Amnion Epithelial Cell Isolation and Characterization for Clinical Use

Sean Murphy1, Sharina Rosli2, Rutu Acharya2, Louisa Mathias1, Rebecca Lim2, Euan Wallace2, Graham Jenkin1

1 Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Victoria, Australia, 2 Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria, Australia
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1E.6
DOI:  10.1002/9780470151808.sc01e06s13
Online Posting Date:  April, 2010
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Abstract

Human amnion epithelial cells (hAECs) are a heterologous population positive for stem cell markers; they display multilineage differentiation potential, differentiating into cells of the endoderm (liver, lung epithelium), mesoderm (bone, fat), and ectoderm (neural cells). They have a low immunogenic profile and possess potent immunosuppressive properties. Hence, hAECs may be a valuable source of cells for cell therapy. This unit describes an efficient and effective method of hAEC isolation, culture, and cryopreservation that is animal product–free and in accordance with current guidelines on preparation of cells for clinical use. Cells isolated using this method were characterized after 5 passages by analysis of karyotype, cell cycle distribution, and changes in telomere length. The differentiation potential of hAECs isolated using this animal product–free method was demonstrated by differentiation into lineages of the three primary germ layers and expression of lineage‐specific markers analyzed by PCR, immunocytochemistry, and histology. Curr. Protoc. Stem Cell Biol. 13:1E.6.1‐1E.6.25. © 2010 by John Wiley & Sons, Inc.

Keywords: isolation; amniotic; amnion; epithelial; stem cells; cryopreservation; culture; characterization; placenta

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation of Amnion Epithelial Cells
  • Basic Protocol 2: Cryopreservation and Thawing of hAECS
  • Support Protocol 1: Characterization of hAECs by Flow Cytometry
  • Support Protocol 2: Differentiation of hAECs
  • Support Protocol 3: CellTiter 96 AQueous One Solution Cell Proliferation Assay
  • Support Protocol 4: Cell Proliferation ELISA
  • Support Protocol 5: Telomerase Assay to Assess Telomere Length
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Amnion Epithelial Cells

  Materials
  • Placentas
  • Hanks' Balanced Salt Solution (HBSS; Invitrogen, cat. no. 14175)
  • TrypZean (animal product–free recombinant trypsin; Sigma‐Aldrich, cat. no. T3449)
  • Soybean trypsin inhibitor (see recipe)
  • EpiLife growth medium (animal product–free medium; see recipe)
  • Anti‐EpCAM‐PE antibody (BD Biosciences, cat. no. 347198)
  • Monoclonal mouse anti‐CD90‐PeCy5 (BD Biosciences, cat. no. 555597)
  • Monoclonal mouse anti‐CD105‐APC (eBioscience, cat. no. 171057)
  • 100‐ to 200‐ml specimen containers, sterile
  • Sterile scissors and forceps
  • 15‐cm petri dishes
  • 37°C shaking water bath
  • 40‐ and 70‐µm filters
  • 15‐ml centrifuge tubes
  • BD FACS Canto flow cytometer
  • Additional reagents and equipment for performing a cell count (unit 1.3)
NOTE: Placentas were collected from healthy women with singleton pregnancies undergoing elective caesarean section delivery at term. Women gave written, informed consent for the collection of their placenta. The collection, and subsequent use, of placentas was performed with approval from the Southern Health Human Research Ethics Committee.

