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Fluorescence‐Based Sorting of Neural Stem Cells and Progenitors

Dragan Maric¹, Jeffery L. Barker¹

¹National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH), Bethesda, Maryland

Publication Name:

Current Protocols in Neuroscience

Unit Number:

Unit 3.18

DOI:

10.1002/0471142301.ns0318s33

Online Posting Date:

November, 2005

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Abstract

Neural stem cells (NSCs) are defined as undifferentiated cells originating from the neuroectoderm that have the capacity both to perpetually self-renew without differentiating and to generate multiple types of lineage-restricted progenitors (LRPs). LRPs can themselves undergo limited self-renewal and ultimately differentiate into highly specialized cells that make up the nervous system. However, this physiologically delimited definition of NSCs and LRPs has become increasingly blurred due to lack of protocols for effectively separating these types of cells from primary tissues. This unit discusses recent attempts using fluorescence-activated cell sorting (FACS) strategies to prospectively isolate NSCs from different types of LRPs as they appear in vivo, and details a protocol that optimally attains this goal. Thus, the strategy presented here provides a framework for more precise studies of NSC and LRP cell biology in the future, which can be applied to all vertebrates, including humans.

Keywords: central nervous system; development; cortex; neural stem cells; lineage-restricted progenitors; fluorescence-activated cell sorting; cell fate

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Unit Introduction
Basic Protocol 1: Identification of Different Types of LRPs in a Telencephalic Tissue Section
Basic Protocol 2: Identification and Sorting of NSCs and LRPs from Telencephalic Dissociates
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Identification of Different Types of LRPs in a Telencephalic Tissue Section

Materials

Timed pregnant Sprague-Dawley rats (Taconic Farms)
Phosphate-buffered saline (PBS; appendix 2A), ice cold
4% (w/v) paraformaldehyde in PBS (see appendix 2A for PBS)
30% (v/v) sucrose in PBS (see appendix 2A for PBS)
Isopentane (Fisher), cooled in liquid N₂
Antibodies and detection reagents (see Table 3.18.1), diluted in NPM with BSA (see recipe for NPM)
- 10 µg/ml mouse IgM anti-JONES
- 10 µg/ml goat anti–mouse IgM–Alexa Fluor 546
- Mixture of 1 µg/ml mouse IgG1 anti-vimentin, 5 µg/ml mouse IgG2a anti-PCNA, and 10 µg/ml TnTx/mouse IgG2b anti-TnTx
- Mixture of 10 µg/ml goat anti–mouse IgG1–Alexa Fluor 488, 10 µg/ml goat anti–mouse IgG2a–biotin, and 10 µg/ml goat anti–mouse IgG2b–Alexa Fluor 647
- 10 µg/ml streptavidin–Alexa Fluor 750 (Molecular Probes)
- 10 µg/ml 4,6-diamidino-2-phenylindole (DAPI; Molecular Probes)
Normal physiological medium (NPM; see recipe) with and without bovine serum albumin (BSA)
70% (v/v) ethanol
Aqua Mount solution (Lerner Laboratories)

Clear nail polish

Table 3.18.1 Primary and Secondary Antibodies/Reagents to Identify Select CNS Cell Types^a^,^b

