Functional Stem Cell Integration Assessed by Organotypic Slice Cultures

互联网2013-12-31

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

Re?formation or preservation of functional, electrically active neural networks has been proffered as one of the goals of stem cell?mediated neural therapeutics. A primary issue for a cell therapy approach is the formation of functional contacts between the implanted cells and the host tissue. Therefore, it is of fundamental interest to establish protocols that allow us to delineate a detailed time course of grafted stem cell survival, migration, differentiation, integration, and functional interaction with the host. One option for in vitro studies is to examine the integration of exogenous stem cells into an existing active neuronal network in ex vivo organotypic cultures. Organotypic cultures leave the structural integrity essentially intact while still allowing the microenvironment to be carefully controlled. This allows detailed studies over time of cellular responses and cell?cell interactions, which are not readily performed in vivo. This unit describes procedures for using organotypic slice cultures as ex vivo model systems for studying neural stem cell and embryonic stem cell engraftment and communication with CNS host tissue. Curr. Protoc. Stem Cell Biol. 23:2D.13.1?2D.13.26. © 2012 by John Wiley & Sons, Inc.

Keywords: organotypic culture; striatum; brainstem; roller drum; Stoppini; neural stem cells; engraftment; transplantation; integration; interaction; neuroprotection

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Introduction
Basic Protocol 1: Organotypic Cultures—Interface Method—Brainstem
Basic Protocol 2: Organotypic Cultures—Roller Drum Method—Striatum/Brainstem
Basic Protocol 3: Neural and Embryonic Stem Cell Preparation
Basic Protocol 4: Time‐Lapse Calcium Imaging
Basic Protocol 5: Dye Spread to Investigate Direct Intercellular Connections
Support Protocol 1: Propidium Iodide Staining
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Organotypic Cultures—Interface Method—Brainstem

Materials

0.05 mg/ml poly‐D‐lysine (Sigma, cat. no. P7405)
70% ethanol
Dissection medium (see recipe )
Culture medium (see recipe )
1 to 8 mice or rat pups embryonic age 18 to postnatal day 14; earlier embryonic as well as adult age can work for both mice and rats
Washing/transportation solution (see recipe )

Millicell culture plate inserts (Millipore, cat. no. PICM03050)
Sterile 6‐well culture plates (Sarstedt, cat. no. 83.1839)
Petri dishes (diameter 60 mm; Sarstedt, cat. no. 83.1801)
Scissors, for decapitation
Stereomicroscope (e.g., Zeiss Stemi DV 4, Carl Zeiss)
Large forceps, for moving the heads
Sharp forceps, for holding tissue
Ewald forceps, for removing skull
Spatulas
Small and larger blunt, rounded spatulas, for dissection and transfer of slices
Scalpel and blade, for cutting through the skull
Double‐edged stainless steel razor blade (e.g., Paragon, cat. no. 10 B.S.2982/Campden Instruments Ltd., Part N. 752/1/SS)
Micro scissors, for cutting nerves
McIllwain tissue chopper: e.g., The Mickle Laboratory Engineering, Co. Ltd. (http://www.micklelab.co.uk/) or Campden Instruments (http://www.campden‐inst.com)
10‐ml sterile syringes
Filter paper (WVR, cat. no. 512‐6099)

Basic Protocol 2: Organotypic Cultures—Roller Drum Method—Striatum/Brainstem

Materials

Xylene
Acetone
Isopropanol
0.05 mg/ml poly‐D‐lysine (Sigma, cat. no. P7405)
Chicken plasma (Sigma, cat. no. P3266)
Tissue‐culture‐grade H₂ O (Invitrogen)
Thrombin from bovine plasma (Sigma, cat. no. T3399‐1KU)
Gey's balanced salt solution (GBSS; Sigma)
OC medium (see recipe )
Sprague‐Dawley rats: postnatal age 2 to 14 or mice embryonic day 18 to postnatal age 14; earlier embryonic as well as adult age may work for both mice and rats.
Dissection medium (see recipe )

12 × 24 mm glass coverslips (No 1; Gribi, http://www.gribi.ch/)
Hot plate
McIlwain tissue chopper (e.g., Campden Instruments)
Stereomicroscope (e.g., Carl Zeiss)
Sterilized surgical cloths
Sterile syringe filter: red FB 30/0 (Merck, cat. no. 265622‐0)
35‐mm and 100‐mm Petri dishes (Corning)
15‐ml polypropylene tubes (Falcon, cat. no. 009‐2095)
Scissors, for decapitation
Forceps, straight, angled, and blunt (e.g., Allgaier Instrumente GmbH, http://www.allgaier‐instrumente.de)
Angled scissors
Double‐edged stainless steel razor blade (e.g., Paragon, cat. no. 10 B.S.2982/Campden Instruments Ltd., Part N. 752/1/SS)
Micropipettor and tips: 1 µl, 100 µl, 1000 µl (e.g., Eppendorf)
Rounded spatula (e.g., Allgaier Instrumente GmbH)
Roller‐drum device (Tissue Culture Roller‐drum, Model TC‐7; New Brunswick Scientific)

