In Vitro Generation of Three‐Dimensional Substrate‐Adherent Embryonic Stem Cell–Derived Neural Aggregates for Application in Animal Models of Neurological Disorders

Gunnar Hargus1, Yi‐Fang Cui2, Marcel Dihné3, Christian Bernreuther4, Melitta Schachner5

1 Department of Neuropathology, University Medical Center Münster, and Max Planck Institute of Molecular Biomedicine, Münster, Germany, 2 Clinical Neurobiology Laboratory, German Primate Center, Leibniz Institute for Primate Research, Göttingen, Germany, 3 Department of Neurology and Epileptology, Center for Neurology, Hertie Institute for Clinical Brain Research, University Hospital Tübingen, Tübingen, Germany, 4 Institute of Neuropathology, University Medical Center Hamburg‐Eppendorf, University of Hamburg, Hamburg, Germany, 5 W.M. Keck Center for Collaborative Neuroscience and Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, New Jersey
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 2D.11
DOI:  10.1002/9780470151808.sc02d11s21
Online Posting Date:  May, 2012
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In vitro−differentiated embryonic stem (ES) cells comprise a useful source for cell replacement therapy, but the efficiency and safety of a translational approach are highly dependent on optimized protocols for directed differentiation of ES cells into the desired cell types in vitro. Furthermore, the transplantation of three‐dimensional ES cell−derived structures instead of a single‐cell suspension may improve graft survival and function by providing a beneficial microenvironment for implanted cells. To this end, we have developed a new method to efficiently differentiate mouse ES cells into neural aggregates that consist predominantly (>90%) of postmitotic neurons, neural progenitor cells, and radial glia−like cells. When transplanted into the excitotoxically lesioned striatum of adult mice, these substrate‐adherent embryonic stem cell−derived neural aggregates (SENAs) showed significant advantages over transplanted single‐cell suspensions of ES cell−derived neural cells, including improved survival of GABAergic neurons, increased cell migration, and significantly decreased risk of teratoma formation. Furthermore, SENAs mediated functional improvement after transplantation into animal models of Parkinson's disease and spinal cord injury. This unit describes in detail how SENAs are efficiently derived from mouse ES cells in vitro and how SENAs are isolated for transplantation. Furthermore, methods are presented for successful implantation of SENAs into animal models of Huntington's disease, Parkinson's disease, and spinal cord injury to study the effects of stem cell−derived neural aggregates in a disease context in vivo. Curr. Protoc. Stem Cell Biol. 21:2D.11.1‐2D.11.19. © 2012 by John Wiley & Sons, Inc.

Keywords: embryonic stem cells; neural aggregates; transplantation; regeneration

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Table of Contents

  • Introduction
  • Basic Protocol 1: In Vitro Differentiation of Mouse Embryonic Stem Cells into SENAs
  • Alternate Protocol 1: Generation of EBs in Hanging Drops
  • Basic Protocol 2: Isolation of SENAs for Transplantation
  • Basic Protocol 3: Transplantation of SENAs into an Animal Model of Huntington's Disease
  • Basic Protocol 4: Transplantation of SENAs into an Animal Model of Parkinson's Disease
  • Basic Protocol 5: Transplantation of SENAs into an Animal Model of Spinal Cord Injury
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: In Vitro Differentiation of Mouse Embryonic Stem Cells into SENAs

  • Pregnant mice, embryonic day 13.5‐14.5
  • PBS, pH 7.4 (Invitrogen)
  • 0.1% trypsin/EDTA (Biochrom AG)
  • Fibroblast medium (see recipe)
  • 0.1% (w/v) gelatin solution (see recipe)
  • 1 mg/ml mitomycin C solution (see recipe)
  • 1 × 106 U/ml leukemia inhibitory factor (LIF; ESGRO, Chemicon)
  • Mouse ES cell line
  • 0.05% and 0.25% trypsin/EDTA (Invitrogen)
  • ES cell propagation medium (see recipe)
  • Stage 2 to Stage 5 differentiation media (see reciperecipes)
  • Poly‐L‐ornithine solution (see recipe)
  • Distilled water, pH 7.4
  • 10‐cm Petri dishes
  • Medium dissecting scissors (Fine Science Tools)
  • Scalpel blade, size 23 (BD Bard‐Parker)
  • 100‐ml Erlenmeyer flask (Fisher Scientific)
  • 6‐mm glass beads (Corning)
  • 3‐inch metal sieve (e.g., Mini Strainer, Oxo)
  • 10‐ and 15‐cm cell culture dishes (Greiner)
  • 6‐well cell culture plates (Greiner)
  • Neubauer counting chamber (Laboroptik)
  • Non‐adherent bacterial Petri dishes (Greiner)
  • 50‐ml Falcon tubes (Greiner)
NOTE: All instruments and self‐made solutions must be sterilized before use. Commercial cell culture materials and solutions must be sterile and should be used as provided by their suppliers.NOTE: Work under sterile conditions in a laminar flow hood when preparing MEFs and when culturing MEFs and ES cells.

Alternate Protocol 1: Generation of EBs in Hanging Drops

  • Terminally differentiated SENAs (see protocol 1)
  • PBS, pH 7.4 (Invitrogen)
  • 0.3 mg/ml collagenase XI (Sigma‐Aldrich) in PBS
  • Light microscope with 4× and 10× objectives (e.g., Zeiss)
  • 1.5‐ml microcentrifuge tubes
NOTE: The PBS used for transplantation of SENAs may be supplemented with 4.5 mg/ml glucose (Sigma‐Aldrich) to support cell survival. Prepare and filter fresh PBS/glucose solution on the day of transplantation.

Basic Protocol 2: Isolation of SENAs for Transplantation

  • Quinolinic acid (QA)−lesioned 8‐week‐old C57BL/6J mice, 3 days after lesion
  • Pre‐anesthetic solution: 1:1 (v/v) atropine (Vedco) and acepromazine (Phoenix Pharmaceutical), prepare fresh
  • Anesthetic solution: dilute 80 mg/ml ketamine‐HCl/xylazine‐HCl solution (Sigma‐Aldrich) 1:10 (v/v) in sterile saline, prepare fresh
  • Eye ointment (Puralube Vet Ointment, Pharmaderm)
  • Iodine solution (Mundipharma)
  • Transplantation solution: terminally differentiated SENAs at 10 SENAs/µl PBS (see protocol 3)
  • Isotonic saline solution (0.9%; Braun), sterile
  • Analgesic solution: dilute 0.3 mg/ml buprenorphine‐HCl (Reckitt Benckiser Pharmaceuticals) 1:10 (v/v) in sterile saline, prepare fresh
  • 1‐ml injection needles (Braun)
  • Stereotaxic frame (David Kopf Instruments)
  • Scalpel (Fine Science Tools)
  • Gauze pads (Hartmann AG)
  • 1‐µl injection cannula (Hamilton syringe 7001N)
  • Microforceps (Fine Science Tools)
  • Suture material (Ethicon)

Basic Protocol 3: Transplantation of SENAs into an Animal Model of Huntington's Disease

  • MPTP‐lesioned 8‐week‐old C57BL/6J mice, 4 days after lesion
  • Pet shaver (Oster)
  • Drill with 0.7‐mm microdrill bits (Fine Science Tools)

Basic Protocol 4: Transplantation of SENAs into an Animal Model of Parkinson's Disease

  • Spinal cord−lesioned 8‐week‐old female C57BL/6J mice, 3 days after lesion
  • Glass micropipet attached to a 1‐µl Hamilton syringe (Hamilton syringe 7001N)
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