Identifying and Quantitating Neural Stem and Progenitor Cells in the Adult Brain

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Abstract

Adult brain contains neural stem and progenitor cells that are capable of generating new neurons. Active continuous neurogenesis is limited to the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus. Newborn neurons gradually become fully functional and integrated into the existing circuitry of the olfactory bulb and the hippocampus. Transition from stem cells to fully differentiation neurons, the neuronal differentiation cascade, occurs through defined steps, and different classes of neuronal precursors can be distinguished by their morphology, expressed markers, and mitotic activity. Cells in these classes can be identified by immunophenotyping, labeling with thymidine analogues, and infection with retro- and lentiviral vectors. We here describe a transgenic approach that allows identification, in vivo visualization, isolation, and accurate enumeration of various classes of stem and progenitor cells in the adult brain. We generated a series of reporter mouse lines in which neural stem and progenitor cells express various fluorescent proteins (GFP, CFPnuc, H2B-GFP, DsRedTimer, and mCherry) under the control of the regulatory elements of the nestin gene. Using these lines, we were able to dissect the neuronal differentiation cascade into several discrete steps and to evaluate the changes induced by various neurogenic and antineurogenic stimuli. In particular, nuclear localization of the fluorescent signal in nestin-CFPnuc mice greatly simplifies the distribution pattern of neural stem and progenitor cells and allows accurate quantitation of changes induced by neurogenic agents in distinct classes of neuronal precursors. We present protocols for applying confocal microscopy, stereology, and electron microscopy to evaluate changes in the neurogenic compartments of the adult brain.

Introduction

New neurons are continuously generated from neural stem and progenitor cells in the brain of adult rodents and primates (Abrous 2005, Alvarez-Buylla 2001, Gage 2000, Kempermann 2006, Kempermann 2004, Lie 2004, Lledo 2006, Ming 2005, Song 2005, Taupin 2002). Neural stem cells are defined as self-renewing, multipotent cells, usually with long life span, that generate neurons, astrocytes, and oligodendrocytes. Progenitor cells have a limited life span, less self-renewal ability, and may be multipotential or unipotential (e.g., only generating neurons). Persistent production of new neurons is limited to two areas of the adult brain: the olfactory bulb (OB) and the dentate gyrus (DG) of the hippocampus. Neurons of the OB originate in the anterior part of the subventricular zone (SVZ) of the lateral ventricles (LVs), whereas neurons of the DG are generated in the subgranular zone (SGZ) of the DG (Fig. 1). Neural precursors in the SVZ migrate through a network of tangential pathways, and converge onto the rostral migratory stream (RMS) before arriving to the OB and differentiating into granule and periglomerular neurons (Alvarez-Buylla 2002, Lois 1994). Neural precursors of the adult DG are born in the SGZ and then migrate locally to the granule cell layer (GCL) and differentiate into granule neurons.

Newly generated neurons are fully functional: for instance, new DG neurons start extending axons several days after the last mitosis and, when fully differentiated, receive synaptic input and project functional connections into the CA3 region of the hippocampus (Ge 2006, Song 2002, Tashiro 2006, van Praag 2002). A full transformation of a neural stem cell into a functional neuron takes 25–30 days. During this time, cells undergo asymmetric and symmetric divisions, exit the cell cycle, express a wide range of markers, change their morphology, and establish connections with other cells. This transition from stem cells to fully integrated neurons, the neuronal differentiation cascade, proceeds through defined steps that can be distinguished through a combination of markers, morphological features, and mitotic activity, and will be discussed in more detail below.

Adult neurogenesis is modulated by a very wide range of intrinsic and extrinsic factors (Abrous 2005, Lledo 2006, Ming 2005). It is regulated by growth factors (e.g., epidermal-, fibroblast-, brain-derived, and insulin-like growth factors), neurotransmitters (e.g., serotonin, dopamine, glutamate, acetylcholine, norepinephrine, and nitric oxide), hormones (e.g., estrogen, prolactin, and corticosteroids), and drugs (e.g., antidepressants, opiates, and lithium). Furthermore, it is influenced by aging, pregnancy, stress, disease, physical activity, enriched environment, dietary restrictions, and learning. To give one set of examples, hippocampal neurogenesis is inhibited by chronic stress, depression, and posttraumatic stress disorder; conversely, it is augmented by antidepressant drugs directed at monoamine neurotransmitters (e.g., the selective serotonine reuptake inhibitor fluoxetine), brain-derived growth factor and insulin-like growth factors (both of which show efficacy in animal models of depression), and electroconvulsive shock (Dranovsky 2006, Malberg 2005, Warner-Schmidt 2006). Moreover, recent evidence indicates that neurogenesis may be an obligatory step in the behavioral action of antidepressants (Santarelli et al., 2003).

