Direct Conversion of Human Pluripotent Stem Cells to Osteoblasts With a Small Molecule

Heemin Kang1, Yu‐Ru V. Shih2, Shyni Varghese3

1 Department of Materials Science and Engineering, University of California, San Diego, La Jolla, California, 2 Department of Bioengineering, University of California, San Diego, 3 Department of Nanoengineering, University of California, San Diego, La Jolla, California
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1F.21
DOI:  10.1002/cpsc.44
Online Posting Date:  February, 2018
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Human pluripotent stem cells (hPSCs), which exhibit unlimited self‐renewal ability and can differentiate into all cell types in the human body, are a promising cell source for cell‐based therapies and regenerative medicine. Small molecules hold great potential in the derivation of tissue‐specific cells from hPSCs owing to their cost‐effectiveness and scalability. Here, we describe a protocol for deriving osteoblasts from hPSCs by using a single, natural small molecule: adenosine. This simple and effective experimental protocol allows one to obtain large numbers of osteoblasts or osteoprogenitor cells, with the ability to form functional bone tissues, from hPSCs, including human embryonic stem cells and induced pluripotent stem cells. This protocol could potentially enable studies of tissue regeneration and skeletal diseases. © 2018 by John Wiley & Sons, Inc.

Keywords: human embryonic stem cells (hESCs); human induced pluripotent stem cells (hiPSCs); small molecule; adenosine; osteogenic differentiation

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Table of Contents

  • Basic Protocol 1:  
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:  

  • 0.1% (w/v) gelatin stock solution (see recipe)
  • Mitotically inactivated (mitomycin C–treated) mouse embryonic fibroblasts (MEFs) (unit 23.2; Conner, )
  • hPSC maintenance medium (see recipe), 37°C
  • Human induced pluripotent stem cells (hiPSCs; IMR90p18‐iPS line, WiCell Research Institute) (Yu et al., ) or human embryonic stem cells (hESCs; HUES9 line, Harvard Stem Cell Institute)
  • Accutase (Millipore, cat. no. SCR005)
  • Matrigel (BD Biosciences, cat. no. 354277; store at –20°C)
  • Dulbecco's Modified Eagle's Medium (DMEM; Thermo Fisher Scientific, cat. no. 11965092), 4°C
  • Ca2+‐ and Mg2+‐free phosphate‐buffered saline (PBS; Thermo Fisher Scientific, cat. no. SH30258.02), 37°C
  • Knockout DMEM (Life Technologies, cat. no. 10829‐018), 37°C
  • hPSC intermediate medium (see recipe), 37°C
  • 2 mM thiazovivin stock solution (see recipe)
  • hPSC differentiation medium (see recipe), 37°C
  • 6‐well culture plate (9.5‐cm2 surface area per well)
  • Humidified 37°C, 5% CO 2 incubator
  • 50‐ml conical tube
  • 12‐well culture plate (3.8‐cm2 surface area per well)
  • Parafilm
  • Standard tabletop centrifuge
  • Hemocytometer
NOTE: All cell culture should be performed in a humidified incubator at 37°C with 5% CO 2.NOTE: The following procedures should be performed in a Class II biological safety cabinet under aseptic conditions using sterile and fresh reagents.
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Literature Cited

Literature Cited
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  Conner, D. A. (2001). Mouse embryo fibroblast (MEF) feeder cell preparation. Current Protocols in Molecular Biology, 51, 23.2.1–23.2.7. doi: 10.1002/0471142727.mb2302s51
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