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

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

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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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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Materials

Basic Protocol 1: In Vitro Differentiation of Mouse Embryonic Stem Cells into SENAs

Materials

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

Materials

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

Materials

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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Figures

Figure 2.D1.1 Schematic presentation of the differentiation of mouse ES cells into substrate‐adherent embryonic stem cell−derived neural aggregates (SENAs). Mouse ES cells expressing enhanced green fluorescent protein (EGFP) under the control of the chicken β‐actin promoter were used for visualization of SENAs after transplantation in mouse models for Parkinson's disease, Huntington's disease, and spinal cord injury. Stage 2 illustrates the hanging drop method (); the same total 4 days are required for the suspension method ().

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Figure 2.D1.2 ES cell−derived SENAs form at stage 4 of differentiation. (A ) Brightfield images show the development of SENAs during the first 15 days after plating (DAP) of neural precursor cells onto poly‐L ‐ornithine‐coated dishes. Neural precursor cells aggregate to form three‐dimensional conglomerates (arrow). (B ) Immunostaining of an EGFP‐positive SENA (green) for β3‐tubulin (red) at 15 DAP. Note the predominant neuronal differentiation. Scale bars: 100 µm (A), 50 µm (B).

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Figure 2.D1.3 Transplantation of SENAs into the striatum and midbrain of adult mice. (A ) Schematic drawing of a mouse brain with sites for injection of SENAs. (B ) EGFP‐positive SENA graft (green) 1 month after transplantation in the host striatum. (C ) EGFP‐positive SENA graft (green) immunostained for the neuronal marker protein NeuN (red). Note the high percentage of NeuN‐positive neurons in the SENA graft. Scale bars: 100 µm (B), 50 µm (C).

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Figure 2.D1.4 Transplantation of SENAs into the compression‐lesioned spinal cord. (A ) Schematic drawing of the spinal cord demonstrating the implanted SENAs (green) both rostral and caudal to the lesion site (gray). (B ) EGFP‐positive SENA grafts (green; asterisks) in the host spinal cord rostral and caudal to the lesion site (arrows). (C ) EGFP‐positive SENA graft immunostained for the neuronal marker protein NeuN (red). A high percentage of NeuN‐positive neurons was found in the SENA grafts. Scale bars: 200 µm (B), 50 µm (C).

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Literature Cited

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