Cell phenotype	Primary antibody/reagent	Secondary antibody/reagent

Immature precursors	Mouse IgG1 anti-nestin^c	Goat anti–mouse IgG1–biotin^d and SA-AMCA^e
	Mouse IgG1 anti-vimentin^f	Goat anti–mouse IgG1–AF 488^g
Neurons	ChTx-biotin^h	SA-PE/Cy5^d
	TnTxⁱ/mouse IgG1 anti-TnTxⁱ	Goat anti–mouse IgG1–PE/Cy5^d
	TnTxⁱ/mouse IgG2b anti-TnTx^j	Goat anti–mouse IgG2b–AF 647^g
	Mouse IgG2b anti-TuJ1^h	Goat anti–mouse IgG2b–AF 647^g
Neuroglial progenitors	Mouse IgM anti-A2B5^f	Goat anti–mouse IgM–PE^e
	Mouse IgM anti-A2B5-PE^k	—
	Mouse IgM anti-JONES^h	Goat anti–mouse IgM–AF 546^g
Oligodendrocytes	Mouse IgM anti-O4^f	Goat anti–mouse IgM–FITC^e
	Mouse IgM anti-O4-FITC^k	—
Astrocytes	Rabbit IgG anti-GFAP^f	Goat anti–rabbit IgG–TRITC^l
Microglia	Mouse IgG2a anti-CD11b^m	Goat anti–mouse IgG2a–FITC^d
Proliferating cells	Mouse IgG2a anti-PCNA^f	Goat anti–mouse IgG2a–biotin^d and SA-AF 750^g
	Mouse IgG1 anti-BrdU-FITCⁿ	—
Apoptotic/necrotic	Annexin V–FITC^o	—

^aDivide all unconjugated primary reagents in small stock aliquots (e.g., 20 µl) and store up to 1 year at –20°C. Thaw required quantity of reagent just before use and do not refreeze. Do not freeze fluorochrome-conjugated antibodies and reagents (e.g., anti-A2B5-PE or secondary reagents). Rather, store these items protected from light up to 6 months at 4°C or until the expiration date supplied by the manufacturer.

^bAbbreviations (in alphabetical order): AF, Alexa Fluor; AMCA, aminomethylcoumarin; BrdU, bromodeoxyuridine; CD11b, cluster differentiation antigen 11b (clone OX42); ChTx, cholera toxin B subunit; FITC, fluorescein isothiocyanate; GFAP, glial fibrillary acidic protein; JONES, anti-9-O-acetylated GD3 ganglioside; PE, phycoerythrin; PE/CY5, phycoerythrin/carbocyanine dye 5 conjugate; PCNA, proliferation cell nuclear antigen; SA, streptavidin; TnTx, recombinant tetanus toxin fragment C; TRITC, tetramethyl rhodamine isothiocyanate; TuJ1, tubulin III.

^cFor availability contact Department of Biological Sciences, University of Iowa, Iowa City, Ia.

^dSupplier: Caltag.

^eSupplier: Jackson ImmunoResearch.

^fSupplier: Chemicon International.

^gSupplier: Molecular Probes.

^hSupplier: Sigma.

ⁱSupplier: Roche Diagnostics.

^jFor availability, contact Dr. William Habig, U.S. Food and Drug Administration, Bethesda, Md.

^kFor availability, contact Dr. Rick I. Cohen, Coriell Institute for Medical Research, Camden, N.J.

^lSupplier: Southern Biotechnology Associates.

^mSupplier: Serotech.

ⁿSupplier: Becton Dickinson Immunocytometry Systems.

^oSupplier: Trevigen, Inc.

Ruler or caliper
Dissecting microscope (e.g., Nikon TMS)
Dissecting instruments (e.g., Roboz Surgical Instrument Co.)
- Dissecting scissors: 7 in. curved; 67-mm blades
- Microdissecting scissors: 3-3/4 in. angular; 14-mm blades
- Microdissecting forceps: 3-1/4 in. straight; fine points
Cryostat (e.g., Jung Frigocut Model 2800E, Leica Instruments)
Poly-l-lysine-precoated 3 × 1–in. Poly-Prep microscope slides (Sigma)
Humidified chamber
Coplin jars
22 × 50–mm coverslips (e.g., Corning)
Fluorescence microscope (e.g., Axiovert 200, Carl Zeiss) equipped with:
- 25× Plan-Neofluar objective (Carl Zeiss)
- High-resolution cooled digital camera (e.g., 12-bit ORCA-ER, Hamamatsu Photonics)
- 100-W mercury arc lamp (Carl Zeiss)
- Optimized excitation/emission filter sets (Omega Optical) for aminomethylcoumarin (AMCA), Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 647, Alexa Fluor 750
- 10 µg/ml 4,6-diamidino-2-phenylindole (DAPI; Molecular Probes)
Image capture and processing software (e.g., Openlab from Improvision Ltd., and Adobe Photoshop)