Factors that lead to a net increase or decrease in the number of new neurons may, in principle, affect any step in the differentiation cascade that converts neural stem cells into fully differentiated neurons, for example, symmetric and asymmetric divisions of different types of precursors, their survival, or their differentiation; both the cell populations that are targeted by neurogenic stimuli and the molecular mechanisms that mediate the action of these stimuli are only beginning to be understood.

A detailed analysis of neurogenesis requires approaches to describe distinct steps in the differentiation cascade that converts stem cells into differentiation neurons, to identify cell types within the cascade, to label these cells types and follow their lineage, and to quantify the changes induced by neurogenic stimuli. Traditionally, the main approaches used to study adult neurogenesis were to label proliferating cells with thymidine analogues (mainly with 8-bromodeoxyuridine, BrdU) and to categorize by phenotype the subclasses of precursors and differentiated cells using immunocytochemistry. More recently, retroviral and lentiviral labeling, double labeling with halogenated thymidine analogues, and generation of transgenic animals with fluorescently labeled subclasses of precursors have been added to the list of experimental approaches.

Labeling of dividing cells with BrdU has become a standard method for studying adult neurogenesis. Its main advantage is that the cells that have undergone DNA synthesis can be detected in days, weeks, and months after the labeling, thus allowing lineage analysis of the newly generated cells. Moreover, in combination with cell-type specific markers, it allows for double and triple labeling and thus, more precise phenotyping of labeled cells. Importantly, the BrdU signal is restricted to the nucleus, facilitating the scoring of the signal and accurate enumeration of labeled cells. Note, however, that BrdU may be incorporated into damaged cells (Kuan et al., 2004); that high doses of BrdU may be toxic to cells; that changes in the length of the cell cycle or S phase and the details of the labeling schedule may have profound effects on the fraction of labeled cells and thus, the interpretation of the results when different treatments are compared (Hayes and Nowakowski, 2002); and that identification of labeled cells can only be achieved with fixed tissue (precluding, for instance, electrophysiological studies of new neurons and their precursors).

Nucleotide labeling of new neurons can be further elaborated by using two halogenated thymidine analogues, 8-chlorodeoxyuridine (CldU) and 8-iododeoxyuridine (IdU) (Burns 2005, Vega 2005). This allows a much more precise temporal discrimination of the cell cycle progression and elimination of new neurons. When combined with immunophenotyping, this method may become a powerful tool for high-resolution analysis of cell proliferation and lineage determination in the adult brain.

Some of the limitations of BrdU labeling can be overcome by using retroviruses carrying reporter transgenes [such as genes for green, cyan, or yellow fluorescent proteins (GFP, CFP, or RFP)] to label dividing cells and their progeny. Infected cells, after undergoing mitosis, become permanently labeled and, most importantly, can be accessed for the morphological and electrophysiological studies (Ge 2006, van Praag 2002). Note, however, that cells have to undergo division to be labeled with the retroviral vectors [this limitation may be overcome through the use of lentiviral vectors (Geraerts et al., 2006)]; that labeling efficiency is low and variable and, therefore, not amenable to quantitative analysis; and that labeling requires an invasive manipulation (stereotaxic injection) which can elicit an inflammatory reaction.

Selected classes of neural stem and progenitor cells can be identified through immunophenotyping. The most primitive precursor cells both in the SVZ and in the DG express glial fibrillary acidic protein (GFAP), vimentin, brain lipid-binding protein (BLBP), Sox2, and nestin; their progeny start to lose these markers and to express Olig2, Tbr2, neurogenin 1, doublecortin (Dcx), Prox-1, and a host of other markers whose combination defines specific steps of the differentiation cascade. When combined with BrdU labeling, immunophenotyping can provide a detailed view of the birth, maturation, and differentiation of newborn neurons. An obvious limitation of the approach is the requirement to fix the tissue which precludes in vivo analyses of these cells, for example, isolation for RNA or protein profiling, imaging, lineage tracing, transplantation, or electrophysiological studies.