Basic Protocol 3: Neural and Embryonic Stem Cell Preparation

Materials

70% ethanol
Dulbecco's Modified Eagle Medium (DMEM; Invitrogen)
Pyruvate with pyridoxine (Invitrogen)
Fetal bovine serum (FBS; Sigma)
Horse serum (Invitrogen)
L‐glutamine (Invitrogen)
Penicillin/streptomycin/fucidin (Invitrogen)
NSC C17.2 clone (Snyder et al., )
DiI (Sigma)
Hanks' balanced salt solution (HBSS; Sigma)
Ca²⁺ and Mg²⁺ –free phosphate‐buffered saline(PBS; Invitrogen)
Animals: the authors have used C57Bl6 and DBA/1LacJ mice and Sprague Dawley or Lewis rats, embryonic day 18 fetal, or newborn (postnatal day 0 to 5) rodents
Ketalar and rompun
Dissection medium (see recipe )
Culture medium (see recipe )
Nerve growth factor (mouse, e.g., Invitrogen)
0.25% trypsin (Invitrogen)
HBSS containing 1% (w/v) trypsin

Petri dishes (Corning)
Bürker chamber for cell counting (e.g., VWR Scientific)
Stereomicroscope (e.g., Carl Zeiss)
Micro scissors
15‐ml conical centrifuge tubes (BD Falcon)
Fire‐polished Pasteur pipets
Forceps, straight, angled and blunt (e.g., Allgaier Instrumente GmbH)
Poly‐D‐lysine coated Petri dishes (see recipe )

Additional reagents and equipment for trypsinization of cells (Phelan, ) and roller drum method for cultures ( protocol 2 )

Basic Protocol 4: Time‐Lapse Calcium Imaging

Materials

NaCl (e.g., Sigma)
KCl (e.g., Sigma)
KH₂ PO₄ (e.g., Sigma)
CaCl₂ (e.g., Sigma)
MgSO₄ (e.g., Sigma)
NaHCO₃ (e.g., Sigma)
Glucose (e.g., Sigma)
Gas mixture 95% O₂ and 5% CO₂ (e.g., AGA Gas AB)
Fluo‐4 AM or Fura‐2 AM (Invitrogen)
Dimethylsulfoxide (DMSO; e.g., Sigma)
Pluronic acid (F‐127, Invitrogen)

For Fluo‐4 recordings: a Leica DM IRBE confocal laser scanning microscope equipped with a 40×/1.25 oil‐immersion objective (Leica Microsystems)
For Fura‐2 recordings: a Zeiss Axiovert 135 epifluorescence microscope equipped with a 40×/1.4 oil‐immersion objective (Carl Zeiss)
Perfusion chamber (e.g., Warner Instruments)
Peristaltic pump (e.g., Ismatec)
Tubing (e.g., Warner Instruments)
Forceps (e.g., Allgaier Instrumente GmbH)
ImageJ software (NIH; http://rsbweb.nih.gov/ij/)

Basic Protocol 5: Dye Spread to Investigate Direct Intercellular Connections

Materials

DiI (Sigma)
Calcein (Sigma)
Hank's balanced salt solution (HBSS; Sigma)
NSC culture (see protocol 3 )
Ca²⁺ Mg²⁺ phosphate‐buffered saline(PBS; Invitrogen)
4% paraformaldehyde (PFA; see recipe )
Fluorescence microscope (e.g., Nikon Eclipse E 800)

Additional reagents and equipment for trypsinization of cell cultures (Phelan, )

Support Protocol 1: Propidium Iodide Staining

Materials

1 mg/ml propidium iodide (PI; Invitrogen)
Culture medium (see recipe )
Organotypic cultures (Basic Protocol protocol 11 or protocol 22 )
4% paraformaldehyde (see recipe )

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Figures

Figure 2.D1.1 Dissection technique for the Stoppini‐interface method—brainstem. (A ) Use a scalpel to cut through the skull; (B ) the dissected brain is placed on a sterile filter paper on the McIlwain tissue chopper; (C ) place the brain on the filter paper and orient it so that the medulla will be cut first and the cerebellum is up; (D ) separate the slices using two spatulas—be very gentle; (E ) transfer the slices with the correct level, from the Petri dish to the insert membranes; (F ) remove all medium that is on top of the insert membrane, but keep 1 ml underneath it.

