成年海马前体细胞的分离，培养和分化

Stefanie N. Bernas; Odette Leiter; Tara L. Walker; Gerd Kempermann

doi:10.21769/BioProtoc.2603

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Isolation, Culture and Differentiation of Adult Hippocampal Precursor Cells

成年海马前体细胞的分离，培养和分化

SB Stefanie N. Bernas ^*

OL Odette Leiter ^*

TW Tara L. Walker

GK Gerd Kempermann email

(*contributed equally to this work) 发布: 2017年11月05日第7卷第21期 DOI: 10.21769/BioProtoc.2603 浏览次数: 19157

评审: Anonymous reviewer(s)

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Abstract

There are two neurogenic niches in the adult mammalian brain: the subventricular zone of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus. Cells from these areas can be isolated and maintained in vitro, using two different culture systems to assess their potential regarding proliferation and differentiation in a reductionist model. While the neurosphere assay is primarily performed to directly study the proliferative and differentiation potential of cells in individual brains, the monolayer culture allows single cell analysis in a rather homogeneous cell population. Here, we describe the isolation, culturing methods and differentiation of neural precursor cells in both systems.

Keywords: Neuroscience (神经科学)

Precursor cell (前体细胞)

Neurosphere (神经球)

Adherent monolayer (粘附单层)

Differentiation (分化)

Subventricular zone (室下区)

Dentate gyrus (齿状回)

Adult mouse (成年老鼠)

Background

In the mammalian brain, adult neural stem cells reside in two main neurogenic niches, the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) and the lateral ventricle of the subventricular zone (SVZ), that allow the generation of new neurons in the adult brain. Neural precursor cells from the neurogenic niches can be isolated and cultured in vitro to model cellular processes, especially proliferation and differentiation. Two standard culture systems, the adherent monolayer culture (Palmer et al., 1995; Ray et al., 1995) and the neurosphere assay (Reynolds and Weiss, 1992 and 1996), both introduced in the 1990s, represent valuable tools to study neural progenitor cell biology in vitro.

Depending on the research question, each system has advantages and disadvantages that should be considered carefully before choosing one or the other culture method. In adherent monolayer cultures cells grow rather isolated and form more homogeneous cultures. Monolayers allow the direct investigation and monitoring of neural precursor cells at the single cell level. Characteristics like morphology, proliferation and differentiation under controlled conditions, can easily be analysed and visualised. However, compared to neurosphere cultures, cells cultured as monolayer represent a more reductionist model as the cells grow with fewer cell-to-cell contacts that are usually present in the niche.

Neurosphere cultures are free-floating aggregate cultures that are easy to obtain from adult tissue. Primary neurospheres are more heterogeneous and presumably represent a more niche-like environment. Neurospheres can be used to model the interaction of different cell types and allow relative comparisons of precursor cell number and potential, but does not allow absolute conclusions about stem cell numbers in vivo. Also, the sphere-forming capacity is not identical to ‘stemness’.

This protocol describes the detailed workflow of the generation and analysis of adult neural precursor cultures as neurospheres and monolayers from both neurogenic regions, the SVZ and the DG. The protocol represents an optimized version of our previously published protocols that have been successfully applied to many research projects within our group and by other groups (Babu et al., 2011; Walker and Kempermann, 2014; Ehret et al., 2016; Hörster et al., 2017).

Materials and Reagents

Animals
Mice: C57BL/6J (8 weeks old)
Note: We recommend three to four mice for establishing a monolayer cell culture. For neurosphere assay experiments we recommend one mouse per 96-well plate.
General materials and reagents

Centrifugation tubes 15 ml and 50 ml
Reaction tubes 1.5 ml
Parafilm
70% ethanol
Double distilled water (ddH₂O)
1x phosphate-buffered saline (PBS)
DMEM/F-12 without glutamine (Thermo Fisher Scientific, Gibco^TM, catalog number: 21331020 )
4% paraformaldehyde (PFA) in 0.1 M phosphate buffer pH 7.4
1. PFA (Merck, catalog number: 1040051000 )
2. Sodium dihydrogen phosphate (Merck, catalog number: 1063421000 )
3. Disodium phosphate dihydrate (Acros Organics, catalog number: 343810025 )
4. Sodium hydroxide (NaOH) (Carl Roth, catalog number: 6771 )
Growth media (see Recipes)
1. Neurobasal^® medium (Thermo Fisher Scientific, Gibco^TM, catalog number: 21103049 )
2. B-27^® supplement (50x) (Thermo Fisher Scientific, Gibco^TM, catalog number: 17504044 )
3. Pen/Strep 100,000 U/ml (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
4. GlutaMAX^TM supplement (100x stock) (Thermo Fisher Scientific, Gibco^TM, catalog number: 35050061 )

