Neural Stem Cell Biology
Postnatal neurogenesis discovery Neural stem cell discovery Embryonic NSCs Adult NSCs BCH/GGB512 Richard Gronostajski History of postnatal neurogenesis discovery
History of postnatal neurogenesis discovery
1800s-1950s no way of measuring proliferation other than mitosis Saw occasional mitoses but couldn't tell if they were neurons 1950s H3 thymidine first used in vivo 1961 3H TdR first applied to adult brain (I. Smart) saw new neurons in 3 day old mice, not adults 1960 (Joseph Altman) 3H TdR adult rats, saw labeling in cortex, hippocampus, olfactory bulb. Ignored for almost 40 years (bias against Altman, he got the last word, didn't get tenure at MIT, did at Purdue) History of postnatal neurogenesis discovery
Injected P20+21, harvested P60 History of postnatal neurogenesis discovery
1960 (Joseph Altman) 3H TdR adult rats, saw labeling in cortex, hippocampus, olfactory bulb. Ignored for almost 40 years 1977 Michael Kaplan's EM studies confirmed neurogenesis. Pasko Rakic's papers found "little or no" adult neurogenesis. 1988 Stanfield and Trice showed adult neurogenesis with fluorescent tracer + 3H TdR Fred Gage and coworkers used BrdU and cell-type markers 1999 Rakic showed neurogenesis with BrdU incorporation and cell-type specific markers. Postnatal neurogenesis
FIG. 2. Newly generated cells in the adult macaque dentate gyrus express neuronal phenotypic markers 32 days after five BrdU injections, as detected by immunofluorescence double-label and confocal microscopy. (ad) Neurons in the dentate gyrus express NeuN (red). The same cell in the GCL that is labeled with BrdU (arrow, green in b) also expresses NeuN (arrow, a). (c and d) An example of a BrdU-labeled nucleus (d, arrow, green) that did not emit a red fluorescence signal (c, arrow), demonstrating that the BrdU fluorescent signal did not bleed into the red channel; this might be a progenitor or new glial cell. (e and f ) A TuJ1-positive cell in the SGZ (arrow, red) colabels with BrdU in its nucleus ( f, arrow, green). Note the slender process (arrowheads) emanating from the cell body, resembling the trailing process of a newly generated migrating neuron. The BrdU in its nucleus confirms its recent generation. (g and h) Two cells in the SGZ expressing TuJ1 in the cytoplasm surrounding their nuclei (red), which are immunopositive for BrdU (h, green). Their close proximity suggests that these two cells might be newly generated siblings. The long thin process (arrowheads), consistent with migratory behavior, is clearly seen in one of the cells. (i and j) A bipolar cell in the SGZ coexpressing TuJ1 (green) and nuclear BrdU (j, orange). Although most double-labeled cells were oriented radially in the GCL, occasionally a cell was oriented parallel to the GCL. This example shows such a BrdU-labeled cell with an extended process on either side of the nucleus. (k) A TuJ1-positive cell (green, arrow) with a BrdU-positive nucleus (orange) has an immature migratory appearance. Note the thin trailing process (arrowheads) and a nearby BrdU-negative neuron, with a mature, apical process (arrowcross). (l) A cell deep in the GCL colabels with TuJ1 (green) and BrdU (orange) with an apical process that is thick and tortuous, similar to the dynamic, exploratory leading process of a migrating neuron (its trailing process is out of the optical plane). Compare this with the straighter apical process of the more mature BrdU-immunonegative granule neuron in k (arrowcross). [Bar (al) 5 10 mm.] Neurobiology: Kornack and Rakic Proc. Natl. Acad. Sci. USA 96 (1999) 5771 Major reasons for 40 year delay
Lack of good markers for both proliferation and cell types. Bias against the idea. Neural Stem Cells in vitro Neural Stem Cells in vitro
Fig. 1. EGF-induced proliferation of cells isolated from the adult mouse striatum. (A) After 2 DIV, cells that hadundergone cell division were first observed. Cell division continued at 3(B) and 4-(C) DIV, although dividing cells beginning to form a cluster migrated slowly across the substrate. (D) After 6 to 8DIV, spheres of cells lifted off the substrate and floated insuspension. Line in substrate (A through C) serves toidentify the field. (E) One hour after plating onto poly-L-ornithine, a 6 DIV sphere attached to the substrate. (F) The cells in(E) were immunostained with antibody to nestin; virtually all cells wereimmunoreactive for nestin. Self renewal shown (G through J) Single cells, derived from dissociated 6- to 8-DIV spheres,were plated in single wells of a 96-well plate; A large, hypertrophic cellafter 2DIV (G) began todivide and form cluster of cells during the subsequent 3 (H), 4 (I), and 6 (J) DIV. Scratches insubstrate serve to den tify the field. Scale bars:(A through D) bar in(D) denotes 50um (E), 50 um;(F), 25 um;(G through J)bar in (J), 50 um. Neurospheres Neural Stem Cells in vitro
FIG. 1. Morphology of neurons generated by culturing adult brain cells with bFGF and then with medium conditioned by Ast-1 cells. Neurons stained by immuno-fluorescence for expression of 150-kDa neurofilament (b, d, f, and h)have various morphologies and, as shown by phase-contrast micrography, their nuclei are labeled with [3H]thymidine (arrowsin a, c, e, and g).The silver grains are more easily seen in g, where the plain of focus is at the emulsion level. (a-f x280;g and h X450.) Neural Stem Cells in vitro Neurosphere assay Primary neurospheres may measure stem and progenitor cells.Initial passage Secondary neurospheres may measure stem cells.Second passage. Assay controversial, spheres and split or merge, best to make at limiting dilution. Question everything you read!
