Protocol 3: PCR‐based Production of IVT Templates From Plasmid Constructs
  • Support Protocol 4: Preparation of Synthetic Messenger RNA
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: cGMP Reprogramming of Human Fibroblasts to Pluripotency

  • Synthemax II‐SC (Corning, cat. no. 3535XX1)
  • Human fibroblasts (see introduction to this protocol)
  • RiPSCell iPSC reprogramming medium (Allele Biotechnology, cat. no. ABP‐SC‐RIPSCMED)
  • Carrier‐free B18R recombinant protein (Affymetrix eBioscience, cat. no. 34‐8185)
  • 6F mRNA reprogramming premix (Allele Biotechnology, cat. no. ABP‐SC‐6FMRNA)
  • SAINT‐RED transfection reagent and HBS, pH 7.4 (Synvolux Therapeutics, cat. no. SR‐1003‐01)
  • HEPES buffered saline (HBS)
  • TeSR‐E8 culture medium (StemCell Technologies, cat. no. 05940)
  • SCLift stem cell detaching reagent (Allele Biotechnology, cat. no. ABP‐SC‐SCLIFT)
  • Dulbecco's phosphate‐buffered saline (DPBS) with calcium and magnesium (Lonza, cat. no. 17‐513F)
  • 12.5‐cm2 flask (Corning, cat. no. 353018)
  • Cell counter (e.g., Orflo Technologies, cat. no. MXC002)
  • Humidified tissue‐culture incubator set to 37°C, 5% CO 2, and (if supported) 5% O 2

Support Protocol 1: Live Staining of iPSC Colonies

  • iPSC colonies ( protocol 1)
  • Dulbecco's phosphate‐buffered saline (DPBS) with calcium and magnesium (Lonza, cat. no. 17‐513F)
  • Directly conjugated fluorescent TRA‐1‐60 or TRA‐1‐81 antibody (e.g., StainAlive DyLight488; Stemgent, cat. no. 09‐0068/09‐0069)
  • TeSR‐E8 culture medium (StemCell Technologies, cat. no. 05940)
  • 6‐well tissue culture plates (BD Falcon, 3046)
  • Fluorescence microscope with FITC filter set and 4× or 10× objectives

Support Protocol 2: Generation of in Vitro Transcription Template Constructs

  • Base vector for cloning (see Critical Parameters)
  • 250‐bp minigene insert sequence (Table 4.6.2)
  • HiFi HotStart ReadyMix PCR master mix (KAPA, cat. no. KK2601)
  • PCR primers (Table 4.6.2)
  • PCR purification Kit (e.g., Qiagen QIAquick, cat. no. 28104)
  • Agarose gels (e.g., 2% SYBR Safe E‐gels ThermoFisher Scientific, cat. no. G521802)
  • Low DNA mass ladder (e.g., ThermoFisher Scientific, cat. no. 10068‐013)
  • Chemically competent E. coli (e.g., ThermoFisher Scientific, One Shot TOP10, cat. no. C4040‐10)
  • 100 mm agar plates with antibiotic(s) of choice
  • LB broth supplemented with antibiotic(s) of choice
  • Plasmid Mini Kit II (e.g., E.Z.N.A. HP Omega Bio‐Tek, cat. no. D7045‐01), or equivalent
Table 4.0.2   MaterialsMinigene and Oligo Sequences

  • 8‐tube 0.2 ml PCR strips and caps (e.g., ThermoFisher Scientific, cat. no. AM12230)
  • 96‐well plate thermocycler (e.g., Bio‐Rad, cat. no. 186‐1096)
  • Agarose gel electrophoresis units (e.g., ThermoFisher Scientific, E‐gel runner and transilluminator, cat. no. G6465)
  • 37°C incubator with shaker
  • DNA sequence analysis software (e.g., CLC Sequence Viewer)

Support Protocol 3: PCR‐based Production of IVT Templates From Plasmid Constructs

  Additional Materials ( protocol 3)
  • 20 ng/μl purified plasmid containing IVT template sequence (see protocol 3)
  • 1 μM stocks of INSERT‐F and TAIL‐120 (Table 4.6.2)

Support Protocol 4: Preparation of Synthetic Messenger RNA

  Additional Materials ( protocol 3)
  • 2.5× NTP mix with cap analog (Allele Biotechnology, cat. no. ABP‐PP‐NTPMIX)
  • MEGAscript T7 IVT Kit (ThermoFisher Scientific, cat. no. AM1333)
  • PCR product templates (Allele Biotechnology, catalog customer order or user‐generated)
  • MEGAclear RNA Purification Kit (ThermoFisher Scientific, cat. no. AM1908)
  • Antarctic phosphatase with 10× buffer (New England Biolabs, cat. No. M0289S)
  • TE buffer, pH 7.0 (ThermoFisher Scientific, cat. no. AM9860)
  • Agarose gels (e.g., 2% SYBR Safe E‐gels; ThermoFisher Scientific, cat. no. G521802)
  • Nanodrop (Thermo Scientific), or equivalent UV spectrophotometer
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