Additional reagents and equipment for euthanasia of the rat (appendix 4H) and cryostat sectioning (unit 1.1)

Basic Protocol 2: Identification and Sorting of NSCs and LRPs from Telencephalic Dissociates

Materials

Timed pregnant Sprague-Dawley rats (Taconic Farms)
Hanks' balanced salt solution (HBSS; Invitrogen), ice cold
HBSS/papain solution (see recipe)
Normal physiological medium (NPM; see recipe) with and without bovine serum albumin (BSA)
Primary antibodies and reagents (see Table 3.18.1), diluted in NPM with BSA (see recipe for NPM):
- ChTx-biotin
- TnTx/mouse IgG1 anti-TnTx
- Mouse IgM anti-A2B5
- Mouse IgM anti-JONES
- PE-conjugated mouse IgM anti-A2B5
- FITC-conjugated mouse IgM anti-O4 (optional)
- Mouse IgG1 anti-nestin (dilute supernatant 1:10 in NPM with BSA)
- Mouse IgG1 anti-BrdU-FITC (dilute commercially available stock 1:10 in NPM with BSA)
- Mouse IgG2b anti-TUJl
- Rabbit IgG anti-GFAP
Secondary antibodies and detection reagents (Table 3.18.1), diluted in NPM with BSA (see recipe for NPM):
- Streptavidin-PE/Cy5
- Goat anti–mouse IgG1–PE/Cy5
- Goat anti–mouse IgM–PE
- Annexin V–FITC
- Goat anti–mouse IgG1–biotin
- Streptavidin-AMCA (Jackson ImmunoResearch)
- Goat anti–mouse IgG2b–Alexa Fluor 647
- Goat anti-rabbit-TRITC
Neurobasal (NB) medium (Invitrogen) supplemented with 1× B27 additives (from 50× stock; Invitrogen) and 1× penicillin/streptomycin/neomycin (PSN; from 100× stock; Sigma)
Medium for self-renewing growth conditions: NB medium supplemented with 1× B27 additives and 1× PSN and containing 10 ng/ml basic fibroblast growth factor (bFGF; Intergen)
Medium for differentiating growth conditions: NB medium supplemented with 1× B27 additives and 1× PSN and containing 10 ng/ml basic fibroblast growth factor (bFGF; Intergen) and 10 ng/ml epidermal growth factor (EGF; Sigma)
5-Bromo-2¢-deoxyuridine (BrdU, Sigma)
4% (w/v) paraformaldehyde in PBS (see appendix 2A for PBS)
70% ethanol
0.4% (v/v) Triton X-100 in PBS (see appendix 2A for PBS)
2 N HCl
0.1 M Na₂O₄B₇ (Sigma), pH 8.5
50 µg/ml unlabeled mouse IgG (Sigma)