Fire polished pipettes
1. Glass Pasteur pipette (1 mm diameter)
Coating
1. Poly-D-Lysine hydrobromide (PDL) (Sigma-Aldrich, catalog number: P7280 )
2. Laminin (Roche Diagnostics, catalog number: 11243217001 )
Brain dissection
Petri dishes (10 cm diameter)
SVZ tissue dissociation
1. Petri dishes (6 cm diameter)
2. Scalpel (#10) (Fisher Scientific, catalog number: 11995756)
  Manufacturer: B. Braun Melsungen, catalog number: 5518059 .
3. Falcon^® 40 µm cell strainer (Corning, Falcon^®, catalog number: 352340 )
4. 0.05% trypsin-EDTA (Thermo Fisher Scientific, Gibco^TM, catalog number: 25300054 )
5. Trypsin inhibitor containing DNAse I (see Recipes)
  1. Trypsin inhibitor (Sigma-Aldrich, catalog number: T6522 )
  2. DNase I (Roche Diagnostics, catalog number: 10104159001 )
DG tissue dissociation
1. Petri dishes (6 cm diameter)
2. Falcon^® 40 µm cell strainer (Corning, Falcon^®, catalog number: 352340 )
3. Neural tissue dissociation kit (P) (Miltenyi Biotech, catalog number: 130-092-628 )
4. Beta-mercaptoethanol (Sigma-Aldrich, catalog number: M7522 )
  Note: This product has been discontinued.
5. Hank’s buffered salt solution (HBSS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 14175 )
Monolayer culture
1. Tissue culture flasks (T25 and T75)
2. 24-well tissue culture plates (growth-enhance treated, gamma-sterilized, free of pyrogens, free of RNA/DNA, DNase, RNase)
3. Coverglass slips 12 mm (Fisher Scientific, catalog number: 12-545-82 )
  Note: This product has been discontinued.
4. Heparin (MP Biomedicals, catalog number: 0210193125 )
5. Human EGF (PeproTech, catalog number: AF-100-15 )
6. Human FGF2 (PeproTech, catalog number: 100-18B )
7. Accutase solution (Sigma-Aldrich, catalog number: A6964 )
8. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
9. Normal donkey serum (Jackson ImmunoResearch Laboratories, catalog number: 017-000-121 )
10. Trypan blue solution, 0.4% (Thermo Fisher Scientific, Gibco^TM, catalog number: 15250061 )
11. BrdU (Sigma-Aldrich, catalog number: B5002 )
12. Freezing mix (see Recipes)
13. Antibody solution (see Recipes)
Neurosphere assay
1. Petri dishes (10 cm diameter)
2. 96-well tissue culture plates (growth-enhance treated, gamma-sterilized, free of pyrogens, free of RNA/DNA, DNase, RNase)
3. 24-well tissue culture plates (growth-enhance treated, gamma-sterilized, free of pyrogens, free of RNA/DNA, DNase, RNase)
4. Coverglass slips 12 mm (Fisher Scientific, catalog number: 12-545-82 )
5. Heparin (MP Biomedicals, catalog number: 0210193125 )
6. Human EGF (Peprotech, catalog number: AF-100-15 )
7. Human FGF2 (PeproTech, catalog number: 100-18B )
8. Blocking solution (see Recipes)

Staining reagents

Microscope slides SuperFrost^® (VWR, Thermo Scientific, catalog number: 631-0706 )
Triton^® X-100 (Carl Roth, catalog number: 3051 )
1 N HCl (from 37% stock solution) (Sigma-Aldrich, catalog number: 435570 )
Note: This product has been discontinued.
0.9% NaCl
Normal donkey serum (Jackson ImmunoResearch Laboratories, catalog number: 017-000-121 )
Hoechst 33342 (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 62249 )
Primary antibodies (see Table 1)
Aqua-Poly/Mount (Polysciences, catalog number: 18606 )

Borate buffer (see Recipes)

Boric acid (Carl Roth, catalog number: 6943.1 )

Sodium hydroxide (NaOH) (Carl Roth, catalog number: 6771 )

Table 1. Primary antibodies for immunocytochemistry

Antibody	Host	Clone	Isotype	Company	Catalog number
β-III-tubulin (β-tubulin)	Mouse	5G8	IgG1	Promega	G7121
5’-bromo-2’-deoxyuridine (BrdU)	Rat	BU1/75 (ICR1)	IgG2a	Bio-Rad Laboratories	OBT0030
Glial fibrillary acidic protein (GFAP)	Rabbit	polyclonal	-	Agilent Technologies	Z0334
Map2ab	Mouse	AP-20	IgG1	Sigma-Aldrich	M1406
Nestin	Mouse	25/NESTIN	IgG1, κ	BD	611658
Oligodendrocyte marker 4 (O4)	Mouse	O4	IgM	R&D Systems	MAB1326
Sox2	Rabbit	polyclonal	-	Merck	AB5603

Equipment

Bunsen burner
Autoclave
Dissection tools
1. Scissors
2. Small spatula (Fine Science Tools, catalog number: 10093-13 )
3. Curved forceps (Fine Science Tools, model: Dumont #7, catalog number: 11271-30 )
4. Angled forceps (Fine Science Tools, model: Dumont #5-45, catalog number: 11253-25 )
5. 27 G ¾ needle (B. Braun Melsungen, catalog number: 4657705-02 )
Vacuum pump
Stereo microscope (Olympus, model: SZ61 )
Inverted microscope (Olympus, model: CKX42 )
Centrifuge with swing bucket rotor for 15 ml and 50 ml centrifuge tubes (Eppendorf, model: 5430 R )
Incubator at 37 °C with 5% CO₂
Sterile laminar flow hood
Hemocytometer (Neugebauer improved)
Fluorescence microscope (ZEISS, model: Axio Imager.M2 )
Freezing containers, Mr. Frosty^TM (Thermo Fisher Scientific, Thermo Scientific^TM, model: Mr. Frosty^TM, catalog number: 5100-0001 )
37 °C water bath
-80 °C freezer
Pipettes
Multistepper pipette (and tips)

Procedure

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Bernas, S. N., Leiter, O., Walker, T. L. and Kempermann, G. (2017). Isolation, Culture and Differentiation of Adult Hippocampal Precursor Cells. Bio-protocol 7(21): e2603. DOI: 10.21769/BioProtoc.2603.