Isolation and Characterization of Adult Neural Stem Cells Chapter 4 Stem Cell Migration

8/17/2019 Isolation and Characterization of Adult Neural Stem Cells Chapter 4 Stem Cell Migration

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Chapter 4

Isolation and Characterization of Adult Neural Stem Cells

Florian A. Siebzehnrubl, Vinata Vedam-Mai, Hassan Azari,Brent A. Reynolds, and Loic P. Deleyrolle

Abstract

It has been thought for a long time that the adult brain is incapable of generating new neurons, or thatneurons cannot be added to its complex circuitry. However, recent technology has resulted in an explo-sion of research demonstrating that neurogenesis, or the birth of new neurons from adult stem cellsconstitutively occurs in two specific regions of the mammalian brain; namely the subventricular zone andhippocampal dentate gyrus. Adult CNS stem cells exhibit three main characteristics: (1) they are “self-renewing,” i.e., they possess a theoretically unlimited ability to produce progeny indistinguishable fromthemselves, (2) they are proliferative (undergoing mitosis) and (3) they are multipotent for the differentneuroectodermal lineages of the CNS, including the different neuronal, and glial subtypes. CNS stemcells and all progenitor cell types are broadly termed “precursors.”

In this chapter, we describe methods to identify, isolate and experimentally manipulate stem cells ofthe adult brain. We outline how to prepare a precursor cell culture from naive brain tissue and how to

test the “stemness” potential of different cell types present in that culture, which is achieved in a three-step paradigm. Following their isolation, stem/progenitor cells are expanded in neurosphere culture.Single cells obtained from these neurospheres are sorted for the expression of surface markers by flowcytometry. Finally, putative stem cells from cell sorting will be subjected to the so-called neural colony-forming cell assay, which allows discrimination between stem and progenitor cells. At the end of thischapter we will also describe how to identify neural stem cells in vivo.

Key words: Neural stem cell, Neurosphere assay, Flow cytometry, Neural colony-forming cell assay,Immunohistochemistry

With the identification of multipotent stem cells in the adultbrain, an assay that allowed the propagation of these cells – theneurosphere assay (NSA) was developed and described (1). TheNSA has become the method of choice not only for the expansionof stem/progenitor cells, but is also widely used to determine

1. Introduction

Marie-Dominique Filippi and Hartmut Geiger (eds.), Stem Cell Migration: Methods and Protocols,Methods in Molecular Biology, vol. 750, DOI 10.1007/978-1-61779-145-1_4, © Springer Science+Business Media, LLC 2011


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stem cell activity in vitro. However, several cell types other thanstem cells can also form neurospheres, including neural progeni-tor cells, O2A cells, oligodendrocyte precursors, and possiblyeven some types of astrocytes (2, 3). This “promiscuity” of sphereformation results in an overestimation of stem cell numbers whencalculating sphere-forming frequency from all plated cells. Whilethe NSA is an appropriate tool to expand stem/progenitor cellsfor experimental manipulation, it is insufficient to discriminatestem cells from other sphere-forming cell types. Even though theNSA is the most popular method to detect neural stem cell activ-ity, it has caveats and cannot be used as an accurate assay to mea-sure neural stem cell (NSC) frequency. As the formation of anindividual neurosphere does not reflect the presence of a singlestem cell, and because progenitors can generate spheres, the one-to-one relationship between neurospheres and neural stem cells isincorrect. Therefore, quantification of the neurosphere-formingfrequency is not an accurate measurement of stem cell enumera-tion. To address this issue, the neural colony-forming cell assay(N-CFCA) was designed (4). This assay discriminates betweenstem and progenitor cells on the basis of their proliferative poten-tial. The N-CFCA is based on the observation that stem cells pres-ent higher proliferative capability compared to progenitor cells;therefore, the size of the clonally derived colonies (i.e., diame-ter) can be used to differentiate its founder cell type. Colonies

were generated with a distinct size range, and subsequentlyfour categories of colonies are identified based on their diameter0.5, 0.5–1, 1–2, and >2 mm (Fig. 3). Only the large colonies(>2 mm) are derived from a cell exhibiting all of the stem cellfeatures. Therefore, the frequency of large colony can be used asa read-out of NSCs frequency. Cogency and validity of the assayhas been established with embryonic and adult stem/precursorcells (4).