Dissecting microscope (e.g., Nikon TMS)
Dissecting instruments (e.g., Roboz Surgical Instrument Co.)
- Dissecting scissors: 7 in. curved; 67-mm blades
- Microdissecting scissors: 3-3/4 in. angular; 14-mm blades
- Microdissecting forceps: 3-1/4 in. straight; fine points
35-mm plastic petri dishes (Thomas Scientific)
15-ml (17 × 120–mm) conical polystyrene centrifuge tubes or 12 × 75–mm round-bottom polystyrene tubes with cell-strainer caps (Becton Dickinson)
Cell strainers (optional)
37°C incubator without CO₂
Platform rocker (e.g., Bellco Biotechnology)
Wide-mouth plastic transfer pipets (PGC Scientific)
Narrow-mouth glass Pasteur pipets (Daigger Scientific)
Tabletop centrifuge, low speed (e.g., Sorvall Legend RT)
Fluorescence-activated cell sorter (e.g., FACSVantage SE, Becton Dickinson) equipped with:
- Argon ion laser tuned to 488-nm excitation wavelength
- Band-pass filters set at 530 ± 30, 575 ± 25, and 675 ± 20 nm (Omega Optical)
CellQuest acquisition and analysis software (Becton Dickinson)
Poly-d-lysine and fibronectin-coated photo-etched coverslips in tissue culture dishes (see recipe)
37°C humidified, 9% CO₂ incubator
Inverted fluorescence microscope (e.g., Axiovert 200, Carl Zeiss) equipped with:
- 25× Plan-Neofluar objective (Carl Zeiss)
- High-resolution cooled digital camera (e.g., ORCA-ER, Hamamatsu Photonics)
- 100-W mercury arc lamp (Carl Zeiss)
- Optimized excitation/emission filter sets (Omega Optical) for aminomethylcoumarin (AMCA), fluorescein isothiocyanate (FITC), phycoerythrin (PE), tetramethyl rhodamine isothiocyanate (TRITC), and Alexa Fluor 647

Additional reagents and equipment for euthanasia of the rat (appendix 4H) and counting cells using a hemacytometer (appendix 3B)

Looking for materials? Suppliers Guide

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Figures

Figure 3.18.1

Heterogeneous LRP populations compose the neuroepithelium at the onset of cortical neurogenesis. A representative sagittal section of the frontal telencephalon taken from an embryonic day 13 (E13) rat embryo was stained with the following: DAPI (A, blue) to reveal the cell nuclei that underlie the cytoarchitecture; anti-PCNA-Alexa Fluor 750 immunoreaction (B, pink) to visualize the actively proliferating cells in the cortical neuroepithelium (NE); JONES-Alexa Fluor 546 (C, orange) to reveal NGRPs distributed throughout the NE and the differentiating zone (DZ), but predominantly lining the lateral ventricle (LV); anti-TnTx–Alexa Fluor 647 immunoreaction (D, red) to identify both the early NRPs that are sparsely dispersed throughout the NE and the postmitotic/differentiating neuronal populations primarily confined to the DZ and the primordial plexiform layer (PPL); and anti-vimentin-Alexa Fluor 488 antibody (E, green) to reveal the radial processes of immature neuronal and glial progenitors spanning the entire tissue section. The images were captured separately under appropriate fluorescence optics and merged to create a composite (F) in order to visualize the multiple cell populations that make up the early rat telencephalon. Scale bar, 50 µm. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole dihydrochloride; JONES, anti-9-O-acetylated GD3 ganglioside; PCNA, proliferative cell nuclear antigen; TnTx, tetanus toxin. For methods of 5-color labeling of the cortical tissue section see Basic Protocol 1. The results illustrate that, even at the early stages of cortical neurogenesis, NSCs, which do not express differentiating neuronal or glial markers (see Fig. 3.18.3), and different types of LRPs are physically apposed to one other in the NE tissue, and their separation is therefore not amenable to microdissection methods.