Nestin (for neuroepithelial stem; Lendahl et al., 1990) is an intermediate filament protein selectively expressed in neural stem and early progenitor cells of the developing and adult nervous system. It was originally identified in neuroepithelial cells as the protein reacting with the monoclonal antibody Rat401 by McKay and coworkers and, so far, has been the best marker that correlates with neural stem cell potential.

Nestin mRNA and protein are abundantly expressed in the developing and adult nervous system. Nestin is also strongly expressed in the myotomes of the embryo. In addition, nestin expression has been reported in several other tissues and cell types, for example, pancreatic islets, the developing testis, tongue, tooth, and heart (Kachinsky 1994, Sejersen 1993, Terling 1995, Zulewski 2001). This relatively wide spectrum of nestin expression reflects the presence of various regulatory elements in the nestin gene: for instance, expression of nestin in the embryonic neuroepithelium is dependent on the presence of transcriptional enhancer which resides in the second intron of the gene (Josephson 1998, Yaworsky 1999, Zimmerman 1994), whereas regulatory elements in the first and third introns direct nestin expression to myotomes (Yaworsky 1999, Zimmerman 1994). The neural enhancer in the second intron of the nestin gene is strong and dominant, being sufficient to direct the expression of an exogenous transgene to the developing neuroepithelium in the transient transgenic assay even when combined with a heterologous promoter (e.g., promoter of the herpes virus thymidine kinase gene; Zimmerman et al., 1994). Importantly, the use of this enhancer element seems to “rectify” the expression pattern of the transgene: for instance, although endogenous nestin is expressed both in the nervous system and in the myotomes of the embryo, expression of the transgene in the myotomes is abrogated if only the second intron is used in the transgenic construct. Thus, regulatory elements residing in the second intron of the nestin gene are both necessary and sufficient to direct the expression of a transgene into neural stem and progenitor cells, and these elements have been used to generate a number of transgenic mouse lines (Encinas 2006, Imayoshi 2006, Kawaguchi 2001, Mignone 2004, Tronche 1999, Yamaguchi 2000).

We used neurospecific regulatory elements of the nestin gene to generate several transgenic mouse lines that allow direct visualization of neural stem and progenitor cells in the developing and adult brain (Encinas 2006, Mignone 2004). In these animals, fragments of the nestin gene (5.8 kb of the promoter region and 1.8 kb of the second intron), combined with a polyadenylation signal from SV40, drive expression of GFP (Fig. 2A), fusion of GFP with H2B histone (H2B-GFP), RFPs DsRedTimer and mCherry, or CFP with nuclear localization signal (CFPnuc, Fig. 2B). Several independent lines were obtained for each transgene and all demonstrated a highly similar pattern of transgene expression in the developing and adult brain [note that similar patterns of expression were obtained with other transgenes containing the second intron of nestin (Kawaguchi 2001, Yamaguchi 2000), even though these transgenes employed heterologous promoters]. No obvious defects were apparent during development and adulthood of the transgenic mice. The transgene was normally transmitted and, in the case of nestin-GFP mice, the expression pattern in the developing and adult CNS remained invariant over the course of at least 20 generations.

Several lines of evidence demonstrate that expression of GFP or CFPnuc marks neural stem and early progenitor cells in our nestin-GFP and nestin-CFPnuc transgenic mice:

  • a

    The transgene is expressed in those areas of the developing embryo that correspond to the neuroepithelial cells of the developing nervous system.

  • b

    The transgene is expressed in those areas of the adult brain (SVZ, RMS, OB, and DG) that are marked by persistent production of new neurons.

  • c

    GFP and CFPnuc expression is absent in those cells that have already undergone differentiation and in those areas of the brain that only contain fully differentiated cells.

  • d

    The sites of the transgene expression in the developing and adult nervous system overlap with the sites of expression of nestin that has served as a reliable marker of neural stem cells.

  • e

    GFP- and CFPnuc-positive cells are capable of forming neurospheres and producing a variety of types of progeny in vitro.

  • f

    GFP-expressing cells are strongly (∼1400 fold) enriched in neurosphere-forming cells, and, conversely, most of the neurosphere-forming cells of the adult brain are present within the fraction of GFP-expressing cells.