Flow cytometry is a very powerful technology that allows forthe purification of cell populations according to size, granularity,and antigens expressed on the cell surface. Unfortunately, adult

neural stem/progenitor cells do not differ very much in size andgranularity, and it is nearly impossible to purify one or the otherpopulation based on any of these characteristics. Over the lastdecade, a variety of such antigens constituting putative stem cellmarkers have been identified (e.g., CD133, LeX, EGFR, Nestin,Musashi and Sox2 (5–10). In addition, assays have been devel-oped to reveal putative stem cell populations based on internalcell characteristics such as the side population (11) or ALDH1activity (12). As flow cytometry can be viewed as live cell immu-nostaining and sorting of stained (or unstained) cells, the tech-

nology is only as good as the markers (i.e., antibodies) targetingthe desired cell populations. Herein also lies the greatest pitfall ofstem cell purification. However, in conjunction with functional


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63Isolation and Characterization of Adult Neural Stem Cells

stemness assays, flow cytometry becomes an indispensable tool inmodern stem cell research. Neurosphere culture is arguably notthe best method of stem cell enrichment (4, 13), but it is a verypracticable culture method allowing for fast expansion of stemand progenitor cells. Cellular subpopulations of this heteroge-neous mixture can be further purified based on their expression ofcertain antigens (we will use CD133 as exemplary marker) andthen tested for their stemness in more complex assays, i.e., theN-CFCA.

Ideally, the identification of stem cells in vivo is based onthe fact that the cells can be labeled as dividing in combination

with the expression of several markers. In this chapter, we willuse Sox2 expression as an example to identify in situ stem/precursor cells.

To avoid inconsistency in experiments due to possible batch-to-batch differences of the in-laboratory prepared medium, opti-mized reagents and medium are available from Stem CellTechnologies (http://www.stemcell.com), Gibco or Sigma. Here

we provide an example list of commercially available reagents thatcan be used to prepare the Neurosphere assay media.

1. Basal medium (NeuroCult NSC basal medium, Stem-CellTechnologies) supplemented or not with Bovine Serum

Albumin (BSA).

2. 10× hormone mix (NeuroCult NSC proliferation supple-ment, StemCell Technologies).

3. Differentiation medium (NeuroCult differentiation supple-ment, StemCell Technologies).

4. Solution of trypsin (0.05%) and ethylenediamine tetraacetic

acid (EDTA). 5. Fetal bovine serum.

6. Trypsin inhibitor solution: add 0.14 g of Trypsin Inhibitor to10 ml of DNase Solution (100 mg DNase dissolved in 100 mlof HEM), then make the volume up to 1 l using HEM. Useratio 1:1 of Trypsin inhibitor solution: Trypsin/EDTA 0.05%or tissue dissociation medium.

To prepare complete NSC medium, combine 450 ml ofNeuroCult NSC basal medium with 50 ml of NeuroCult NSC

proliferation supplement and then add required amount ofgrowth factors (20 ng/ml EGF, 10 ng/ml bFGF, and 0.679 U/mlheparin).

2. Materials

2.1. Culture Medium

for Neurosphere Assay


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1. BSA is dissolved at 2% BSA in phosphate-buffered saline(PBS) (20 g of BSA in 1 l of PBS). Filter sterilize.

2. 200 mM EDTA solution: dissolve 584.5 mg of EDTA in10 ml of PBS; then filter sterilize.

3. Rat anti-mouse CD133 monoclonal antibody, conjugated to Allophycocyanin (APC).

4. Fluorescence-activated Cell Sorter (e.g., BD FACSAria II).

5. Propidium Iodide is dissolve at 1 mg/ml in sterile water.

1. Complete NeuroCult® Proliferation Medium (StemCellTechnologies).

2. NeuroCult® NCFC Serum-Free Medium without Cytokines(StemCell Technologies).

3. Mouse NeuroCult® NSC Proliferation Supplements (StemCellTechnologies).

4. Collagen Solution (StemCell Technologies).

5. Light microscope with 5× and 10× objectives.

6. 40 mm cell strainer.

1. Paraformaldehyde (PFA) is made up at 4% in PBS; pH 7.4.

2. Sucrose (20% in PBS, 30% in PBS); BP-220-1.

3. OCT tissue embedding compound (Tissue-Tek).

4. Triton-X 100.

5. Blocking solution (5–15% normal serum in PBS. The blocker varies with the source of antibodies to be used).

6. Positively charged Superfrost glass slides.

7. Forceps.

8. Primary and secondary antibodies.

In the following section, we describe the isolation and expansionmethod for adult murine neural stem cells by means of growth fac-tor stimulation. Sacrifice of animals, removal and dissection of brainare performed outside the laminar flow hood. Particular cautionand sterile techniques should be exercised to avoid contamination.

