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Amnion Epithelial Cell Isolation and Characterization for Clinical Use

Sean Murphy1,2,  Sharina Rosli1,  Rutu Acharya1,  Louisa Mathias2,  Rebecca Lim1,  Euan Wallace1,  Graham Jenkin2

1Department of Obstetrics and Gynaecology, Monash University, Clayton, Victoria, Australia, Australia
2Monash Immunology and Stem Cell Laboratories, Monash University, Clayton, Victoria, Australia, Australia


Publication Name: 
Current Protocols in Stem Cell Biology
Unit Number: 
Unit 1E.6
DOI: 
10.1002/9780470151808.sc01e06s13
Online Posting Date: 
November, 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 1C.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 (Basic 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 1C.3)

Support Protocol 1: Characterization of hAECs by Flow Cytometry

 Materials
  • Fresh isolates of hAECs (Basic Protocol 1) or cultures of hAECs after 5 passages (Basic Protocol 2)
  • FACS buffer (see recipe)
  • Panel of monoclonal antibodies (Table 1E.6.1) provided by BD Biosciences
  • 96-well tissue culture plates
  • BD FACS Calibur flow cytometer
     
    Table 1E.6.1 List of Monoclonal Antibodies Used to Interrogate hAECs
    BD mAbHostIsotype
    CD1aMouseIgG1
    CD1bMouseIgG1
    CD1dMouseIgG1
    CD2MouseIgG1
    CD3MouseIgG2a
    CD4MouseIgG1
    CD5MouseIgG1
    CD6MouseIgG1
    CD7MouseIgG1
    CD8MouseIgG1
    CD9MouseIgG1
    CD10MouseIgG1
    CD11aMouseIgG1
    CD11bMouseIgG1
    CD11cMouseIgG1
    CD13MouseIgG1
    CD14MouseIgG1
    CD15MouseIgM
    CD15sMouseIgG1
    CD16MouseIgG1
    CD18MouseIgG1
    CD19MouseIgG1
    CD20MouseIgG2b
    CD21MouseIgG1
    CD22MouseIgG1
    CD23MouseIgG1
    CD24MouseIgG2a
    CD25MouseIgG1
    CD26MouseIgG1
    CD27MouseIgG1
    CD28MouseIgG1
    CD29MouseIgG2a
    CD30MouseIgG1
    CD31MouseIgG1
    CD32MouseIgG2b
    CD33MouseIgG1
    CD34MouseIgG1
    CD35MouseIgG1
    CD36MouseIgM
    CD37MouseIgG1
    CD38MouseIgG1
    CD40MouseIgG1
    CD41aMouseIgG1
    CD41bMouseIgG3
    CD42aMouseIgG1
    CD42bMouseIgG1
    CD43MouseIgG1
    CD44MouseIgG2b
    CD45MouseIgG1
    CD45RAMouseIgG2b
    CD45RBMouseIgG1
    CD45ROMouseIgG2a
    CD46MouseIgG2a
    CD47MouseIgG1
    CD48MouseIgM
    CD49aMouseIgG1
    CD49b (1)MouseIgG1
    CD49b (2)MouseIgG2a
    CD49cMouseIgG1
    CD49dMouseIgG1
    CD49e (1)MouseIgG1
    CD49e (2)MouseIgG2a
    CD49fMouseIgG2b
    CD50MouseIgG1
    CD51/61MouseIgG1
    CD53MouseIgG1
    CD54MouseIgG1
    CD55MouseIgG2a
    CD56MouseIgG2b