Together, these results indicate that GFP- and CFPnuc-positive cells in the nestin-GFP and nestin-CFPnuc transgenic animals accurately represent neural stem and early progenitor cells in the developing and adult nervous system.

We have used the nestin-GFP and the nestin-CFPnuc reporter lines to define discrete steps in the neuronal differentiation cascade in the DG (leading from stem/progenitor cells to differentiated granule neurons), based on the morphology of the cells, the marker proteins that they express, and their mitotic activity (measured by BrdU incorporation) (Encinas et al., 2006). We identify six classes of cells in the neuronal lineage in the DG of nestin-CFPnuc mice; these classes encompass and partially overlap with the categories of neuronal precursors defined by other approaches (Fukuda 2003, Kempermann 2004, Kronenberg 2003, Mignone 2004, Seri 2004).

The first class is represented by GFAP/nestin/vimentin/BLBP/Sox2-positive nestin-GFP and nestin-CFPnuc cells. The triangular soma and the nuclei of these cells reside in the SGZ; they extend a single or double apical process radially across the GCL, terminating as an elaborated arbor of very fine leaf-like processes in the molecular layer. Cells of this class correspond to the most primitive, stem-like population in the DG; note, however, that not all of the criteria of stem cells, for example ability to self renew, have been demonstrated for these cells (Seaberg and van der Kooy, 2003). Only a small fraction of these cells (less than 2%) are labeled by BrdU after a short (2 h) pulse, indicating their low rate of division and consistent with the quiescent state of these cells; we therefore designate these cells as quiescent neural progenitors (QNPs).

The second class is represented by small (somatic diameter ∼10 μm) round or oval cells located in the SGZ. These cells also express nestin-GFP or nestin-CFPnuc but they do not stain for GFAP or vimentin and stain very weakly for nestin (this may indicate that reporter fluorescent proteins persist in these cells longer than nestin, or that the nestin is unequally distributed during cell division); they also do not stain for Dcx, for PSA-NCAM, or for markers of differentiated neurons. These cells are labeled with BrdU at high frequency (20–25%, 2 h after a single injection of BrdU) indicating that most of them are involved in mitotic activity; we designate these cells as amplifying neural progenitors (ANP). They are often seen in clusters extending along the SGZ; when the plane of division of cells in these clusters is visible, it is most often perpendicular to the SGZ such that the daughter cells remain in the SGZ. Importantly, a fraction of these cells are seen separating from QNPs after mitosis; in each case, the division plane is parallel or slightly oblique to the SGZ such that the daughter cell is deposited beneath the QNP cell (the plane of division may explain why these cells do not inherit GFAP, vimentin, or nestin which are predominantly localized to the apically positioned processes of the QNPs but not to their soma). Together, our results suggest that QNP cells undergo asymmetric divisions and give rise to ANP cells, which then propagate in the SGZ through a series of symmetric divisions.

The next class of precursor cells, still located in the SGZ, ceases to express nestin or nestin-driven reporters and starts to express Dcx and PSA-NCAM. A small subclass (∼1% of cells in this class) morphologically resembles ANPs, carries short (1–5 μm) horizontal processes, and is the final population in the differentiation cascade that is labeled by BrdU. Most of the cells in this class are represented by larger (10–15 μm somatic diameter) cells which extend longer (10–30 μm) horizontal processes in the plane of the SGZ and do not incorporate BrdU. These cells stain for Dcx, PSA-NCAM, and Prox-1. Thus, the bulk of this class is represented by postmitotic neuronal precursors; we designate them as type 1 neuroblasts (NB1).

Cells of the next class, type 2 neuroblasts or NB2, are larger than NB1 cells (somatic diameter ∼15 μm) and remain confined to the SGZ. They extend longer (20–40 μm) processes horizontally and obliquely to the plane of the SGZ. They do not express nestin, nestin-GFP, or CFPnuc, and express Dcx, PSA-NCAM, Prox-1, and NeuN.

The next class of cells corresponds to immature neurons (IN). They are larger than the cells of the previous classes (somatic diameter 15–20 μm), and their morphology resembles that of mature granule cells of the DG. Their soma is round or oval and can be found both in the SGZ and, mainly, in the GCL. These cells carry a single apical process that branches in its distal part located in the molecular layer. They express Dcx, PSA-NCAM, Prox-1, and NeuN.

The next class represents differentiated granule neurons, with developed apical dendrites and axons forming the mossy fiber. They cease to express PSA-NCAM and Dcx, but express NeuN and Prox-1.

