Revealing Neuronal Circuitry Using Stem Cell‐Derived Neurons

Isabella Garcia1, Cynthia Kim2, Benjamin R. Arenkiel3

1 Medical Scientist Training Program, Baylor College of Medicine, Houston, Texas, 2 Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, Texas, 3 Jan and Dan Duncan Neurological Research Institute at Texas Children's Hospital, Houston, Texas
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2D.15
DOI:  10.1002/9780470151808.sc02d15s25
Online Posting Date:  May, 2013
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Abstract

Mouse embryonic stem cell (mESC)‐derived neurons are a renewable cell source for investigation of neuronal circuits. Engineering circuit‐tracing components into stem cells facilitates studies on mechanisms of synaptic coupling and circuitogenesis. This unit details methods for the generation of mESC‐derived neurons harboring trans‐synaptic viral tracing elements, which are used for investigation of synaptic connections within circuits in vitro, ex vivo, and in vivo. The first protocol describes procedures for feeder‐free passaging of mESCs, modified to carry reporter and rabies virus tracing elements. The second protocol describes in vitro generation of neurons from these ESCs. The last protocols describe the use of ESC‐derived neurons as “source cells” for rabies virus circuit‐tracing to identify inputs onto synaptically connected neurons. Given the broad applicability, these protocols can be applied to investigate the ability of in vitro‐derived neurons to establish/maintain synaptic connections in disease models, and/or with human‐induced pluripotent stem cells. Curr. Protoc. Stem Cell Biol. 25:2D.15.1‐2D.15.18. © 2013 by John Wiley & Sons, Inc.

Keywords: monosynaptic; embryonic stem cells; retrograde; rabies; circuit; neuron; synapse; TVA; rabies‐G

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Expansion and Passaging of mESCs on Feeder‐Free, Gelatin‐Coated Culture Plates
  • Support Protocol 1: Preparation of Gelatin‐Coated Tissue Culture Dishes
  • Basic Protocol 2: In Vitro Neuronal Induction of mESCs Harboring Transsynaptic Tracing Elements
  • Support Protocol 2: Preparation of Poly‐D‐Lysine (PDL)‐Coated Glass Coverslips
  • Basic Protocol 3: Seeding mESC‐Derived Neurons with Primary Neurons and In Vitro Transsynaptic Viral Tracing
  • Basic Protocol 4: Seeding mESC‐Derived Neurons onto Brain Slices for Transsynaptic Viral Tracing
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Expansion and Passaging of mESCs on Feeder‐Free, Gelatin‐Coated Culture Plates

  Materials
  • mESCs harboring viral tracing elements and fluorescent marker (Rabies‐G, TVA receptor, tdTomato reporter; from here on referred to as Tom‐G‐TVA ESCs); such lines can be generated via homologous recombination, transfection, lentiviral transduction, or electroporation
  • Embryonic stem cell (ESC) medium (see recipe), prewarmed to 37°C
  • Tissue culture grade phosphate‐buffered saline (PBS; Lonza), prewarmed to 37°C
  • 0.05% trypsin/EDTA (Hyclone), prewarmed to 37°C
  • Fetal bovine serum (FBS; Hyclone)
  • Gelatin‐coated 10‐cm tissue culture dishes (see protocol 2)
  • Stereomicroscope with fluorescent capability
  • 37°C water bath to warm media
  • 37°C humidified incubator with 5 % CO 2
  • 50 ml conical tubes
  • Benchtop centrifuge
  • 1‐ml pipet tips

Support Protocol 1: Preparation of Gelatin‐Coated Tissue Culture Dishes

  Materials
  • Tissue culture grade 0.1% gelatinized water (Millipore)
  • Tissue culture grade phosphate‐buffered saline (PBS; Lonza)
  • 10‐cm plastic tissue culture dishes

Basic Protocol 2: In Vitro Neuronal Induction of mESCs Harboring Transsynaptic Tracing Elements

  Materials
  • mESCs harboring viral tracing elements and fluorescent marker (rabies‐G, TVA receptor, tdTomato reporter, referred to as Tom‐G‐TVA ESCs); such lines can be generated via homologous recombination, transfection, lentiviral transduction, or electroporation
  • ESC medium (see recipe), prewarmed to 37°C
  • Gelatinized tissue culture plates (see protocol 2)
  • Trypan blue
  • Neurosphere (NS) medium (see recipe), prewarmed to 37°C
  • Retinoic acid (Sigma)
  • Dimethyl sulfoxide (DMSO; ATCC)
  • Tissue culture grade PBS (Lonza), prewarmed to 37°C
  • 0.05% trypsin/EDTA (Hyclone), prewarmed to 37°C
  • Neurobasal (NB) medium (see recipe), prewarmed to 37°C
  • Poly‐D‐lysine (PDL)‐coated glass coverslips (see protocol 4)
  • 70‐µm cell strainer, optional
  • Hemacytometer
  • 37°C humidified incubator with 5 % CO 2
  • 10‐cm bacteriological petri dishes (Greiner, cat. no. 633102)
  • 50‐ml conical tubes
  • 5‐, 10‐, and 15‐ml pipets
  • 37°C water bath to warm media and other solutions
  • 1‐ml pipet tips
  • Benchtop centrifuge
  • Additional reagents and equipment for counting cells using a hemacytometer (Phelan, )