    CD57MouseIgM
    CD58MouseIgG2a
    CD59MouseIgG2a
    CD61MouseIgG1
    CD62eMouseIgG1
    CD62LMouseIgG1
    CD62PMouseIgG1
    CD63MouseIgG1
    CD64MouseIgG1
    CD66MouseIgG1
    CD66bMouseIgM
    CD66fMouseIgG1
    CD69MouseIgG1
    CD70MouseIgG3
    CD71MouseIgG2a
    CD72MouseIgG2b
    CD73MouseIgG1
    CD74MouseIgG2a
    CD77MouseIgM
    CD79bMouseIgG1
    CD80MouseIgG1
    CD81MouseIgG1
    CD83MouseIgG1
    CD84MouseIgG1
    CD85JMouseIgG2b
    CD86MouseIgG1
    CD87MouseIgG1
    CD88MouseIgG1
    CD89MouseIgG1
    CD90MouseIgG1
    CD91MouseIgG1
    CD94MouseIgG1
    CD95MouseIgG1
    CD97MouseIgG1
    CD98MouseIgG1
    CD99MouseIgG2a
    CD99RMouseIgM
    CD100MouseIgG1
    CD103MouseIgG1
    CD104MouseIgG1
    CD106MouseIgG1
    CD108MouseIgG2a
    CD109MouseIgG1
    CD110MouseIgG1
    CD117MouseIgG1
    CD123MouseIgG1
    CD134MouseIgG1
    CD135MouseIgG1
    CD137 (1)MouseIgG1
    CD137 (2)MouseIgG1
    CD138MouseIgG1
    CD140aMouseIgG2a
    CD140bMouseIgG2a
    CD141MouseIgG1
    CD142MouseIgG1
    CD146MouseIgG1
    CD147MouseIgG1
    CD150MouseIgG1
    CD151MouseIgG1
    CD152MouseIgG2a
    CD153MouseIgG1
    CD154MouseIgG1
    CD158aMouseIgM
    CD158bMouseIgG2b
    CD161MouseIgG1
    CD162MouseIgG1
    CD163MouseIgG1
    CD164MouseIgG2a
    CD165MouseIgG1
    CD166MouseIgG1
    CD172bMouseIgG1
    CD177MouseIgG1
    CD180MouseIgG2a
    CD181MouseIgG2a
    CD182MouseIgG1
    CD183MouseIgG1
    CD184MouseIgG2b
    CD195MouseIgG1
    CD200MouseIgG1
    CD201RatIgG1
    CD206MouseIgG1
    CD209MouseIgG2b
    CD210RatIgG1
    CD212RatIgG1
    CD220MouseIgG1
    CD221MouseIgG1
    CD226MouseIgG1
    CD227MouseIgG1
    CD229MouseIgG1
    CD235aMouseIgG2b
    CD244MouseIgG1
    B7-H2MouseIgG2b
    CMRF-44MouseIgG1
    CMRF-5MouseIgG1
    B2-MICROGLOBULINMouseIgG1
    CLIPMouseIgG1
    gd-TCRMouseIgG1
    Vb8-TCRMouseIgG1
    CLARatIgG1
    EGF-RMouseIgG2a
    fMLP-RMouseIgG1
    fII-RMouseIgG1
    HLA-ABCMouseIgG1
    HLA-A2MouseIgG2b
    HLA-DQMouseIgG2a
    HLA-DRMouseIgG2a
    HLADRDPDMouseIgG2a
    abTCRMouseIgM
    IntegrinB7MouseIgG2b
    M-calpainMouseIgG1
    LAIR-1MouseIgG1
    NL-B1MouseIgG1
    NK-G2dMouseIgG1
    NK-P46MouseIgG1
    ABC-G2MouseIgG2b
    Blood group AMouseIgG3
    NP<-ALK/ALMouseIgG3
    B glycoproteinMouseIgG2b
    Invariant NKMouseIgG1
    MUC2MouseIgG1
    NGF-RMouseIgG1
    PRR2MouseIgG1
    Siglec-6MouseIgG1
    Siglec-7MouseIgG1
    B5-TCRMouseIgG2a
    Common gamma chainRatIgG2b
    Stro-1MouseIgG1
    CD105MouseIgG1

