Zeigler, Frank and Hall, Stephen G. (2007) Isolation of Oligodendroglial Cells From Cultured Neural Stem /Progenitors, Stem Cell Assays (Ed: Mohan C. Vemuri) for the series Methods in Molecular Biology (series Ed: John Walker), Humana Press, NJ. More information on the book here.

Quantitative phenotyping of oligodendroglial cells

Intracellular FACS for oligodendroglial phenotypes can be applied to the study of oligodendroglial lineage differentiation from cultured CNS stem/progenitor cells. AlphaGenix's stem cell assay kits and reagents save time for assay development work, allowing researchers more time to focus on functional biology, and provide an excellent starting point for phenotyping oligodendroglial cell populations in vitro (Figure 1).

Figure 1.  Quantitative Phenotyping of Cultured Rodent Oligodendroglial Cells Using Multi-parameter Intracellular Flow Cytometry. Cultured rodent NSC’s (AlphaGenix Cat. #77024) were withdrawn from bFGF, and exposed to 1.0uM retinoic acid, 10 ng/ml T3 hormone, and 1% FBS to induce oligodendroglial differentiation and lineage commitment. Viable cells were identified retrospectively using oxidative metabolic activity as before. Cultures were analyzed for nestin, GFAP, TUJ1, and MBP at time 0, and days 2, 5, and 7 using AlphaGenix rNSC intracellular FACS kit Cat. # 77026. Controls included unstained samples and irrelevant IgG’s from the appropriate species’ primary Ab. FACS plots show the percent positive above the IgG controls.

While the exact location of multipotent neural stem cells (NSCs) in the adult brain is somewhat controversial, it is accepted that one of the areas with highest NSC concentration is in the subventricular zone (SVZ) of the lateral ventricular walls (1-3). One can consistently distinguish multipotent NSCs from other precursor cells as well as differentiated cells present in the CNS.  The addition of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) has been shown to expand neurospheres in undifferentiated state (4).  In contrast, retinoic acid (RA) can induce stem cells to differentiate into glial cells (astrocytes and oligodendrocytes) and neurons (5).

  The existence of a glial precursor in both the developing and adult CNS was first demonstrated with the isolation and culture of a common glial progenitor to oligodendrocytes and type-2 astrocytes, termed O-2A progenitor or oligodendroglial progenitor cells (OPC) (6). In culture this precursor cell could directly give rise to either oligodendrocytes or type-2 astrocytes, or divide to form another OPC. Subsequently cells with similar attributes have been isolated from various regions of both rodent and human CNS (7), and more recently growth factor responsive CNS stem/progenitor cells grown as non-adherent cultures of “neurospheres” have also been shown to contain OPC’s (8).

These studies have primarily relied on semi-quantitative determinations, such as RT-PCR for gene expression analysis and immuno-phenotyping with manual microscopic counting. Although RT-PCR is exquisitely sensitive, it cannot distinguish the precise number of cells of a given phenotype, while microscopic hand counting of immunostained cells is prone to both inaccuracy and variability, unless great effort is taken to count sufficient cell numbers to obtain a representative sample size. This type of quantitation is also subjective, and often depends on individual operators whose relative experience and “trained eye” can vary widely. Additionally, manual immuno-phenotyping methods are not easily adapted to multi-parameter analyses, particularly when more than two markers are desired within a single population of cells. A method is therefore required to address the need for quantitative cellular phenotyping of cultured CNS stem/progenitors, OPC’s, and terminally differentiated oligodendrocytes.

Flow cytometry provides an analytical technology that can supply the required phenotypic information, while also providing the necessary accuracy, sensitivity, and robustness. Its multi-parameter capability allows for the detection and enumeration of multiple phenotypes simultaneously on thousands of cells per second in just a few minutes. This provides a large amount of high content information regarding a population of cells at the single-cell level, and is ideally suited for studies of CNS stem/progenitor cells, which display a strong inherent potential for multi-lineage differentiation. However, many of the markers commonly used to phenotype OPC’s and oligodendrocytes are in fact intracellular, e.g. CNPase, GFAP, nestin, and the transcription factors Olig 1/2.

Intracellular FACS requires that cells are first dissociated into a single-cell suspension. In our experience trypsin is the best enzyme for efficient preparation of a viable single-cell suspension of cultured cells in many applications, including rodent and human cultured neural stem cells.  However, care must be taken since trypsin can vary significantly in activity, and we have found that both considerable effort can be required to optimize the use of trypsin, including optimization of reaction time and screening different manufacturer’s and production lots. CNS cells are fragile and have been reported to lose viability after trypsin dissociation, but since they will be fixed for intracellular FACS in any case, this is not problematic. 

Once the method of cell dissociation has been optimized, cells must be fixed to preserve both cellular morphology and antigenicity, and permeabilized to allow antibody penetration within the cell. Formaldehyde is the most widely used and effective fixative for preserving cell and tissue morphology and antigenicity, and although many different formulations and commercial kits exist for intracellular FACS, they generally rely on the covalent cross-linking of primary amines in cellular macromolecules, mostly proteins and nucleic acids, and generally contain between 1 and 4% formaldehyde.  Some protocols use methanol as both fixative and permeabilizer, however issues with cell losses and incompatibility can occur. Permeabilization can be accomplished using a variety of detergents, such as saponin and Tween series non-ionic detergents, with effective concentrations of 0.01 to 0.1% in most cases.

With the basic techniques for cell dissociation and intracellular FACS preparation complete, attention must next be paid to the choice of both phenotypic markers, and the antibody probes used to identify them. Regarding the choice of phenotypic marker, each study may have its own unique requirements and a full discussion of the relevance of the many possible markers is outside the scope of this work. However, a brief review of the literature shows that many phenotypic markers are used in the majority of studies involving cultured CNS stem/progenitors, OPC’s, and oligodendrocytes derived from them. Nestin is commonly used to mark non-differentiated stem/progenitors, b-tubulin III identifies neuronal cells, GFAP for astroglial, and for committed oligodendroglial progenitors and terminally-differentiated oligodendrocytes CNPase, MBP, and the O1/O4 glycolipid antigens. The biological relevance of each of these must of course be empirically determined through rigorous experimentation.

Many primary antibodies are available commercially, however the degree to which they have been validated and in some cases even their specificity is variable or unknown. Each antibody-antigen combination needs to be evaluated for compatibility with the intracellular FACS reagents, as well as for specificity for the target antigen. Dual-parameter flow cytometry can be helpful in this regard, e.g. using both CNPase and MBP to qualify both antibodies by dual localization on primary spinal cord isolated oligodendrocytes. Cell lines that have been shown to express the antigen can also be useful in some cases, or more frequently, as a source of a non-expressing cell type for the particular antigen (i.e. if your MBP antibody reacts with KG1a hematopoietic cells then it cannot be trusted). We have also found it important to visualize the pattern of reactivity with each antibody and cell type by immunofluorescence, to confirm the appropriate cellular localization (e.g. nestin and GFAP should look like intermediate filaments microscopically) when qualifying an antibody.  

Since FACS is an extremely sensitive detection methodology, and since one cannot visualize the phenotypic signal in intracellular FACS, the appropriate negative controls are absolutely required. Each cell type and preparation needs to have a negative control using either an isotype matched IgG, or irrelevant IgG from the same species as the primary antibody and at same concentration, time, etc. For multi-parameter studies using 2 different primary antibodies, tests should be run with the secondary fluorochrome conjugated antibodies to confirm specificity of dual-labeling.