Neural Differentiation of P19 Carcinoma Cells and Primary Neurospheres: Cell Morphology, Proliferation, Viability, and Functionality

Priscilla D. Negraes1, Telma T. Schwindt1, Cleber A. Trujillo2, Henning Ulrich2

1 These authors contributed equally to this work, 2 Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2D.9
DOI:  10.1002/9780470151808.sc02d09s20
Online Posting Date:  March, 2012
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Abstract

This unit describes the culture and induction of in vitro models of neural differentiation and strategies to evaluate the participation of extrinsic and intrinsic factors in modulation of this process. Protocols focus on large‐scale expansion of pluripotent P19 murine embryonic carcinoma cells and their induction to neural differentiation in the presence of retinoic acid, closely resembling conditions of early neuroectodermal differentiation. Procedures are also described for obtaining rat neural precursor cells (NPCs) or neurospheres and for differentiating them in the absence of growth factors. Experimental strategies are reported using P19 cells and NPCs as in vitro models for studying the actions of extrinsic and intrinsic factors on morphology, proliferation, viability, neural phenotype determination, and progress of differentiation, as well as the functionality of ion channels and metabotropic receptors in inducing calcium fluxes at different developmental stages. The methods described here may be useful for optimizing in vitro protocols for stem cell differentiation into defined neural populations, as well as for studying mechanisms that underlie neurogenesis and gliogenesis. Curr. Protoc. Stem Cell Biol. 20:2D.9.1‐2D.9.22. © 2012 by John Wiley & Sons, Inc.

Keywords: P19 murine embryonic carcinoma cells; rat neurospheres; neural precursor cells (NPCs); neural differentiation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Maintenance and Expansion of P19 Cells in Culture by Enzymatic Passaging
  • Basic Protocol 2: Induction of Embryonic Body Formation and Neural Differentiation in P19 Cells
  • Basic Protocol 3: Culture of Rat Neural Precursor Cells Derived from Embryonic Cortex and Mesencephalon
  • Basic Protocol 4: Passaging and Expansion of Rat Neurospheres
  • Support Protocol 1: Freezing Cells
  • Basic Protocol 5: Immunocytochemistry
  • Basic Protocol 6: Flow Cytometry
  • Basic Protocol 7: BrdU Proliferation Assay
  • Basic Protocol 8: Detection of Cell Viability and Apoptosis
  • Basic Protocol 9: Calcium Imaging of Single Cells by Confocal Microscopy
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Maintenance and Expansion of P19 Cells in Culture by Enzymatic Passaging

  Materials
  • P19 murine embryonic carcinoma cells (ATCC CRL‐1825)
  • P19GM (see recipe)
  • Phosphate‐buffered saline (PBS; Life Technologies)
  • 0.25% trypsin/EDTA solution (Life Technologies)
  • 15‐ml conical centrifuge tubes
  • T25, T75, and T150 filter‐top culture flasks
  • Inverted microscope (e.g., Axiovert 25, Zeiss)

Basic Protocol 2: Induction of Embryonic Body Formation and Neural Differentiation in P19 Cells

  Materials
  • 70% (v/v) ethanol
  • Pregnant Sprague‐Dawley or Wistar rats (Harlan Industries)
  • Saline solution: 0.9% (w/v) NaCl
  • NPCBM, NPCGM, and NPCDM (see reciperecipes)
  • 0.25% trypsin/EDTA solution (Life Technologies)
  • Fetal bovine serum (FBS, Life Technologies)
  • 0.4% trypan blue (Sigma‐Aldrich)
  • Microdissecting instruments (sterilized): small dissecting scissors, Dumont forceps (straight and angled), curved microdissecting scissors, spatula
  • CO 2 chamber
  • 10‐cm culture dishes
  • Dissecting microscope (e.g., MZ6, Leica)
  • 0.2‐ and 1.0‐ml pipet tips
  • 40‐µm cell strainer
  • 15‐ml conical centrifuge tubes
  • Neubauer Improved Bright‐Line chamber
  • T75 filter‐top culture flasks coated with poly‐HEMA (see recipe)
NOTE: Harvest of appropriate embryonic tissues requires the use of timed pregnant rats (E12.5‐E14.5), which can be obtained by overnight mating. The efficiency of mating should be confirmed by the presence of sperm after vaginal smear.