Support Protocol 2: Preparation of Poly‐D‐Lysine (PDL)‐Coated Glass Coverslips

  Materials
  • Autoclaved deionized water
  • Poly‐D‐lysine (PDL) aliquots (see recipe)
  • Tissue culture grade phosphate‐buffered saline (PBS; Lonza)
  • 18‐mm circular glass coverslips kept in 100% ethanol
  • Forceps, sterile
  • 12‐well tissue culture plates
  • Tissue culture hood with UV capability
  • 37°C humidified incubator with 5% CO 2

Basic Protocol 3: Seeding mESC‐Derived Neurons with Primary Neurons and In Vitro Transsynaptic Viral Tracing

  Materials
  • Dissection medium (DM; see recipe), ice‐cold
  • Digestion solution (DS; see recipe), prewarmed to 37°C
  • 2.5% trypsin (Hyclone), prewarmed to 37°C
  • Timed‐pregnant C57BL/6 females (pups E18 on dissection day)
  • Isoflurane, optional
  • DNase aliquots (see recipe), prewarmed to 37°C
  • Trypsin inhibitor (Sigma)
  • Trypan blue
  • Neurobasal (NB) medium (see recipe), prewarmed to 37°C
  • PDL‐coated glass coverslips (see protocol 4)
  • FUdR (see recipe)
  • SADΔG‐EGFP rabies virus (produced as described in Wickersham et al., , b)
  • TOM‐G‐RITVA ESC‐derived neurons (see protocol 3)
  • 37°C water bath to warm media and other solutions
  • Sterile sharp and blunt forceps and scissors for dissection
  • 10‐cm tissue culture dishes
  • 0.20‐µm syringe filter
  • Centrifuge
  • 37°C humidified incubator with 5% CO 2
  • 1‐ml pipet tips
  • Hemacytometer
  • 12‐well plates
  • 50‐ml conical tubes
  • Additional reagents and equipment for euthanizing the mice (Donovan and Brown, ) and counting cells using a hemacytometer (Phelan, )

Basic Protocol 4: Seeding mESC‐Derived Neurons onto Brain Slices for Transsynaptic Viral Tracing

  Materials
  • C57BL/6 wild‐type mice or any disease model of interest
  • Isoflurane
  • 10‐ml syringe (28‐G needle) filled with ice‐cold GBSS
  • Modified Gey's balanced salt solution (GBSS; see recipe), ice‐cold
  • Slice culture medium (see recipe), prewarmed to 37°C
  • Neurobasal (NB) medium (see recipe), prewarmed to 37°C
  • TOM‐G‐RITVA ESC‐derived neurons (see protocol 3)
  • SADΔG‐EGFP rabies virus (produced as described in Wickersham et al., , b)
  • Dissecting tray
  • Sterile dissection scissors, bone scissors, and blunt and sharp forceps
  • Vibratome (Leica)
  • 30‐mm Millicell‐CM culture membranes (Millipore)
  • 6‐well tissue culture plates
  • 37°C, 5% CO 2 incubator
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Figures

Videos

Literature Cited

Literature Cited
   Bissonnette, C.J., Lyass, L., Bhattacharyya, B.J., Belmadani, A., Miller, R.J., and Kessler, J.A. 2011. The controlled generation of functional basal forebrain cholinergic neurons from human embryonic stem cells. Stem Cells 29:802‐811.
   Caiazzo, M., Dell'Anno, M.T., Dvoretskova, E., Lazarevic, D., Taverna, S., Leo, D., Sotnikova, T.D., Menegon, A., Roncaglia, P., Colciago, G., Russo, G., Carninci, P., Pezzoli, G., Gainetdinov, R.R., Gustincich, S., Dityatev, A., and Broccoli, V. 2011. Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature 476:224‐227.
   Callaway, E.M. 2008. Transneuronal circuit tracing with neurotropic viruses. Curr. Opin. Neurobiol. 18:617‐623.
   Cho, E.G., Zaremba, J.D., McKercher, S.R., Talantova, M., Tu, S., Masliah, E., Chan, S.F., Nakanishi, N., Terskikh, A., and Lipton, S.A. 2011. MEF2C enhances dopaminergic neuron differentiation of human embryonic stem cells in a parkinsonian rat model. PLoS One 6:e24027.
   Donovan, J. and Brown, P. 2006. Euthanasia. Curr. Protoc. Immunol. 73:1.8.1‐1.8.4.
   Garcia, I., Huang, L., Ung, K., and Arenkiel, B.R. 2012. Tracing synaptic connectivity onto embryonic stem cell‐derived neurons. Stem Cells 30:2140‐2151.
   Phelan, M.C. 2006. Techniques for mammalian cell tissue culture. Curr. Protoc. Mol. Biol. 74:A.3F.1‐A.3F.18.
   Takahashi, K. and Yamanaka, S. 2006. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663‐676.
   Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. 2007. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861‐872.
   Tonnesen, J., Parish, C.L., Sorensen, A.T., Andersson, A., Lundberg, C., Deisseroth, K., Arenas, E., Lindvall, O., and Kokaia, M. 2011. Functional integration of grafted neural stem cell‐derived dopaminergic neurons monitored by optogenetics in an in vitro Parkinson model. PLoS One 6:e17560.
   Wickersham, I.R., Finke, S., Conzelmann, K.K., and Callaway, E.M. 2007a. Retrograde neuronal tracing with a deletion‐mutant rabies virus. Nat. Methods 4:47‐49.
   Wickersham, I.R., Lyon, D.C., Barnard, R.J., Mori, T., Finke, S., Conzelmann, K.K., Young, J.A., and Callaway, E.M. 2007b. Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons. Neuron 53:639‐647.
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