1. Warm the culture medium and tissue dissociation medium to

37°C in a water bath. 2. Anesthetize mice using 3–4% Isoflurane and sacrifice them by

cervical dislocation. Pool tissues from two or four mice (4–8 weeks old) to start a culture.

2.2. Flow Cytometry

2.3. Neural Colony-

Forming Cell Assay

2.4. Immuno-

histochemistry

3. Methods

3.1. Establishment

of Primary Adult

Neural Stem Cells

Using the Neurosphere

Assay

3.1.1. Dissection


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3. Using large scissors excise the head just above the cervicalspinal cord region. Rinse the head with 70% ethanol. Thenusing small pointed scissors make a median caudal–rostral cutand expose the skull.

4. Using the skin to hold the head in place, place each blade ofsmall scissors in orbital cavity, so as to make a coronal cutbetween the orbits.

5. Using the foramen magnum as an entry point, make a longitu-dinal cut through the skull along the sagittal suture. Be cautiousnot to damage the underlying brain by making small cuts ensur-ing the angle of the blades is as shallow as possible. Cut theentire length of the skull to the coronal cut between the orbits.

6. Using curved, pointed forceps grasp and peel the skull of theeach hemisphere outward to expose the brain, then using a

small wetted curved spatula, scoop the brain into a 50 mltube containing HEM.

7. Wash brains three times using HEM to remove blood and/orhairs and transfer them to 100-mm Petri dishes containingHEM.

8. To dissect the forebrain subventricular region, place the dishcontaining the brain under the dissecting microscope (×10)magnification. Position the brain flat on its ventral surfaceand hold it from the caudal side using fine curved forceps

placed on either side of the cerebellum. Use scalpel to make acoronal cut just behind the olfactory bulbs.

9. After the removal of the olfactory bulbs, rotate the brain toexpose the ventral aspect. Make a 90° coronal cut at the levelof the optic chiasm, discarding the caudal aspect of the brain.

10. Switch to a (×25) magnification. Rotate the rostral aspect ofthe brain with the presumptive olfactory bulb facing down-

ward. Using fine curved microscissors, first remove the sep-tum and discard and then cut the thin layer of tissuesurrounding the ventricles, excluding the striatal parenchyma

and the corpus callosum. Pool dissected tissue in a newlylabeled 35-mm Petri dish.

11. Upon harvesting the periventricular regions from all brains,transfer dish to tissue culture laminar flow hood. Continue touse strict sterile technique.

1. Mince tissue for ~ 1–2 min using a scalpel blade until only very small pieces remain (see Note 1). Add a total volume of3 ml of tissue dissociation medium (Trypsin–EDTA); transferall of the minced tissues into of a 15-ml tube. 3 ml dissociationmedium is enough for good digestion of tissues harvestedfrom up to eight mice. Then incubate the tube for 7 min in a37°C water bath.

3.1.2. Tissue Dissociation


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2. At the end of the enzymatic incubation, add an equal volumeof trypsin inhibitor (3 ml).

3. Avoid generation of air bubbles, mix well and pellet the sus-pension by centrifugation at 100 × g for 5 min.

4. Aspirate the supernatant and discard it, then resuspend thecells in 150 ml of sterile basal medium containing BSA, resetthe pipettor to 200 ml. Pipette up and down gently to breakthe clumps up until a milky single cell suspension is achieved(see Note 2).

5. Add medium for a total volume of 1 ml and pass the suspen-sion through a 40-mm pore size strainer into a 15-ml tube, soas to remove debris or undissociated pieces, and then pelletthe cells by centrifugation at 100 × g for 5 min (see Note 3).

6. Transfer one brain into a T25 flask (containing 5 ml of com-plete media). The cells are then incubated at 37°C, 5% CO2

for 7–10 days by which time neurospheres should haveformed. Tissue harvested from one brain usually can generate400–600 spheres but a count of over 300 spheres is accept-able from a T 25 flask.

Usually, neurospheres with a variety of diameters are apparent inthe culture. To determine if spheres are ready to be passaged, themean neurospheres diameter should be about 100–150 mm (seeNotes 4 and 5). If neurospheres are allowed to grow too large,they become dark colored because of cell death at the center ofthe spheres, difficult to dissociate and eventually begin to differ-entiate in situ (attaching to the substrate and migrating towardthe periphery).