Basic Protocol 2: Cryopreservation and Thawing of hAECS

  Materials
  • Freshly isolated or FACS‐sorted hAECs ( protocol 1)
  • Cryopreservation medium (see recipe)
  • Liquid nitrogen storage system
  • EpiLife growth medium (animal product–free medium; see recipe)
  • O‐ring cryopreservation vials
  • Mr. Frosty freezing container (Thermo Fisher Scientific, cat. no. C1562)
  • −80°C freezer
  • 37°C water bath
  • 15‐ml centrifuge tubes
  • Additional reagents and equipment for counting the cells using trypan blue exclusion (unit 1.3)

Support Protocol 1: Characterization of hAECs by Flow Cytometry

  Materials
  • Fresh isolates of hAECs ( protocol 1) or cultures of hAECs after 5 passages ( protocol 2)
  • FACS buffer (see recipe)
  • Panel of monoclonal antibodies (Table 1.6.1) provided by BD Biosciences
  • 96‐well tissue culture plates
  • BD FACS Calibur flow cytometer
    Table 1.0.1   MaterialsList of Monoclonal Antibodies Used to Interrogate hAECs

    BD mAb Host Isotype
    CD1a Mouse IgG1
    CD1b Mouse IgG1
    CD1d Mouse IgG1
    CD2 Mouse IgG1
    CD3 Mouse IgG2a
    CD4 Mouse IgG1
    CD5 Mouse IgG1
    CD6 Mouse IgG1
    CD7 Mouse IgG1
    CD8 Mouse IgG1
    CD9 Mouse IgG1
    CD10 Mouse IgG1
    CD11a Mouse IgG1
    CD11b Mouse IgG1
    CD11c Mouse IgG1
    CD13 Mouse IgG1
    CD14 Mouse IgG1
    CD15 Mouse IgM
    CD15s Mouse IgG1
    CD16 Mouse IgG1
    CD18 Mouse IgG1
    CD19 Mouse IgG1
    CD20 Mouse IgG2b
    CD21 Mouse IgG1
    CD22 Mouse IgG1
    CD23 Mouse IgG1
    CD24 Mouse IgG2a
    CD25 Mouse IgG1
    CD26 Mouse IgG1
    CD27 Mouse IgG1
    CD28 Mouse IgG1
    CD29 Mouse IgG2a
    CD30 Mouse IgG1
    CD31 Mouse IgG1
    CD32 Mouse IgG2b
    CD33 Mouse IgG1
    CD34 Mouse IgG1
    CD35 Mouse IgG1
    CD36 Mouse IgM
    CD37 Mouse IgG1
    CD38 Mouse IgG1
    CD40 Mouse IgG1
    CD41a Mouse IgG1
    CD41b Mouse IgG3
    CD42a Mouse IgG1
    CD42b Mouse IgG1
    CD43 Mouse IgG1
    CD44 Mouse IgG2b
    CD45 Mouse IgG1
    CD45RA Mouse IgG2b
    CD45RB Mouse IgG1
    CD45RO Mouse IgG2a
    CD46 Mouse IgG2a
    CD47 Mouse IgG1
    CD48 Mouse IgM
    CD49a Mouse IgG1
    CD49b (1) Mouse IgG1
    CD49b (2) Mouse IgG2a
    CD49c Mouse IgG1
    CD49d Mouse IgG1
    CD49e (1) Mouse IgG1
    CD49e (2) Mouse IgG2a
    CD49f Mouse IgG2b
    CD50 Mouse IgG1
    CD51/61 Mouse IgG1
    CD53 Mouse IgG1
    CD54 Mouse IgG1
    CD55 Mouse IgG2a
    CD56 Mouse IgG2b
    CD57 Mouse IgM
    CD58 Mouse IgG2a
    CD59 Mouse IgG2a
    CD61 Mouse IgG1
    CD62e Mouse IgG1
    CD62L Mouse IgG1
    CD62P Mouse IgG1
    CD63 Mouse IgG1
    CD64 Mouse IgG1