Basic Protocol 3: Culture of Rat Neural Precursor Cells Derived from Embryonic Cortex and Mesencephalon

  Materials
  • Rat neurospheres in culture (see protocol 3)
  • NPCGM and NPCDM (see reciperecipes)
  • Narrow‐profile 60‐mm culture dish (lid only)
  • Pasteur pipets
  • 0.2‐ml pipet tips
  • McIlwain tissue chopper (Mickle Engineering)
  • Double‐edge razor blade (e.g., Fisher)
  • 15‐ and 50‐ml conical centrifuge tubes
  • T75 filter‐top culture flasks coated with poly‐L‐lysine and laminin (see recipe)

Basic Protocol 4: Passaging and Expansion of Rat Neurospheres

  Materials
  • Undifferentiated P19 cells, P19 EB, or neurospheres in culture (see Basic Protocols 1‐3)
  • Phosphate‐buffered saline (PBS; Life Technologies)
  • 0.25% trypsin‐EDTA solution (Life Technologies)
  • P19GM (see recipe)
  • Freezing medium: P19BM or NPCBM containing 10% (v/v) dimethyl sulfoxide (DMSO)
  • 15‐ml conical centrifuge tubes
  • Cryovials
  • −80°C freezer
  • Liquid nitrogen tank

Support Protocol 1: Freezing Cells

  Materials
  • Undifferentiated/differentiated P19 cells or neurospheres in culture
  • Culture medium: P19GM, P19DM, NPCGM, or NPCDM (see reciperecipes)
  • 0.25% trypsin/EDTA solution (Life Technologies; optional)
  • Phosphate‐buffered saline (PBS; Life Technologies)
  • 4% (w/v) paraformaldehyde in PBS
  • Blocking solution:
    • For P19 cells: PBS/0.2% Triton X‐100/0.2% Tween 20/2% FBS
    • For neurospheres: PBS/0.1% Triton X‐100/5% FBS
  • Primary antibodies, e.g., anti‐nestin (Millipore) for progenitor cells; anti‐β‐3‐tubulin (Sigma‐Aldrich) for neurons; anti‐GFAP (glial fibrillary acidic protein, Dako) for astrocytes; anti‐galactocerebroside (Gal‐C; Sigma‐Aldrich) for oligodendrocytes
  • Secondary antibodies, e.g., conjugated with Alexa488 and Alexa555 (Life Technologies)
  • 1 µg/ml DAPI (Sigma‐Aldrich) in PBS
  • DPX mountant (Sigma‐Aldrich)
  • T75 culture flasks
  • 13‐mm round coverslips coated with poly‐L‐lysine/laminin in 24‐well culture plates (see )
  • Plastic film (e.g., Saran Wrap)
  • Microscope slides
  • Fluorescence microscope (e.g., Axiovert 200, Zeiss)

Basic Protocol 5: Immunocytochemistry

  Materials
  • Undifferentiated/differentiated P19 cells or neurospheres in culture
  • Phosphate‐buffered saline (PBS)
  • 0.25% trypsin/EDTA solution (Life Technologies; optional)
  • Fetal bovine serum (FBS, Life Technologies)
  • 1% (w/v) paraformaldehyde in PBS
  • PBS/2% FBS
  • Blocking solution:
    • For P19 cells: PBS/0.2% Triton X‐100/0.2% Tween 20/2% FBS
    • For neurospheres: PBS/0.05% Triton X‐100/2% FBS
  • Primary antibodies, e.g., anti‐nestin (Millipore) for progenitor cells; anti‐β‐3‐tubulin (Sigma‐Aldrich) for neurons; anti‐GFAP (glial fibrillary acidic protein; Dako) for astrocytes; anti‐galactocerebroside (Gal‐C; Sigma‐Aldrich) for oligodendrocytes
  • Secondary antibodies, e.g., conjugated with Alexa555 (Life Technologies)
  • 15‐ and 50‐ml conical centrifuge tubes
  • 40‐µm cell strainer
  • 1.5‐ml microcentrifuge tubes
  • Cytometer (e.g., Beckman Coulter Fc500) with:
    • Argon laser line (550‐600 nm excitation)
    • FL2 575‐nm bandpass filter
    • Cytometer tubes
    • Cyflogic software (http://www.cyflogic.com) or WinMDI 2.8 software (http://facs.scripps.edu/software.html)