Lack of good markers for both proliferation and cell types. Bias against an idea doesn't mean it isn't true. Summary of embryonic and adult neurogenesis
Protoplasmic or fibrous astrocytes DCX+ Nestin- GFAP- GLAST+ Nestin+ GFAP+ Pax6+ Tbr2+ Figure 1 | Patterns of gliogenesis in embryonic and adult progenitor zones. The progression from the embryo to the adult is shown from left to right (a to c). Black arrows indicate self-renewal or differentiation from one cell type to another. Markers of macroglia and their precursors are listed. a, Self-renewing neuroepithelial cells line the ventricles throughout the neuraxis at the stages of neural tube closure. These cells may generate some neurons. Neuroepithelial cells are transformed into radial glial cells as neurogenesis begins. b, Radial glia produce intermediate progenitor cells and oligodendrocyte precursor cells (OPCs), which in turn produce neurons and oligodendrocytes, respectively. Radial glia can also become astrocytes, as well as producing intermediate progenitors that expand in number before producing astrocytes. Protoplasmic astrocytes and fibrous astrocytes might arise from common or independent progenitors. Radial glia also produce ependymal cells. c, In adults, oligodendrocytes are produced by two independent pathways: type B cells in the cortical subventricular zone produce transit-amplifying cells (known as type C cells), which in turn produce OPCs as well as neurons. The OPCs subsequently generate oligodendrocytes, and OPCs that are already resident in the grey matter also produce oligodendrocytes. ALDH1L1, aldehyde dehydrogenase 1 family, member L1; APC, adenomatous polyposis coli; GFAP, glial fibrillary acidic protein; MBP, myelin basic protein; PDGFR-, platelet-derived growth factor receptor-; PLP, proteolipid protein 1. All green cells are intermediate progenitors, with type C cells being a subset of these, and all blue cells are neural stem cells (even though each blue cell is a different type). Subgranular zone hippocampus Nestin+, GFAP+ Pax6+ Blue cells - stem cellsGreen cells - intermediate progenitor cells Orange cells neuronal progenitors and neurons Modified from: Developmental genetics of vertebrate glial-cell specification.Rowitch DH, Kriegstein AR. Nature Nov 11;468(7321):214-22 Evidence GFAP-GFP transgene expression
Fig. 1. Green fluorescent radial glial cells of 94-4 transgenic mice, in which the human GFAP-promoter drives expression of the S65Tmutant green fluorescent protein (GFP) (Zhuo et al., 1997). (A) GFP-immunostained and (B) GLAST-immunoreactive radial glial cells in frontal sections from E16 (A) and E14 (B) mouse cortex (pial surface upwards in A and towards the upper right corner in B). (C) depicts GFP-immunostained cells from acutely dissociated E14 cortex double stained with GLAST antiserum (D). Filled arrowheads depict double-positive cells and open arrowheads double-negative cells in the corresponding fluorescent micrographs. This shows that GFP is localized in GLAST-containing precursor cells with radial glial morphology. Scale bars: 25 mm. GFAP-GFP transgene is expressed in GLAST+ cells that form radial pattern Sorted GFP+ and put in culture
Fig. 2. Characterization of green fluorescent cells isolated from the cortex of hGFAP-GFP transgenic mice and their progeny. Green fluorescent cells were isolated from the transgenic mouse line 94-4 expressing GFP under the control of the human GFAP promoter (Zhuo et al., 1997; Fig. 1). (A-C) depict examples of sort profiles of cells from E14 (A), E16 (B) and E18 (C) cortex. The left columns in (A-C) show the dot plots of cells in forward scatter (FSC) and side scatter (SSC) with a polygon indicating the gate selecting the healthy cells. The histograms in the right columns in (A-C) show the number of cells (events, y-axis) with a fluorescent intensity indicated on the x-axis of wild-type controls (upper panels) and hGFAP-GFP transgenic littermates (lower panels). The percentage of fluorescent cells in the sort gate (grey shading, A-C) is indicated. (A-C) depict the composition of cells 2 hours (2h) or 5-7 days after the sort (days in vitro, div). A few cells sorted from mouse cortex were plated on a rat cortex feeder layer and identified by their M2M6 immunoreactivity, as depicted in Fig. 3. Clusters of M2M6-positive cells after 2 hours were mostly (97%) single cells and were double stained with antiserum directed against b-tubulin-III as a neuronal and