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Figure 2.D1.2 Stoppini‐interface method. Brain slices prepared from fetal or neonatal rodents are maintained in culture at the interface between air and a culture medium. Brain, cerebellar, or brainstem slices are placed on a sterile, transparent, and porous membrane and kept in Petri dishes in an incubator.

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Figure 2.D1.3 Morphological integrity of the organotypic auditory brainstem slice—300‐µm thick brainstem slices monocultured for periods of 1 day up to 3 weeks using the Stoppini method. The slices comprise the cochlear nucleus (CN). Panel (A ) illustrates a brainstem slice maintained in culture for 1 day. Note the sharp morphological integrity of the slice including the area of the cochlear nucleus. In panels (B ), (C ), and (D ), the slices were cultured for 1, 2, and 3 weeks, respectively. Due to spread of the tissue into the surrounding culture medium, the thickness of the slices eventually thinned down to about 150 µm, but with well preserved morphology. Scale bar = 500 µM. Figure 2D.13.3 from Thonabulsombat et al. ().

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Figure 2.D1.4 With the Stoppini method, organotypic brainstem nucleii are preserved. (A ‐I ) Beta‐tubulin III (Tuj1) expression in brainstem slice cultures from mice and rats exhibit preserved cytoarchitectural organization after culturing ex vivo. At the slice edges, Tuj1 negative cells are seen due to the reactive gliosis after chopping and migration of these (GFAP positive) cells. A, B, D, and G focus on the ventrolateral region, and C, F, and I are higher magnifications of these areas. E and H show expression of Tuj1 in distinct cell groups around the 4th ventricle, indicated by hatched lines. DAPI (4′,6‐diamidino‐2phenylindole); marks all nuclei blue.

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Figure 2.D1.5 Roller drum method organotypic striatal slice. (A ) Striatum culture after 1 day in culture has an opaque appearance and distinct border. (B ) Subsequently, the organotypic slice thins down and spreads out during increasing time in culture to a multilayer of cells (2‐ to 5‐cell layer), with less distinct borders. (C ) Beta‐tubulin III (Tuj1, red) expression in striatum slice culture after 7 DIV. Panels A and B modified from Jäderstad et al. ().

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Figure 2.D1.6 Dye injection and intercellular connections. The functionality of the gap‐junctional couplings between graft and host cells can be confirmed by Lucifer Yellow (LY) dye transfer experiments. Shown here is a grafted cell, recognized by GFP expression, that is whole‐cell patch clamped using standard electrophysiological techniques (A ) with a glass pipet filled with the gap‐junctional permeable dye Lucifer Yellow. Dye spread to neighboring organotypic culture cells was studied with subsequent three‐dimensional reconstruction (B ). The asterisk in (A) marks the patching pipet. The white arrows in (A and B) mark the same cell. Scale bars are 20 µM in A and B. Figure 2D.13.6B is partly adapted from Jäderstad et al. ().

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Figure 2.D1.7 Calcein dye transfer. Two exogenous murine neural stem cells (NSCs) filled with the gap junction‐permeable dye calcein (green) and the gap junction‐impermeable dye DiI (red) following engraftment to an organotypic striatal tissue culture. Cell nuclei are displayed by DAPI nuclear counterstain (blue). Already after 4 hr, engrafted NSCs had formed gap‐junctional couplings to the host cells, indicated by the spread of calcein from NSCs to surrounding cells. These couplings allowed early functional and beneficial interactions with the host even before electrophysiologically mature neuronal differentiation. See Jäderstad et al. ().

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Figure 2.D1.8 Grafted neural stem cells survive and integrate throughout the organotypic culture. A striatal organotypic culture after 7 DIV. Neural stem cells (C17.2‐NT3‐GFP+) grafted within 1 mm from the slice migrated and integrated into the striatal slice. Grafted neural stem cells recognized by GFP+ fluorescence (green). Although many of the surrounding host striatal cells in this area express GFAP (red), none of the engrafted NSCs expressed this marker for immature NSC and reactive gliosis. Nuclear stain DAPI (blue). DAPI: 4′,6‐diamidino‐2‐phenylindole; GFAP: glial fibrillary acidic protein; NSC: neural stem cell; GFP: green fluorescent protein. Scale bar, 20 µm.

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Functional Stem Cell Integration Assessed by Organotypic Slice Cultures

Abstract

Table of Contents

Materials

Basic Protocol 1: Organotypic Cultures—Interface Method—Brainstem

Basic Protocol 2: Organotypic Cultures—Roller Drum Method—Striatum/Brainstem

Basic Protocol 3: Neural and Embryonic Stem Cell Preparation

Basic Protocol 4: Time‐Lapse Calcium Imaging

Basic Protocol 5: Dye Spread to Investigate Direct Intercellular Connections

Support Protocol 1: Propidium Iodide Staining

Figures

Videos

Literature Cited