Basic Protocol 6: Flow Cytometry

  Materials
  • Undifferentiated/differentiated P19 cells or neurospheres in culture
  • Culture medium: P19GM, P19DM, NPCGM, or NPCDM (see reciperecipes)
  • 0.2 mM BrdU solution (Sigma‐Aldrich)
  • 0.25% trypsin/EDTA solutions (Life Technologies; optional)
  • Fetal bovine serum (FBS; Life Technologies)
  • Ice‐cold methanol
  • Phosphate‐buffered saline (PBS; Life Technologies)
  • 1.5 M HCl
  • 4% (w/v) paraformaldehyde in PBS
  • 10%, 20%, and 30% (w/v) sucrose solution in PBS
  • TissueTek OCT
  • Blocking solution:
    • For P19 cells: PBS/0.2% Triton X‐100/0.2% Tween 20/2% FBS
    • For neurospheres: PBS/0.1% Tween 20/2% FBS
  • Anti‐BrdU monoclonal antibody (Axyll)
  • Anti‐rat Alexa488 secondary antibody (Life Technologies)
  • 1 µg/ml DAPI solution (Sigma‐Aldrich) in water
  • DPX mountant (Sigma‐Aldrich)
  • 13‐mm coverslips coated with poly‐L‐lysine and laminin (see recipe) in 24‐well culture dishes
  • 50‐ml conical centrifuge tube
  • 10 × 10 × 10−mm mold for tissue embedding
  • Cryostat (e.g., CM1900, Leica)
  • Silanized glass microscope slides
  • Plastic film (e.g., Saran Wrap)
  • Fluorescence microscope (e.g., Axiovert 200, Zeiss)

Basic Protocol 7: BrdU Proliferation Assay

  Materials
  • Undifferentiated/differentiated P19 cells or neurospheres in culture
  • Phosphate‐buffered saline (PBS; Life Technologies)
  • 0.25% trypsin/EDTA solution (Life Technologies)
  • Fetal bovine serum (FBS; Life Technologies)
  • 2% (w/v) paraformaldehyde in PBS
  • Blocking solution:
    • For P19 cells: PBS/0.2% Triton X‐100/0.2% Tween 20/2% FBS
    • For neurospheres: PBS/0.05% Triton X‐100/2% FBS
  • In Situ Cell Death Detection Kit, fluorescein or TMR red (Roche)
  • DNase I (Ambion)
  • 40‐µm cell strainer
  • 15‐ml conical centrifuge tubes
  • 1.5‐ml microcentrifuge tubes
  • Cytometer (e.g., Beckman Coulter Fc500) with:
    • Argon laser line (550‐600 nm excitation)
    • FL2 575‐nm bandpass filter
    • Cytometer tubes
    • Cyflogic software (http://www.cyflogic.com) or WinMDI 2.8 software (http://facs.scripps.edu/software.html)

Basic Protocol 8: Detection of Cell Viability and Apoptosis

  Materials
  • Undifferentiated/differentiated P19 cells or neurospheres in culture
  • Culture medium: P19GM, P19DM, NPCGM, or NPCDM (see reciperecipes)
  • 5 µg/µl Fluo‐3 AM (lyophilized; Life Technologies) in cell culture−grade DMSO (store at −20°C)
  • 20% pluronic acid in DMSO
  • Designed drugs to stimulate differentiation (based on experimental design)
  • Ionomycin (calcium ionophore; Sigma‐Aldrich)
  • Ethylene glycol tetraacetic acid (EGTA)
  • 40 × 11−mm dishes
  • Confocal microscope (e.g., LSM 510 Meta, Zeiss)
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Figures

Videos

Literature Cited

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