    CD66 Mouse IgG1
    CD66b Mouse IgM
    CD66f Mouse IgG1
    CD69 Mouse IgG1
    CD70 Mouse IgG3
    CD71 Mouse IgG2a
    CD72 Mouse IgG2b
    CD73 Mouse IgG1
    CD74 Mouse IgG2a
    CD77 Mouse IgM
    CD79b Mouse IgG1
    CD80 Mouse IgG1
    CD81 Mouse IgG1
    CD83 Mouse IgG1
    CD84 Mouse IgG1
    CD85J Mouse IgG2b
    CD86 Mouse IgG1
    CD87 Mouse IgG1
    CD88 Mouse IgG1
    CD89 Mouse IgG1
    CD90 Mouse IgG1
    CD91 Mouse IgG1
    CD94 Mouse IgG1
    CD95 Mouse IgG1
    CD97 Mouse IgG1
    CD98 Mouse IgG1
    CD99 Mouse IgG2a
    CD99R Mouse IgM
    CD100 Mouse IgG1
    CD103 Mouse IgG1
    CD104 Mouse IgG1
    CD106 Mouse IgG1
    CD108 Mouse IgG2a
    CD109 Mouse IgG1
    CD110 Mouse IgG1
    CD117 Mouse IgG1
    CD123 Mouse IgG1
    CD134 Mouse IgG1
    CD135 Mouse IgG1
    CD137 (1) Mouse IgG1
    CD137 (2) Mouse IgG1
    CD138 Mouse IgG1
    CD140a Mouse IgG2a
    CD140b Mouse IgG2a
    CD141 Mouse IgG1
    CD142 Mouse IgG1
    CD146 Mouse IgG1
    CD147 Mouse IgG1
    CD150 Mouse IgG1
    CD151 Mouse IgG1
    CD152 Mouse IgG2a
    CD153 Mouse IgG1
    CD154 Mouse IgG1
    CD158a Mouse IgM
    CD158b Mouse IgG2b
    CD161 Mouse IgG1
    CD162 Mouse IgG1
    CD163 Mouse IgG1
    CD164 Mouse IgG2a
    CD165 Mouse IgG1
    CD166 Mouse IgG1
    CD172b Mouse IgG1
    CD177 Mouse IgG1
    CD180 Mouse IgG2a
    CD181 Mouse IgG2a
    CD182 Mouse IgG1
    CD183 Mouse IgG1
    CD184 Mouse IgG2b
    CD195 Mouse IgG1
    CD200 Mouse IgG1
    CD201 Rat IgG1
    CD206 Mouse IgG1
    CD209 Mouse IgG2b
    CD210 Rat IgG1
    CD212 Rat IgG1
    CD220 Mouse IgG1
    CD221 Mouse IgG1
    CD226 Mouse IgG1
    CD227 Mouse IgG1
    CD229 Mouse IgG1
    CD235a Mouse IgG2b
    CD244 Mouse IgG1
    B7‐H2 Mouse IgG2b
    CMRF‐44 Mouse IgG1
    CMRF‐5 Mouse IgG1
    B2‐MICROGLOBULIN Mouse IgG1
    CLIP Mouse IgG1
    gd‐TCR Mouse IgG1
    Vb8‐TCR Mouse IgG1
    CLA Rat IgG1
    EGF‐R Mouse IgG2a
    fMLP‐R Mouse IgG1
    fII‐R Mouse IgG1
    HLA‐ABC Mouse IgG1
    HLA‐A2 Mouse IgG2b
    HLA‐DQ Mouse IgG2a
    HLA‐DR Mouse IgG2a
    HLADRDPD Mouse IgG2a
    abTCR Mouse IgM
    IntegrinB7 Mouse IgG2b
    M‐calpain Mouse IgG1
    LAIR‐1 Mouse IgG1
    NL‐B1 Mouse IgG1
    NK‐G2d Mouse IgG1
    NK‐P46 Mouse IgG1
    ABC‐G2 Mouse IgG2b
    Blood group A Mouse IgG3
    NP<‐ALK/AL Mouse IgG3
    B glycoprotein Mouse IgG2b
    Invariant NK Mouse IgG1
    MUC2 Mouse IgG1
    NGF‐R Mouse IgG1
    PRR2 Mouse IgG1
    Siglec‐6 Mouse IgG1
    Siglec‐7 Mouse IgG1
    B5‐TCR Mouse IgG2a
    Common gamma chain Rat IgG2b
    Stro‐1 Mouse IgG1
    CD105 Mouse IgG1