1. If the neurospheres are ready to be passaged, remove medium with suspended spheres and place in an appropriate size ster-ile tissue culture tube. Wash the flasks out with 2 ml of warmbasal medium (to prevent the cells from being shocked) andadd that to the centrifuge tube(s). Centrifuge cells at 100 × g

for 5 min at room temperature. 2. Remove supernatant and resuspend the spheres in 1 ml of

dissociation medium, then incubate at 37°C in the water bathfor 2–3 min, then inactivate the trypsin using an equal vol-ume of trypsin inhibitor.

3. Mix well to ensure that all the trypsin has been completelyinactivated, then spin at 100 × g for 5 min.

4. Remove by aspiration the supernatant down to the actual pel-let and resuspend the cells in 1 ml of basal medium and mix

well, but gently. 5. Transfer 10 ml of this suspension into a 0.6-ml tube that con-

tains 90 ml of trypan blue. Perform a cell count. At this stageit is easy to see if the cells are single or are still aggregated.

3.1.3. Passaging

Neurospheres


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If they are not “a single cell suspension” then it will be necessaryto resuspend the cells a little more vigorously. Perform thecell count again.

6. Cells are seeded at a concentration of 2.5 × 105 cells in 5 ml of

complete medium in a T25 Flask.

1. Transfer the contents of a primary or passaged neurosphereculture to an appropriately sized sterile tissue culture tube.Centrifuge at 30 × g for 5 min.

2. Aspirate essentially the entire growth medium, then gentlyresuspend (so as not to dissociate any neurospheres) with anappropriate volume of basal medium.

3. Transfer neurosphere suspension (~500 spheres/ml) intoindividual wells of 24- or 96-well tissue culture plate with a

poly-L -ornithine coated surface in neurosphere medium.

4. Centrifuge plate at 700 rpm for 10 min.

5. Leave the plate in the 37°C incubator for 20 min.

6. Carefully aspirate the entire medium (so as not to dislodgeany neurospheres), then gently add appropriate volume of 4%paraformaldehyde (in PBS, pH 7.2) and leave it for 20 min atroom temperature.

7. Remove the paraformaldehyde solution by aspiration.

8. Add PBS (pH 7.2) to the samples and incubate for 5 min. Aspirate PBS and repeat this washing procedure two moretimes for a total of three wash steps before immunolabeling.

When cultured in the presence of EGF and/or bFGF, neural stemcells and progenitor cells proliferate and give rise to neurospheres

which, when harvested at the appropriate time-point and usingthe appropriate methods as described here, can be passed practi-cally indefinitely, demonstrating long-term self renewal, and cangenerate a large number of progenies. However, upon removal ofgrowth factors, neurosphere-derived cells are induced to differen-tiate into neurons, astrocytes, and oligodendrocytes indicative ofmultipotency (Fig. 1e, f ). In general, two methods have beendescribed for the differentiation of neurospheres: as whole spheres(typically used to demonstrate individual spheres are multipotent)or as dissociated cells (used to determine the relative percentageof differentiated cell types generated).

1. Once primary or passaged neurospheres reach 150 mm indiameter, transfer the contents of the flask to an appropriatesize sterile tissue culture tube. Centrifuge at 30 × g for 5 min.

2. Aspirate essentially the entire growth medium, then gentlyresuspend (so as not to dissociate any neurospheres) with anappropriate volume of basal medium.

3.1.4. Undifferentiated

Whole Neurosphere

Preparation for Cellular

Characterization

3.1.5. Neural Stem Cell

Differentiation

3.1.5.1. Whole

Neurosphere

Differentiation


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3. Transfer neurosphere suspension to a 60-mm dish (or othersized vessel) to enable the harvesting/plucking of individual

neurospheres with a disposable plastic pipette.

4. Transfer approximately ten neurospheres into individual wellsof 24- or 96-well tissue culture plate with a poly-L -ornithinecoated surface in neurosphere medium containing 1% sterilefetal calf serum.

5. After 5–8 days in vitro, individual neurospheres should haveattached to the substrate and dispersed in such a manner so asto appear as a flattened monolayer of cells.

6. Proceed to fix cells with the addition of 4% paraformaldehyde

(in PBS, pH 7.2) for 20 min at room temperature and thenprocess the adherent cells for immunocytochemistry asrequired.

Fig. 1. Immunocytochemistry on undissociated neurospheres: (a) Phase contrast, (b) DAPI, (c) Nestin, and (d) Merged

Nestin-DAPI. Differentiated neural stem cells: (e) Double labeling showing astrocyte (GFAP-green ) and neurons (bIII-tubulin,red ), (f) Triple labeling showing astrocytes (GFAP, blue ), neurons (bIII-tubulin, red ) and oligodendrocytes (MBP, green ).