View Image
Figure 3.18.2

Accessing CNS development by FACS. Selected cortical (CX); subcortical (hypothalamus, HT; thalamus, TH; mesencephalon, MC; cerebellum, CB); and spinal cord (cervical, SC_c; thoracic, SC_t; lumbosacral, SC_l) regions of the embryonic/early postnatal CNS can be microdissected and then dissociated into uniform single-cell suspensions with optimal recovery using papain digestion (see Basic Protocol 2, steps to ). The resulting heterogeneous mixture of unlabeled cells can then be selectively stained using a panel of primary antibodies, reagents, and/or indicator dyes that target specific cell phenotypes or cellular compartments (see Table 3.18.2). Appropriate fluorochrome-conjugated secondary antibodies/reagents selectively reactive with the primary antibodies/reagents are used to render the different cell phenotypes or their biological properties fluorescent (see Table 3.18.1). Fluorochrome-labeled cells in a test tube are placed on the sample holder platform of the FACS instrument, and a slender capillary is inserted to the bottom of the test tube. Gentle pressure (1 to 2 psi) is applied to the closed system of the test tube in order to force the cells into the capillary, which conveys the cells to the 70-µm nozzle tip at a flow rate of ³1000 cells/sec. The cells depart the nozzle enclosed in a narrow stream of fluid suspended in air (i.e., stream-in-air), which is then illuminated at a precise point and time with a coherent laser light tuned to a specific wavelength (e.g., 488 nm), also known as the laser intercept point (LIP). The resulting light-scattering properties and fluorescence signals emitted by individual cells are then collected via appropriate filters mounted in front of photomultiplier tubes, which amplify and convey these signals to a computer (e.g., Power Macintosh G4) for multiparameter signal analysis using appropriate flow cytometry software (e.g., CellQuest). In order to physically sort the cells of interest, the nozzle is electronically induced to vibrate at 31 kHz, thus breaking off a continuous stream-in-air into a steady single column of uniform microdroplets, which then act as vehicles for individual cells. This stream break-off point (SBP) is adjusted to occur at a specific fixed distance from the LIP, at which time only those microdroplets containing the cells of interest are selectively charged (+) or (–). The whole process is computer-coupled to charged deflection plates in order to accurately sort two populations of interest at a time by precisely deflecting them into assigned collection tubes. Cells that do not exhibit specific light scatter and fluorescence signals can also be negatively selected or played into a waste receptacle, which is located between the receiving collection tubes.

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Figure 3.18.3

Negative and positive selection FACS strategies to sort cortical NSCs and LRPs. E13 telencephalic cells were selectively labeled based on their differential surface expression of ganglioside and/or apoptotic epitopes (see Basic Protocol 2, steps to ). Labeled cells were then visualized using appropriate fluorochrome-conjugated secondary antibodies or reagents and their individual emission and light-scattering properties acquired by FACS using CellQuest acquisition and analysis software. The resulting data were plotted as bivariate dot density scatter plots in pseudocolor (A1 and A2) depicting the accumulated information from 100,000 cells. Panel A1 is a representative density plot showing the expression or lack of expression of surface ganglioside markers by telencephalic neural precursors and progenitors differentiating along the neuronal or neuroglial lineages. Early and late neuron restricted progenitors (ENRP, LNRP) are identified by their expression of the neuron-specific GM1 and GT1b ganglioside markers, as revealed by cholera toxin (ChTx) and tetanus toxin (TnTx) binding, respectively. Neuroglial and oligoglial restricted progenitors (NGRP, OGRP) are identified by their expression of low (NGRP) or high (OGRP) levels of GQ1c, GT3, GD3, and 9-O-acetylated GD3 gangliosides, as revealed by A2B5/JONES staining. Characteristically, both NGRP and OGRP populations exhibit a complete lack of ChTx/TnTx labeling, whereas a subpopulation of LNRPs expresses both neuronal and neuroglial ganglioside epitopes (ChTx⁺TnTx⁺A2B5⁺JONES⁺). Cells devoid of all of these differentiation markers are thus identified as putative neural stem cells (NSCs). Panel A2 illustrates annexin V binding to plasma membrane phosphatidylserine residues, used as an additional surface marker together with forward-angle light scatter (FALS), a measure of cell/particle size, to distinguish cell debris (low FALS, no annexin V) from necrotic (low FALS, high annexin V), apoptotic (high FALS, low annexin V), and nonapoptotic or vital (high FALS, no annexin V) cells. In order to define and quantify the aforementioned populations, the two plots in A1 and A2 are empirically subdivided into 10 distinct annotated regions according to the relative intensities of the cell fluorescence signals, and, in A2, FALS values. These regions also serve to define multiparameter logical electronic gates to physically sort NSCs and select subpopulations of LRPs into designated collection tubes. Thus, the NSCs are sorted as a quintuple-negative (i.e., CnTx^–TnTx^–A2B5^–JONES^–annexin V^–) population of vital cells using a negative selection process by only including cells in regions 1 and 10, while at the same time excluding ganglioside-expressing cells in regions 2 to 6, cell debris in region 7, and apoptotic/necrotic cells in regions 8 and 9. In contrast, different types of LRPs are sorted by positive selection using logical gating that combines one ganglioside-expressing population in regions 2 to 6 together with region 10, but at the same time excluding dead or dying cells and debris in regions 7 to 9. In panel B1, reanalysis of the sorted NSCs reveals that 99% of these cells are devoid of surface ganglioside markers that were used for sorting, reflecting the high degree of purity in the negative selection strategy. In panel B2, immunocytochemical staining of the FACS-purified and newly adherent NSCs demonstrates that most are nestin⁺, reflecting their immature state, and actively proliferating, having incorporated the thymidine analog bromodeoxyuridine (BrdU) during a 2-hr labeling period in vivo prior to harvesting the cells. In addition, only minute fractions (<5%) of these cells exhibit either surface (ChTx, TnTx, A2B5, JONES, O4, OX42) or intracellular (TuJ1, GFAP) differentiation markers, indicating that the NSC population contains a relatively insignificant number of committed cells. Bars represent percentage (mean ± SEM) of immunopositive cells in the NSC fraction immediately after sorting. Panels C1 and C2 are phase-contrast and fluorescence photomicrographs of acutely plated NSCs showing highly variable morphologies characteristic of motile cells. The great majority of these cells are nestin⁺ (blue) and many are BrdU⁺ (green). Calibration bars: 20 µm. Parts of this figure are adapted from Maric et al. (2003). Fluorochromes used in visualizing surface-labeled cells for FACS analysis are as follows: FITC, fluorescein isothiocyanate; PE, phycoerythrin; and PE/CY5, PE/carbocyanine dye 5. For methods on FACS analysis and cell sorting, see Basic Protocol 2.