The differentiation cascade in the DG of nestin-CFPnuc mice can thus be divided into discrete steps based on the expression of markers, morphology, and mitotic activity (Fig. 3).

Accurate enumeration of neural precursors using immunocytochemistry is often problematic: high cell density, complex cell morphology, and uncertainties in defining distinct boundaries between subclasses of cells present a real challenge when precise counts are required (for instance, when evaluating the action of a neurogenic stimulus). This reduces the precision of evaluating changes in particular subclasses of neuronal precursors (e.g., in contrast to BrdU or thymidine labeling of cell nuclei, where great precision can be achieved); this problem is particularly acute in the young brain, where the number of neural stem and progenitor cells is particularly high or when the changes evoked by a stimulus are low (note that most of the known inducers of neurogenesis increase the number of newly generated cells only by 30–50%).

For experiments which require both morphological and quantitative analysis of neurogenesis, use of two reporter lines, nestin-GFP and nestin-CFPnuc, is particularly helpful. In nestin-GFP mice, the fluorescent signal highlights all of the soma and the processes of stem and early progenitor cells (Fig. 2A) and these mice are very well suited for the studies of the morphology of neuronal precursors in the developing and adult brain. In contrast, in nestin-CFPnuc mice, the signal is localized in the cell nucleus and the distribution of the stem and progenitor cells is visualized as a punctuate pattern; this nuclear representation of stem and progenitor cells greatly reduces the complexity of their distribution pattern and permits their unambiguous enumeration (thus capturing the power of BrdU- or thymidine-based enumeration of nuclei) (Fig. 2B). Thus, these two reporter lines complement each other and allow visualization and counting of neural stem and progenitor cells. In our pilot experiments, we carefully compared the structures of the SVZ and DG as revealed by immunochemistry for nestin and by expression of nestin-CFPnuc or nestin-GFP. Whereas we were unable to generate accurate counts of nestin- or nestin-GFP-positive cells (particularly in the young brain), we were able to unambiguously count all of the labeled nuclei in the SVZ and DG of the nestin-CFPnuc mice. Importantly, crosses between these two lines allow simultaneous visualization of the soma and the nuclei of stem and progenitor cells, thus we were able to follow the morphological changes in these cells while enumerating them (Encinas, Chiang, and Enikolopov, unpublished data).

In summary, our approach with transgenic reporter lines that circumvents several obstacles is assessing changes in cell number during neurogenesis, for example, high cell density which hinders precise counts or uncertainty in attributing precursor cells to a particular class. It reduces the complex distribution pattern of precursor cells to a readily quantifiable punctuate pattern of labeled nuclei. It allows unambiguous enumeration of cells in a particular precursor class and can be used to analyze changes induced by a wide range of stimuli in the developing or adult brain.

Section snippets

Protocol I: Immunofluorescence Microscopy of Nestin-GFP and Nestin-CFPnuc Cells

Confocal microscopy is a crucial tool for the analysis of transgenic reporter mice, allowing quantification of neuroprogenitors and visualization of their anatomical and morphological features. In the nestin-GFP mice, GFP fluorescence reveals the entire stem or progenitor cell, helps to distinguish between the subtypes of neuroprogenitors, and, when combined with immunodetection of other cell-specific markers, makes it possible to investigate changes in protein expression patterns and

Protocol II: The Use of Confocal Stereology to Quantify Changes in Defined Classes of Neuronal Precursors

Quantitative analysis of the changes in different classes of neuronal precursors in response to neurogenic stimuli is crucial for identifying the classes that respond to the stimulus, that is, the steps within the differentiation cascade targeted by the stimulus. Accurate cell enumeration can be achieved through the use of design-based stereology (Gundersen 1999, Howell 2002, Peterson 1999, Schmitz 2005); this approach is particularly powerful when combined with confocal microscopy which

Protocol III: Electron Microscopy of Nestin-GFP/CFPnuc Cells

The following protocol is developed for the optic-electronic microscopy transfer technique (Fig. 8), whose main feature is immunostaining of the samples at the preembedding stage. This is our method of choice for ultrastructural analysis of adult neural stem and progenitor cells because preembedding immunostaining allows visualization and tracking of cells of interests throughout the entire processing of the tissue, and because of the strength of the signal, two features that are absent in

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