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1. Once primary or passaged neurospheres reach 150 mm, transferthe contents of the flask to an appropriate size sterile tissueculture tube. Spin at 30 × g for 5 min.

2. Remove supernatant, resuspend the cells in 1 ml of trypsin/

EDTA and incubate for 3–4 min. 3. Add 1 ml of trypsin inhibitor to each tube, mix well, centri-

fuge at 100 × g for 5 min and remove the supernatant beforeto resuspend the cells by the addition of 1 ml of basal medium.Dissociate the cells until suspension appears milky and nospheres can be seen (~ five to seven times pipetting).

4. Combine a 10 ml aliquot from the cell suspension with 90 mlof Trypan blue in a microcentrifuge tube, mix, and thentransfer 10 ml to a hemocytometer so as to perform a cellcount.

5. Seed individual wells of 24-well tissue culture plate containinga poly-L -ornithine coated glass coverslip with 5 × 105 cells.

6. After 4–6 days in vitro, neurosphere-derived cells will havedifferentiated sufficiently. Proceed to fix the cells with theaddition of 4% paraformaldehyde (in PBS, pH 7.2) for 20 minat room temperature.

7. Remove the paraformaldehyde, add PBS (pH 7.2) to thesamples and incubate for 5 min. Aspirate PBS and repeat this

washing procedure two more times for a total of three wash

steps before to process the cells for immunocytochemistry asrequired.

1. Block and permeabilize (if the antigen is intracellular) for60 min in PBS-0.1% Triton-X100 + 10% Normal Goat Serumat 37°C.

2. Incubate the cells for 60–90 min at room temperature withthe primary antibodies diluted in blocking solution (or over-night at 4°C) (see Table 1).

3. Wash the cells three times with PBS and incubate 45–60 minat 37°C with fluorochrome-conjugated secondary antibodydiluted in blocking buffer at 1:700.

4. Wash the cells three times with PBS; include DAPI (1:1,000)in second wash for nuclear counter-stain.

5. Mount on slides using DAKO fluorescent mounting media(S3023).

6. Visualize the immunostaining using a fluorescent microscopeusing appropriate filters.

Figure 1a–d shows undifferentiated/undissociated neuro-spheres stained for nestin (marker to identify neural stem cells).Figure 1e shows the differentiation in neurons (bIII-tubulin) and

3.1.5.2. Dissociated

Neurosphere

Differentiation

3.1.6. Immuno-

cytochemistry


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astrocytes (GFAP) and Fig. 1f shows neuronal (bIII-tubulin),astrocytic (GFAP), and oligodendrocytic (MBP) triple-labeling indifferentiated-dissociated neurosphere culture.

1. Prepare a single cell solution from a neurosphere culture (seeNote 6).

2. Wash the cell suspension once with PBS, count, and pellet thecells.

3. Adjust the cell suspension with PBS/2 % BSA to 1–5 × 106 cells/ml. Add 2.5 ml of 200 mM EDTA per ml suspension(final conc. 0.5 mM). Split cell suspension into a smaller neg-ative control (approx. 2 × 105 cells) and the proper sample.

4. Add appropriate volume of primary antibody to the cell sus-pension (CD133) and incubate for 30 min on ice. Incubate thenegative control with isotype control antibody (see Note 7).

5. Wash with PBS (resuspend the pellet in PBS and spin downagain).

6. Resuspend the final pellet in an appropriate volume of PBScontaining 0.5 mM EDTA (cell concentration should beabout 1 × 107 cells/ml for faster sorting) and 1 ml/ml pro-pidium iodide (PI) solution.

7. Run the samples on sorter; use the negative control to adjust

voltage for forward/side scatter so the cells form a cloudthat is roughly centered in the dot plot. Set the first gate (P1)to include the cloud (Fig. 2a). Adjust voltage for specific

3.2. Neural Stem Cell

Enrichment Using

Flow Cytometry

Table 1

Suggested primary antibodies and targeted antigens for the different neural

lineages

Antigen Working dilution Source

Neurons bIII-tubulin 1:2,000 Promega#G7121Microtubule-associated

protein-2 (MAP-2)1:300 Chemicon # MAB3418

Doublecortin 1:1,000 Chemicon # AB5910PSA-NCAM 1:300 Chemicon # MAB5324

Astrocytes Glial fibrillary acidic protein(GFAP)

1:700 Dako Cytomation #Z0334

Oligodendrocytes O4 1:300 Chemicon # MAB345Gal-c 1:300 Chemicon # MAB342Myelin basic protein (MBP) 1:300 Chemicon # AB980


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antibody (depending on the fluorochrome) so that the eventsin gate 1 do not exceed a fluorescence intensity of greaterthan 102. Set second gate (P2, Fig. 2b) for all events of gate 1that are negative for propidium iodide (i.e., live cells). Setthird gate (P3) for all events with fluorescence intensitiesgreater than the negative control (Fig. 2c, d).