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Figure 3.18.4

Clonal expansion of NSCs reveals their self-renewing and multipotential lineage-restricted properties, confirming their phenotypic identity. FACS-purified NSCs were plated at clonal density on poly-d-lysine- and fibronectin-coated glass coverslips and then cultured under self-renewing or differentiating growth conditions in order to determine their individual developmental potentials (see Basic Protocol 2, steps to ). After 7 days in culture, the resulting clones were cumulatively labeled with 10 µM BrdU for 24 hr, then processed for 5-color immunostaining with anti-nestin-AMCA, anti-BrdU-FITC, anti-A2B5-PE, anti-GFAP-TRITC, and anti-TUJ1-Alexa Fluor 647 antibodies in order to detect the proliferative and differentiation status of the NSC-derived progeny (see Basic Protocol 2, steps to ). In panels A1 to A3, NSCs self-renew in NB/B27/PSN medium supplemented with 10 ng/ml bFGF. Panel A1 illustrates an individual NSC photographed under phase-contrast optics after 2 hr in culture (HIC). In panel A2, after 7 days in culture (DIC) under self-renewing growth conditions, the progeny of the NSC founder in A1 number ~70 cells, and all exhibit homogeneous phase-dark and epithelioid morphologies characteristic of immature cells. In panel A3, immunostaining reveals that virtually all cells of the progeny are nestin⁺ and most are actively proliferating (BrdU⁺), but none express any epitopes characteristic of differentiating neuronal or glial phenotypes. In panels B1 to B3, NSCs exhibit multilineage differentiation potential in NB/B27/PSN medium supplemented with 10 ng/ml bFGF together with 10 ng/ml EGF. Panel B1 shows an individual NSC founder cell photographed at 2 HIC. Panel B2 shows the same cell cultured under differentiating growth condition for 7 days, generating a multipotential and heterogeneous progeny of ~90 cells, which exhibit either phase-dark or phase-bright morphologies. In panel B3, immunostaining further reveals the presence of numerous neuronal (nestin⁺TUJ1⁺), astroglial (nestin⁺GFAP⁺), and oligodendroglial (nestin⁺A2B5⁺TUJ1^–GFAP^–) LRPs, as well as post-mitotic differentiating neurons (nestin^–BrdU^–TUJ1⁺) and astrocytes (nestin^–BrdU^–GFAP⁺), in addition to undifferentiated, self-renewing (nestin⁺BrdU⁺A2B5^–GFAP^–TUJ1^–) precursor cells. For a complete account of clonal expansion potentials of NSCs under self-renewing or differentiating growth conditions see Maric et al. (2003). The fluorescently conjugated markers used to identify the various phenotypes are color-coded and identified at the bottom of the merged micrographs of fluorescence signals: nestin (blue), BrdU (green), A2B5 (yellow), GFAP (orange), and TUJ1 (red). Calibration bar: 20 µm.

