Rapid Screening of the Endodermal Differentiation Potential of Human Pluripotent Stem Cells

Richard Siller1, Gareth J. Sullivan2

1 Norwegian Center for Stem Cell Research, Blindern, Oslo, 2 Hybrid Technology Hub–Centre of Excellence, Institute of Basic Medical Sciences, University of Oslo, Blindern, Oslo
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1G.7
DOI:  10.1002/cpsc.36
Online Posting Date:  November, 2017
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Abstract

Human pluripotent stem cells (hPSCs) hold tremendous promise for regenerative medicine, disease modeling, toxicology screening, and developmental biology. These applications are hindered due to inherent differences in differentiation potential observed among different hPSC lines. This is particularly true for the differentiation of hPSCs toward the endodermal lineage. Several groups have developed methods to screen hPSCs for their endodermal differentiation potential (EP). Particularly notable studies include (i) the use of WNT3A expression as a predictive biomarker, (ii) an embryoid body–based screen, and (iii) a transcriptomics‐based approach. We recently developed a rapid screen to access the EP of hPSCs solely based on morphological analysis. The screen takes 4 days to perform and yields results that are easy to interpret. As the screen is based on our recently developed small molecule protocol for hepatocyte like cell (HLC) differentiation of hPSCs, this method is extremely cost‐effective compared to the aforementioned approaches. © 2017 by John Wiley & Sons, Inc.

Keywords: definitive endoderm; differentiation potential; hPSCs; hESCs; hiPSCs; small molecules

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Screen for the Endodermal Differentiation Potential (EP) of Human Pluripotent Stem Cell Lines
  • Support Protocol 1: Preparation of Geltrex‐Coated Tissue Culture Plates
  • Support Protocol 2: Feeder‐Independent Culture and Maintenance of Human Pluripotent Stem Cells on Geltrex
  • Support Protocol 3: Preparation of E8 Medium
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Screen for the Endodermal Differentiation Potential (EP) of Human Pluripotent Stem Cell Lines

  Materials
  • E8 medium (see protocol 4)
  • Human pluripotent stem cells (hPSCs; maintained in E8 medium; see protocol 3):
  • hESC line:
  • H1 (purchased from WiCell)
  • hiPSC lines:
  • Retroviral‐derived Detroit RA (Detroit 551 fibroblast line purchased from ATCC)
  • Sendai‐derived CRL S23 (fibroblast line CRL 2097 purchased from ATCC)
  • Sendai AG15 (AG05836B fibroblast line purchased from Coriell Cell Repositories)
  • Dulbecco's PBS without calcium and magnesium (DPBS−/−; Life Technologies, cat. no. 14190)
  • 0.5 mM EDTA (see recipe)
  • RPMI/B‐27 medium with and without insulin (see reciperecipes)
  • 3 mM CHIR99021 stock (see recipe)
  • Geltrex‐coated 6‐ and 12‐well tissue culture plates (see protocol 2)
  • Phase‐contrast microscope

Support Protocol 1: Preparation of Geltrex‐Coated Tissue Culture Plates

  Materials
  • Geltrex™ LDEV‐Free, hESC‐qualified, reduced growth factor basement membrane matrix (Life Technologies, cat. no. A1413302)
  • Advanced Dulbecco's modified Eagle medium/Ham's F‐12 (Advanced DMEM/F‐12; Life Technologies, cat. no. 12634‐028)
  • Sterile 1‐ml tubes
  • 6‐well tissue culture plates (VWR, cat. no. 7340991)
  • 12‐well tissue culture plates (VWR, cat. no. 10098870)

Support Protocol 2: Feeder‐Independent Culture and Maintenance of Human Pluripotent Stem Cells on Geltrex

  Materials
  • E8 medium (see protocol 4)
  • 10 mM (1000×) ROCK Inhibitor Y‐27632 stock (see recipe)
  • Human pluripotent stem cells (hPSCs):
  • hESC line:
  • H1 (purchased from WiCell)
  • hiPSC lines:
  • Retroviral‐derived Detroit RA (Detroit 551 fibroblast line purchased from ATCC)
  • Sendai‐derived CRL S23 (fibroblast line CRL 2097 purchased from ATCC)
  • Sendai AG15 (AG05836B fibroblast line purchased from Coriell Cell Repositories)
  • 70% (v/v) ethanol
  • Dulbecco's PBS without calcium and magnesium (DPBS−/−; Life Technologies, cat. no. 14190)
  • 0.5 mM EDTA (see recipe)
  • Dimethylsulfoxide (DMSO; Sigma‐Aldrich, cat. no. 472301)
  • Liquid nitrogen
  • Geltrex‐coated 6‐well plates ( protocol 2)
  • 15‐ml conical tubes (e.g., BD Falcon)
  • Centrifuge
  • Phase‐contrast microscope
  • Cryovials (Thermo Scientific, cat. no. CLS4488‐200EA)
  • Mr. Frosty or other controlled cooling system
  • Liquid nitrogen storage setup

Support Protocol 3: Preparation of E8 Medium

  Materials
  • Dulbecco's modified Eagle medium/F12 (DMEM/F12) medium (Life Technologies, cat. no. 1130057)
  • NaHCO 3 (Sigma‐Aldrich, cat. no. S6041)
  • 5 N NaOH (see recipe)
  • 1000× L‐ascorbic acid 2‐phosphate stock (see recipe)
  • 10,000× sodium selenite stock (see recipe)
  • Insulin solution, human (Sigma‐Aldrich, cat. no. I9278—ready to use)
  • 1000× human recombinant transferrin stock (see recipe)
  • 1000× bFGF2 stock (see recipe)
  • 1000× TGFβ1 stock (see recipe)
  • Glass bottles (10 liter)
  • Magnetic stirring bars and stir plate
  • Graduated cylinder
  • Magnetic stirring plate
  • Osmometer
  • pH meter
  • 500‐ml filter unit (VWR, cat. no. 513‐3355)
  • 6‐well tissue culture plate
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Figures

Videos

Literature Cited

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