8. After adjusting all voltages and acquiring 10,000 events ofnegative control (Fig. 2c), run proper sample (Fig. 2d).

Acquire 10,000 events and check the gates. Cells should

form a cloud on the FSC/SSC blot that falls into gate 1. A significant portion of events from gate 1 should be measur-able in P2.

Fig. 2. Neural stem cell isolation. (a) Representative dot plot scatter of cells from neurosphere culture derived from adult

periventricular area. Gating for cells in population 1 (P1) exclude the debris. (b) Representative dot plot comparing Side

scatter and Propidium Iodide (PI) staining of the P1. A gate is determined around the PI negative population (P2) to

exclude PI positive dead cells for further analysis. (c, d) Dot plot distribution of viable cells based on side scatter and

CD133 staining intensity. CD133 positive gate is set on the dot plot using the background level of fluorescence of the

unstained negative control (containing only the fluorochrome-conjugated secondary antibody without the primary or with

isotype control).


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9. Before beginning the sort, set sorter to sort all events fromP3 into collection tube filled with 2 ml of complete growthmedium.

10. After sort is finished, spin down the collected cells; count

using a hematocytometer and plate in the NSA and NCFCA.

1. Neurosphere-derived sorted cells (CD133 immunoreactivecells) are diluted to a concentration of 2.2 × 105 cells/ml inComplete NeuroCult® Proliferation Medium and plated at2,500 cells/35 mm culture dish with 1.5 ml.

2. To prepare a solution for two replicates, mix the followingcomponents: (1) 1.7 ml of NeuroCult® NCFC Serum-FreeMedium without Cytokines, (2) 0.33 ml of Mouse NeuroCult® NSC Proliferation Supplements, (3) 6.6 ml of Recombinant

Human Epidermal Growth Factor (rhEGF) (10 mg/ml),3.3 ml of Recombinant Human Basic Fibroblast GrowthFactor (10 mg/ml) and 6.6 ml of Heparin Solution (0.2%).

3. Mix the medium containing the cells and transfer 1.3 ml ofcold Collagen Solution to the tube and mix again. Remove1.5 ml of the final culture mixture and dispense this vol-ume into a 35 mm culture dish. Dispense another 1.5 mlin the same manner into a second 35 mm dish (see Notes 8and 9).

4. Place the 35 mm culture dishes in a 100 mm petri dish (seeNote 10) and replace the lid of the 100 mm petri dish.

5. Transfer the plates to an incubator set at 37°C, 5% CO2 and

>95% humidity. Gel formation will occur within approxi-mately 1 h. Incubate the cultures for 21–28 days.

6. Due to the prolonged culture period, the medium need to bereplenished by depositing 60 ml of complete liquid mediumsupplemented with concentrated EGF (0.5 mg/ml) plus fibro-blast growth factor (0.25 mg/ml) and heparin (0.01%) in thecenter of the dish once every week for the total of 3–4 weeks.

7. Visually assess the cultures regularly for overall colony growthand morphology using an inverted microscope (see Note 11).

A number of the colonies stop growing after approximately 10–14days while other colonies continue to expand. By day 21–28, fourcategories of colony size can be classified: (1) less than 0.5 mm indiameter, (2) 0.5–1 mm in diameter, (3) 1–2 mm in diameter,and (4) 2.0 or >2 mm in diameter.

The original cell that forms a large colony (2.0 or >2 mm indiameter) is referred to as a Neural Stem Cell, while colonies


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Immunohistochemistry (IHC) is a commonly used method thatdefinitively demonstrates not only the presence, but also the loca-tion of proteins in tissue sections. IHC is a less sensitive methodthan some immunoassays; however, it allows for examination ofintact tissue. For optimal immunohistochemical studies, quickprocessing of brain tissue is very important.

1. Animals are deeply anesthetized and then transcardially per-fused with saline (0.9%) followed by ice-cold 4% paraformal-dehyde (PFA). Volumes will have to be adjusted dependingon the size of the animal.

2. The brain of the animal is removed from the skull, and is post-fixed in 4% PFA overnight, and is then placed in a phosphate-buffered sucrose solution (usually 20–30%) for 24 h.