RC2, GLAST, BLBP, Ki67 or nestin as precursor markers. Neurons are depicted in blue, precursor cells in green, indicating their differential RC2, RC2/GLAST, RC2/GLAST/BLBP or GLAST/BLBP immunoreactivity as depicted in the figure. Note that almost all sorted precursor cells are GLAST immunoreactive. Clusters after 7 div contained, in most cases, several cells and were interpreted as clones derived from single plated precursor cells. Clusters were classified as pure neuronal when all cells of a cluster were b-tubulin-III immunoreactive (see example in Fig. 3E,F) and their proportion is depicted in blue, as pure non-neuronal when no cell of a clone was b-tubulin-III immunoreactive (depicted in red), but cells were nestin or GFAP positive (see examples in Fig. 3G-J) and as mixed neuronal and non-neuronal when clones contained b-tubulin- III-positive and -negative cells (depicted in orange). Note the large increase in the number of neuronal clusters at E14 and E16 (A,B), suggesting that most GLAST-positive precursors generate neurons at this stage. In contrast, the progeny were mostly astrocytes when cells were isolated from an E18 cortex (C). GFAP-GFP+ cells made neurons, glia and mixed colonies when put into culture of 5-7 days.Some contaminating neurons present in starting material Types of colonies made in vitro
Filled arrowheads indicate double-labeled cells, open arrowheads indicate single-labeled cells in corresponding micrographs. Note that GLAST-positive precursor cells generate neurons and astrocytes in two separate lineages. Scale bars: 25um. Fig. 3. Examples of the progeny of hGFAP-GFP- and GLASTpositive precursor cells isolated by fluorescence-activated cell sorting. Cells were sorted from E14 (A-H) and E18 (I,J) mouse cortex by green fluorescent protein content driven from the human GFAP promoter (Zhuo et al., 1997; see Fig.1, sort gates as in Fig. 2). The sorted cells were cultured for 5-7 days. In C-J, sorted cells were cultured on a rat cortex feeder layer of the corresponding age and identified by the mouse-specific antibody M2M6 (Lagenaur and Schachner, 1981; Lund et al., 1985) (C,E,G,I). Clusters of labeled cells were considered as clones derived from a single sorted precursor cell, as illustrated in the overview in C,D. Cell-typespecific antibodies were used as indicated in the panels to identify the composition of the clones. Pure neuronal clones were composed exclusively of b-tubulin-III-positive cells extending neurites marked by arrows (E,F). Neurons were generated in vitro and incorporated BrdU (red in B). An example of a non-neuronal clone generated from E14 precursors containing a GFAP-positive cell (filled arrowhead) is depicted in G,H. (I,J) A non-neuronal clone composed exclusively of GFAP-positive astrocytes generated by cells sorted from E18 cortex. Filled arrowheads indicate double-labeled cells, open arrowheads indicate single-labeled cells in corresponding micrographs. Note that GLAST-positive precursor cells generate neurons and astrocytes in two separate lineages. Scale bars: 25mm. Fig. 3. Examples of the progeny of hGFAP-GFP- and GLAST positive precursor cells isolated by fluorescence-activated cell sorting. Cells were sorted from E14 (A-H) and E18 (I,J) mouse cortex by green fluorescent protein content driven from the human GFAP promoter. The sorted cells were cultured for 5-7 days. In C-J, sorted cells were cultured on a rat cortex feeder layer of the corresponding age and identified by the mouse-specific antibody M2M6 (Lagenaur and Schachner, 1981; Lund et al., 1985) (C,E,G,I). Clusters of labeled cells were considered as clones derived from a single sorted precursor cell, as illustrated in the overview in C,D. Cell-type specific antibodies were used as indicated in the panels to identify the composition of the clones. Pure neuronal clones were composed exclusively of b-tubulin-III-positive cells extending neurites marked by arrows (E,F). Neurons were generated in vitro and incorporated BrdU (red in B). An example of a non-neuronal clone generated from E14 precursors containing a GFAP-positive cell (filled arrowhead) is depicted in G,H. (I,J) A non-neuronal clone composed exclusively of GFAP-positive astrocytes generated by cells sorted from E18 cortex. Patterns of embryonic neurogenesis