Support Protocol 2: Differentiation of hAECs

  Materials
  • Freshly isolated hAEC ( protocol 1)
  • EpiLife growth medium (see recipe)
  • EpiLife coating matrix (Invitrogen, cat. no. R‐011‐K)
  • Neural differentiation medium (see recipe)
  • Small airway growth medium (see recipe)
  • Adipogenic differentiation medium (see recipe)
  • Osteogenic differentiation medium (see recipe)
  • Hanks' Balanced Salt Solution (HBSS; Invitrogen, cat. no. 14175)
  • Poly‐D‐lysine/laminin‐coated glass coverslips (BD Biosciences, cat. no. 354087)
  • or 12‐well multiwell plates (BD Biosciences, cat. no. 351143)

Support Protocol 3: CellTiter 96 AQueous One Solution Cell Proliferation Assay

  Materials
  • Cryopreserved and thawed hAECs
  • EpiLife growth medium (see recipe)
  • CellTiter 96 AQ ueous One Solution Cell Proliferation Assay (Promega, cat. no. G3582) containing:
    • Cell Titer 96 Aqueous One Solution Reagent
  • 96‐well opaque‐walled tissue culture plates compatible with fluorometer
  • Multichannel pipettor
  • Humidified 37°C, 5% CO 2 incubator
  • Fluorescence plate reader with 490‐nm and 690‐nm filters
NOTE: Protect CellTiter 96 AQ ueous One Solution Reagent from direct light to prevent increased background readings.

Support Protocol 4: Cell Proliferation ELISA

  Materials
  • Cultures of hAEC
  • Appropriate culture medium
  • Cell Proliferation ELISA, BrdU (colorimetric; Roche, cat. no. 11647229001) containing:
    • BrdU Labeling Reagent
    • FixDenat (ready to use)
    • Anti‐BrdU‐POD
    • Antibody Dilution Solution (ready to use)
    • Washing Buffer PBS, 10×
    • Substrate Solution TMB (ready to use)
  • 96‐well opaque‐walled tissue culture plates compatible with fluorometer
  • Multichannel pipettor
  • 37°C, 5% CO 2 humidified incubator
  • Fluorescence plate reader with 370‐nm and 490‐nm filters
  • Additional reagents and equipment for cell counting (unit 1.3)

Support Protocol 5: Telomerase Assay to Assess Telomere Length

  Materials
  • hAEC cells, freshly isolated and/or cultured for 5 passages
  • Kit or reagents for genomic DNA isolation (phenol, chloroform, sodium acetate, ethanol)
  • TeloTAGGG Telomere Length Assay (Roche, cat. no. 12209136001) containing:
    • HinfI
    • RsaI
    • Digestion buffer, 10×
    • Water, nuclease free
    • Control DNA
    • DIG molecular weight marker
    • Loading buffer
    • DIG Easy Hyb granules
    • Telomerase probe
    • Washing buffer
    • Maleic acid buffer
    • Blocking buffer
    • Anti‐DIG AP
    • Detection buffer
    • Substrate solution (CDP‐Star, ready to use)
  • HCl solution (0.25 M HCl)
  • Denaturation solution (0.5 M NaOH, 1.5 M NaCl)
  • Neutralization solution (0.5 M Tris⋅Cl, pH 7.5, 3 M NaCl)
  • Stringent wash buffer I (2× SSC, 0.1% SDS)
  • Stringent wash buffer II (0.2× SSC, 0.1% SDS)
  • 42°C shaking incubator
  • Absorbent paper
  • Hybridization bag
  • Bio‐Rad ChemiDoc
  • Densitometer
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, ) and Southern blotting (Brown, )
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Figures

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Literature Cited

Literature Cited
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