3. The brain is now ready to be trimmed and frozen

1. Blocks of frozen tissue are sectioned in a coronal plane on acryostat using OCT compound as an adhesive. The cryostatchuck should be placed in dry ice till it is chilled thoroughly.OCT compound can then be used to cover the surface of thechuck. Once the OCT has frozen and turned white, the block

of frozen tissue can be attached to it. 2. It is important to let the temperature of the chuck equilibrate

with that of the cryostat in order to avoid fracturing the tissue.

3.4. In Vivo

Identification of

Neural Stem Cells

Using Immuno-

histochemistry

3.4.1. Brain Perfusion

3.4.2. Tissue Sectioning

Fig. 3. Neural colony-forming cell assay. Four size categories are identified based on the diameter of the colonies: (a) 2 mm. Photos reproduced with permission from STEMCELL Technologies Inc.


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3. Sections should optimally be 5–14 mm thick.

4. The sections are collected with a fine brush, and stored inPBS with 0.1% sodium azide.

5. At this point, sections can be stored for several weeks before

immunohistochemistry is performed on them.

1. Before beginning this procedure frozen sections need to betaken out of the freezer, and equilibrated to roomtemperature.

2. It is recommended to block sections with serum (BovineSerum Albumin BSA, or Normal Donkey Serum NDS); 5% inPBS for about an hour.

3. Then, the primary antibody (Table 2) is appropriately diluted

and applied onto the sections. 4. The secondary antibody (anti-mouse, anti-rabbit, anti-guinea

pig, or anti-sheep) is coupled to different fluorochromes(e.g., Cy3, Cy5, Alexa Fluor 355, 385, 405, etc.).

5. Incubate the sections in primary antibody (appropriatelydiluted) at 4°C overnight. Figure 4 shows an example of asection of a mouse brain stained for Sox-2.

6. After the overnight incubation wash off the primary antibodyusing PBS 3 × 5 min.

3.4.3. Immunolabeling

Table 2

Suggested primary antibodies and targeted antigens to identify neural stem cells

Antibody Properties Source Working dilution

Nestin Intermediate filament protein; expressed byneuronal precursor cells of the SVZ

Millipore, MAB353 1:500

Sox2 Transcription factor essential for maintenance

of self-renewal of stem cells

R&D, MAB2018 1:250

Ki67 Cellular marker for proliferation; is presentduring all active phases of the cell cycle

NovoCastra, NCLKi67p

1:500

PCNA Protein synthesized in early G1 and S phases;detectable in nuclei of proliferating cells

DAKO Cytomation,M0879

1:1,000

MCM2 Is involved in the initiation of eukaryoticgenome replication

Cell SignalingTechnologies,D7G11

1:500

GFAP Intermediate filament protein; thought to bespecific for astrocytes in the CNS

DAKO Cytomation,Z0334

1:500

Musashi-1 RNA-binding protein expressed in neuralprogenitor and stem cells

Chemicon 1:250


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7. Incubate the sections in species-specific fluorescent secondaryantibody for 2–3 h, at room temperature, in a dark box.

8. Wash off the secondary antibody using PBS 3 × 5 min.

9. Keep the sections wet by mounting onto slides (if not alreadymounted) and coverslipping.

10. Once the slides have dried, the sections are ready to be viewedunder either a fluorescent microscope, or a laser scanningconfocal microscope. It is recommended that the imaging isdone systematically in order to eliminate any errors or omis-sions in data collection. Images are captured and stored indigital format for analysis.

1. Ensure that the scalpel blade does not cut into the plastic ofthe petri dish, as this could be toxic to the cells. It would bepreferable to use a 10 cm glass petri dish.

2. Make sure to avoid generating air bubbles, as this reduces thenumber of viable cells and makes for inefficient dissociation.

Also, the expulsion of cells during the dissociation should notbe too forceful, as this will also significantly reduce viability.

3. In primary cultures from adult brain significant debris is nor-mally present, together with adherent cells. In general, debrisand adherent cells are eliminated after about two passages.

4. Notes

Fig. 4. Low magnification of a coronal section of adult mouse brain showing the lateral ventricle, ( a) nuclei are stained

blue with DAPI, (b) expression of Sox2, a key transcription factor required in pluripotent stem cells. Inset shows a higher

magnification of the same along the wall of the LV.


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4. Spheres must be rounded but not compacted; they shouldmeasure between 100 and 150 mm (Fig. 1a).