Support Protocol 2: Differentiation of hAECs

 Materials
  • Freshly isolated hAEC (Basic 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 AQueous 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% CO2 incubator
  • Fluorescence plate reader with 490-nm and 690-nm filters

NOTE: Protect CellTiter 96 AQueous 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% CO2 humidified incubator
  • Fluorescence plate reader with 370-nm and 490-nm filters
  • Additional reagents and equipment for cell counting (unit 1C.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, 2000) and Southern blotting (Brown, 1999)
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Figures

  • Figure 1E.6.1
    Comparison of the previously established animal product–containing method (unit 1E.3) and our method suitable for clinical use. (A) Comparison of average hAEC number and viability [mean stated above histograms, error bars denote SEM, n = 92 (standard), n = 19 (clinical)]. (B) Comparison of epithelial marker expression (EpCAM) (percentage of EpCAM+ cells stated). (C) Comparison of mesenchymal marker (CD90/CD105) expression (number of CD90+CD105+ cells <1%).

    View Image
  • Figure 1E.6.2
    FACS plot of hAECs stained for HLA-DQ. Freshly isolated hAECs were stained with HLA-DQ and analyzed by flow cytometry. Cells from the same amnion were cultured for 5 passages, stained with HLA-DQ, and analyzed by flow cytometry.

    View Image
  • Figure 1E.6.3
    Comparison of several serum-free cryopreservation and culture media. (A) Post-thaw viability of hAECs cryopreserved in animal product–free cryopreservation media Cryostor CS5, Synth-a-Freeze, and a commonly used serum-based media (FBS + 10% DMSO), expressed as percentage live cell recovery (ANOVA, error bars denote SEM, n = 10). (B) MTS assay measuring hAEC metabolism 7 days post-thaw. Cryostor CS5 was shown to be the preferred cryopreservation media for cell recovery and maintenance in vitro (ANOVA, error bars denote SEM, n = 20).

    View Image
  • Figure 1E.6.4
    Characterization of cultured hAECs. (A) Giemsa band karyogram showing chromosomes of passage 5 hAECs. (B) Flow cytometry of passage 5 hAECs showing DNA stained with DyeCycle Violet. G1 and G2/M indicate 2n and 4n cellular DNA content, respectively; S indicates cells undergoing DNA synthesis, intermediate in DNA content between 2n and 4n. (C) Telomere lengths of bone marrow–derived mesenchymal stromal cells (BM MSC), hESC, and hAECs when freshly isolated (P0) and at passage 5 (P5). High length control (H) and low length control (L) telomere standards are provided in the assay kit. Average telomere lengths are indicated (C).

    View Image
  • Figure 1E.6.5
    Multipotent differentiation potential of hAECs isolated with animal product–free method. Differentiation potential was demonstrated by the culture of hAECs in conditions developed to grow and maintain neurons, small airway epithelial cells, osteocytes, and adipocytes. PCR was used to detect expression of lung epithelial (A), osteogenic (B), adipogenic, glial, and neural (C) specific gene expression. Immunocytochemistry was performed to determine expression of Pro-SP-C (D) and GFAP and MAP2 (F). Alizarin Red staining was used to detect calcified deposition by cells, and Oil-Red-O staining was used to detect lipid in cells (E). Human brain and lung were used as negative and positive controls for PCR and immunocytochemistry.

