Isolation of Human Melanoma Stem Cells Using ALDH as a Marker

Yuchun Luo1, Nicholas Nguyen1, Mayumi Fujita2

1 University of Colorado Denver, Aurora, Colorado, 2 Denver Veterans Affairs Medical Center, Denver, Colorado
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 3.8
DOI:  10.1002/9780470151808.sc0308s26
Online Posting Date:  September, 2013
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This unit describes a protocol for establishing a patient‐derived tumor xenograft model (PDTX model) of human melanoma and isolating human melanoma stem cells from human melanoma specimens using aldehyde dehydrogenase (ALDH) as a marker. One major limitation of analyzing a small fraction of cancer stem cells from patient tumor samples is that substantial quantities of fresh tumor tissues are not available. To overcome this challenge, we have established a PDTX model of human melanoma. In this model, human tumor tissues obtained from melanoma patients are dissected into small pieces and subsequently implanted into immunocompromised mice. The PDTX tumors retain fundamental genotypic and phenotypic features of the original tumors and are suitable for further biological analyses. Using the PDTX model, we have analyzed ALDH‐labeled human melanoma stem cells. This unit will describe how to establish a PDTX model using human tumor samples. We will also describe how to isolate and analyze ALDH‐labeled human melanoma stem cells using this model. Curr. Protoc. Stem Cell Biol. 26:3.8.1‐3.8.10. © 2013 by John Wiley & Sons, Inc.

Keywords: melanoma; patient‐derived tumor xenograft; cancer stem cell; aldehyde dehydrogenase

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Table of Contents

  • Introduction
  • Basic Protocol 1: Establishing a PDTX Model Using Human Melanoma Tumor Samples
  • Basic Protocol 2: Analysis of ALDH‐Labeled Human Melanoma Stem Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Establishing a PDTX Model Using Human Melanoma Tumor Samples

  • Human melanoma tumor specimens
  • RPMI‐1640 medium
  • Matrigel (BD Biosciences)
  • Nude mice (five‐ to six‐week‐old)
  • Isofluorane
  • Drysol (20% aluminum chloride in anhydrous ethyl alcohol)
  • 60‐cm2 sterile culture dishes
  • Sterile surgical blade, forceps, micro‐dissecting forceps, and scissors
  • 1.5‐ml microcentrifuge tubes
  • Anesthesia machine
  • Surgical clippers (9‐mm), clip applier and removal device (Roboz)
  • Cotton buds

Basic Protocol 2: Analysis of ALDH‐Labeled Human Melanoma Stem Cells

  • Xenografted melanoma tissues or original human melanoma specimens
  • Tissue digestion solution (see recipe)
  • 1 × red blood cell lysis buffer (eBioscience)
  • Phosphate‐buffered saline (PBS; Cellgro, cat. no. 21‐040‐CV)
  • Trypan blue (Sigma‐Aldrich; also see unit 1.3)
  • Aldefluor kit (Stem Cell Technologies) containing:
    • Aldefluor buffer
    • DEAB
  • PE‐cy7‐labeled anti‐human CD45 (eBioscience)
  • PE‐cy7‐labeled anti‐human CD31 (eBioscience)
  • Allophycocyanin (APC)‐labeled anti‐mouse MHC Class I (H‐2Kd if BALB/c nude mice are used, eBioscience)
  • PE‐cy7‐labeled anti‐mouse CD45 (eBioscience)
  • PE‐cy7‐labeled anti‐rat IgG2b (eBioscience)
  • PE‐cy7‐labeled anti‐rat IgG2a (eBioscience)
  • APC‐labeled anti‐mouse IgG2a (eBioscience)
  • Ice
  • DAPI (Sigma‐Aldrich)
  • 100‐mm sterile petri dishes
  • Sterile surgical razor blades, forceps, and scissors
  • 15‐ml conical tubes
  • 10‐ml serological pipets
  • Water bath at 37°CVortex mixer
  • Centrifuge
  • 40‐ and 70‐µm cell strainers
  • Hemacytometer
  • MoFlo machine (DakoCytomation) to sort cells
  • Summit (DakoCytomation) software for data analysis
  • Additional reagents and equipment for trypan blue test of cell viability (unit 1.3)
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Literature Cited

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