5. Primary neurospheres are often associated with cellular debris;however, subculturing will effectively select for proliferating

precursor cells and remove cell aggregates, debris, and deadcells.

6. The spheres should preferably be of early passage primarycells, between 2 and 8.

7. Direct conjugated primary antibodies are preferred for flowcytometry, but a primary/secondary staining is possible as well.In case secondary staining is necessary, wash twice with PBSafter the primary and incubate with the secondary for 30 minon ice. Proceed with step 7 of the FACS-sorting protocol. Anegative control for the secondary antibody is recommended.

8. Remove any air bubbles by gently touching bubble with theend of the pipette.

9. The collagen starts to polymerize within several minutes afterto be added to the cell suspension. Gently tip each culturedish using a circular motion to allow the mixture in the dishesto spread evenly over the surface.

10. The 100 mm petri dish should contain an open 35 mm cul-ture dish filled with 3 ml of sterile water to maintain optimalhumidity during the incubation period.

11. Do not leave cultures at room temperature for extended peri-ods of time as the collagen gel will begin to liquefy.

Acknowledgments

The authors would like to thank Dr. Mohammad G. Golmoha-mmadi for kindly providing the neurosphere pictures. This work

was supported by the Overstreet foundation.

References

1. Reynolds, B.A., and Weiss, S. (1992)Generation of neurons and astrocytes from iso-lated cells of the adult mammalian central ner-

vous system Science 255, 1707–10.

2. Engstrom, C.M., Demers, D., Dooner, M.,McAuliffe, C., Benoit, B.O., Stencel, K., Joly,M., Hulspas, R., Reilly, J.L., Savarese, T., Recht,L.D., Ross, A.H., and Quesenberry, P.J. (2002)

A method for clonal analysis of epidermal

growth factor-responsive neural progenitors J Neurosci Methods 117, 111–21.

3. Gritti, A., Parati, E.A., Cova, L., Frolichsthal,P., Galli, R., Wanke, E., Faravelli, L., Morassutti,

D.J., Roisen, F., Nickel, D.D., Vescovi, A.L.(1996) Multipotential stem cells from theadult mouse brain proliferate and self-renew inresponse to basic fibroblast growth factor

J Neurosci 16, 1091–100.

4. Louis, S.A., Rietze, R.L., Deleyrolle, L., Wagey, R.E., Thomas, T.E., Eaves, A.C.,Reynolds, B.A. (2008) Enumeration of neuralstem and progenitor cells in the neuralcolony-forming cell assay Stem Cells 26,988–96.

5. Pastrana, E., Cheng, L.C., Doetsch, F. (2009)Simultaneous prospective purification of adult


17/17


subventricular zone neural stem cells and theirprogeny Proc Natl Acad Sci USA 106,6387–92.

6. Capela, A., Temple, S. (2002)LeX/ssea-1 isexpressed by adult mouse CNS stem cells,identifying them as nonependymal Neuron 35,865–75.

7. Uchida, N., Buck, D.W., He, D., Reitsma, M.J.,Masek, M., Phan, T.V., Tsukamoto, A.S., Gage,F.H., Weissman, I.L. (2000) Direct isolation ofhuman central nervous system stem cells ProcNatl Acad Sci USA 97, 14720–5.

8. Gilyarov, A.V. (2008) Nestin in central nervous system cells Neurosci Behav Physiol 38,165–9.

9. Sakakibara, S., Nakamura, Y., Satoh, H.,Okano, H. (2001) Rna-binding protein

Musashi2: developmentally regulated expres-sion in neural precursor cells and subpopulations

of neurons in mammalian CNS J Neurosci 21,8091–107.

10. Graham, V., Khudyakov, J., Ellis, P., Pevny, L.(2003) SOX2 functions to maintain neuralprogenitor identity Neuron 39, 749–65.

11. Kim, M., Morshead, C.M. (2003) Distinctpopulations of forebrain neural stem andprogenitor cells can be isolated using side-pop-ulation analysis J Neurosci 23, 10703–9.

12. Corti, S., Locatelli, F., Papadimitriou, D.,Donadoni, C., Salani, S., Del Bo, R., Strazzer,S., Bresolin, N., Comi, G.P. (2006)Identification of a primitive brain-derivedneural stem cell population based on aldehydedehydrogenase activity Stem Cells 24,975–85.

13. Reynolds, B.A., Rietze, R.L. (2005) Neuralstem cells and neurospheres--re-evaluating therelationship Nat Methods 2, 333–6.

Isolation and Characterization of Adult Neural Stem Cells Chapter 4 Stem Cell Migration

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