    View Image

Literature Cited

Literature Cited
    Bailo, M., Soncini, M., Vertua, E., Signoroni, P.B., Sanzone, S., Lombardi, G., Arienti, D., Calamani, F., Zatti, D., Paul, P., Albertini, A., Zorzi, F., Cavagnini, A., Candotti, F., Wengler, G.S., and Parolini, O. 2004. Engraftment potential of human amnion and chorion cells derived from term placenta. Transplantation 78:1439-1448.
    Brown, T. 1999. Southern blotting. Curr. Protoc. Mol. Biol. 68:2.9.1-2.9.20.
    Fernandes, M., Sridhar, M.S., Sangwan, V.S., and Rao, G.N. 2005. Amniotic membrane transplantation for ocular surface reconstruction. Cornea 24:643-653.
    Hou, Y., Huang, Q., Liu, T., and Guo, L. 2008. Human amnion epithelial cells can be induced to differentiate into functional insulin-producing cells. Acta Biochim. Biophys. Sin. 40:830-839.
    Ilancheran, S., Michalska, A., Peh, G., Wallace, E.M., Pera, M., and Manuelpillai, U. 2007. Stem cells derived from human fetal membranes display multi-lineage differentiation potential. Biol. Reprod. 77:577-588.
    Kakishita, K., Elwan, M.A., Nakao, N., Itakura, T., and Sakuragawa, N. 2000. Human amniotic epithelial cells produce dopamine and survive after implantation into the striatum of a rat model of Parkinson's disease: A potential source of donor for transplantation therapy. Exp. Neurol. 165:27-34.
    Kakishita, K., Nakao, N., Sakuragawa, N., and Itakura, T. 2003. Implantation of human amniotic epithelial cells prevents the degeneration of nigral dopamine neurons in rats with 6-hydroxydopamine lesions. Brain Res. 980:48-56.
    Laws, P.J. and Hilder, L. 2008. Australia's mothers and babies 2006. Perinatal statistics series no. 22. Cat. no. PER 46. Sydney. AIHW National Perinatal Statistics Unit.
    Li, H., Niederkorn, J.Y., Neelam, S., Mayhew, E., Word, R.A., Mcculley, J.P., and Alizadeh, H. 2005. Immunosuppressive factors secreted by human amniotic epithelial cells. Invest. Ophthalmol. Vis. Sci. 46:900-907.
    Parolini, O., Alviano, F., Bagnara, G.P., Bilic, G., Buhring, H.J., Evangelista, M., Hennerbichler, S., Liu, B., Magatti, M., Mao, N., Miki, T., Marongiu, F., Nakajima, H., Nikaido, T., Portmann-Lanz, C.B., Sankar, V., Soncini, M., Stadler, G., Surbek, D., Takahashi, T.A., Redl, H., Sakuragawa, N., Wolbank, S., Zeisberger, S., Zisch, A., and Strom, S.C. 2008. Concise review: Isolation and characterization of cells from human term placenta: Outcome of the first international Workshop on Placenta Derived Stem Cells. Stem Cells 26:300-311.
    Reubinoff, B.E., Pera, M.F., Fong, C.-Y., Trounson, A., and Bongso, A. 2000. Embryonic stem cell lines from human blastocysts: Somatic differentiation in vitro. Nat. Biotech. 18:399-404.
    Sankar, V. and Muthusamy, R. 2003. Role of human amniotic epithelial cell transplantation in spinal cord injury repair research. Neuroscience 118:11-17.
    Therapeutic Goods Act. 1989. C2009C0028.
    Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. 1998. Embryonic stem cell lines derived from human blastocysts. Science 282:1145-1147.
    Trelford, J.D. and Trelford-Sauder, M. 1979. The amnion in surgery, past and present. Am. J. Obstet. Gynecol. 134:833-845.
    Trounson, A. and Pera, M. 2001. Human embryonic stem cells. Fertility and Sterility 76:660-661.
    Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1-2.5A.9.
    Wei, J.P., Zhang, T.S., Kawa, S., Aizawa, T., Ota, M., Akaike, T., Kato, K., Konishi, I., and Nikaido, I. 2003. Human amnion-isolated cells normalize blood glucose in streptozotocin-induced diabetic mice. Cell Transplant. 12:545-552.
    Yu, J., Vodyanik, M.A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J.L., Tian, S., Nie, J., Jonsdottir, G.A., Ruotti, V., Stewart, R., Slukvin, I.I., and Thomson, J.A. 2007. Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917-1920.
     
 
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