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Figure 3.18.5

Clonal expansion of different types of LRPs confirms their lineage-restricted identities. Select LRP populations were sorted according to their differential expressions of surface ganglioside markers (see Fig. 3.18.3). Early neuronal-restricted progenitors (ENRPs) were identified by their low levels of cholera toxin (ChTx) and tetanus toxin (TnTx) binding. Neuroglial-restricted progenitors (NGRPs) were identified by their low levels of staining with A2B5/JONES antibodies, while oligoglial-restricted progenitors (OGRPs) were identified by their high levels of A2B5/JONES labeling and complete absence of ChTx/TnTx markers (see Fig. 3.18.3). Reanalysis of each LRP population after sorting shows ³99% purity with respect to ganglioside markers used for sorting (A1, B1, C1). Sort-purified ENRPs, NGRPs, and OGRPs were plated at clonal density and photographed after 2 hr in culture (HIC) under phase-contrast optics (A2, B2, C2), then maintained for 7 days in culture (DIC) in NB/B27/PSN supplemented with 10 ng/ml bFGF in order to determine their developmental potentials (see Basic Protocol 2, steps to ). At the end of culture, the progeny of the resulting clones (A3, B3, C3) were cumulatively labeled with 10 µM BrdU for 24 hr, then processed for 5-color immunostaining with anti-nestin-AMCA, anti-BrdU-FITC or anti-O4-FITC, anti-A2B5-PE, anti-GFAP-TRITC, and anti-TUJ1-Alexa Fluor 647 antibodies in order to detect their proliferative and differentiation status (see Basic Protocol 2, steps to ). Panel A4 shows that clonally expanded progeny derived from ENRPs are typically composed of process-bearing nestin⁺TUJ1⁺ neuronal progenitors and nestin^–TUJ1⁺ postmitotic neurons. Panel B4 shows that progeny derived from NGRPs are more heterogeneous, containing both nestin⁺A2B5⁺TUJ1⁺ immature neurons and nestin⁺A2B5⁺TUJ1^– glial progenitors, with some of these still actively proliferating (BrdU⁺). Panel C4 shows that clonally expanded progeny derived from OGRPs typically exhibit cells at different stages of oligodendroglial lineage progression, including immature nestin⁺A2B5⁺O4^– and differentiating nestin⁺A2B5⁺O4⁺ oligoglial progenitors and transitional nestin^–A2B5^–O4⁺ oligodendrocytes. For a complete accounting of clonal expansion potentials of ENRPs, NGRPs and OGRPs, see Maric et al. (2003). Phenotypic markers in A4, B4, and C4 are color-coded at the bottom of the merged micrographs of fluorescence signals: nestin (blue), BrdU (green) in A4 and B4, O4 (green) in C4, A2B5 (yellow), GFAP (orange), and TUJ1 (red). Calibration bar: 20 µm.

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Literature Cited

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