Neural tube E11-12 Neural tube E11-12 ~E14-E15 ~E14-E15 Figure 2 | Patterning of the neural tube generates unique domains for neuronal and glial progenitors. a, The primitive neuroepithelium of the neural tube is patterned by organizing signals. These signals emanate from the ventral floor plate (such as SHH, purple) and roof plate (BMPs and WNTs, green). b, A cross-sectional view of the neural tube is shown. Progenitors of motor neurons and interneurons are formed within distinct regionally restricted domains of the ventral neural tube: the p0, p1, p2 and p3 domains for interneuron subtypes, and the pMN domain for motor neurons. Dorsal domains are also similarly parcelled (not shown). Signalling mediated by SHH (gradient denoted by purple circles) regulates the expression of transcription factors (for example, NKX2.2, OLIG2, PAX6 and SCL) in the ventral neural tube. The interactions of these factors sharpen and maintain the domain boundaries. Embryonic OPCs are derived mainly from the pMN domain. OPCs are recognized by expression of PDGFR-, SOX10 and NG2. Three astrocyte subtypes have been identified: VA1 astrocytes (which express PAX6 and reelin, derived from p1) are the most dorsal; VA3 astrocytes (which express NKX6.1 and SLIT1, derived from p3) are the most ventral; and VA2 astrocytes (which express PAX6, NKX6.1, reelin and SLIT1, derived from p2) are located in an intermediate white-matter domain. c, Organizing centres of the forebrain are shown. These include the cortical hem (purple), which is a dorsal source of BMPs and WNTs; a ventral centre (green), which is a source of SHH; and rostral (pink) and anti-hem (blue) regions, which are sources of growth factors such as FGF8, and transforming growth factor- (TGF-) and FGF7, respectively. d, A coronal view of the embryonic (~E14.5) forebrain showing its division into dorsal and ventral regions that are specialized for producing different neuron and glial-cell types. The dorsal region includes the cortex, a source of pyramidal neurons and astrocytes. The ventral region includes the lateral and medial ganglionic eminences and the pre-optic area, which are sources of GABA (-aminobutyric acid)-containing interneurons and oligodendrocytes. The green, blue and red shaded areas represent the pre-optic area, medial/lateral ganglionic eminences and neocortex, respectively (see Fig. 3). Transcription factors that are associated with dorsal (NGN1, NGN2, GLI3) and ventral (ASCL1, DLX1, DLX2) patterning and cell fate specification are indicated. NGN, neurogenin. Multiple types of embryonic neural progenitors
Similar to what you saw in the retina lecture, Interkinetic nuclear migration Neural progenitor cell not Neural Stem cell Symmetric vs. Asymmetric cell divisions! Several types of progenitors contribute to neurogenesis in the mammalian cortex [1214]. (a) At the beginning of neurogenesis (around E11E12 in the mouse), neuroepithelial cells located in the ventricular zone (VZ) and undergoing interkinetic nuclear migration either divide symmetrically to generate two new neuroepithelial cells or divide asymmetrically to generate either a neuroepithelial cell and a neuron, which migrates to the preplate (PP), or a neuroepithelial cell and a basal progenitor, which divides symmetrically on the basal side of the VZ (bVZ) to generate two neurons. (b) As neurogenesis progresses (around E13E17 in the mouse), several signalling pathways induce the expression of glial markers by neuroepithelial cells, which thus become radial glial cells. These cells also divide either symmetrically, to generate two radial glial cells, or asymmetrically, to generate a radial glial cell and either a neuron, which migrates through the intermediate zone (IZ) into the cortical plate (CP), or a basal progenitor, which moves to the subventricular zone (SVZ) and divides symmetrically to generate two neurons. MZ, marginal zone. Neural progenitor cell not Neural Stem cell Niches of adult neurogenesis
Newly generated NSCs TACs Fig. 1. Adult neurogenesis occurs primarily in the subventricular zone (SVZ) and subgranular zone (SGZ). In this sagittal view of the adult mouse brain, the neurogenic regions are indicated in blue. In the SVZ, stem cells (green) reside in the wall of the lateral ventricle, just below the ependymal layer (gray), and give rise to transit amplifying cells (light blue) and neuroblasts (purple). These neuroblasts migrate in chains in the rostral migratory stream (RMS) to reach the olfactory bulb (OB), where they mature into functionally integrated granule (peach) and periglomerular (red) neurons. In the SGZ of the hippocampal dentate gyrus (DG), stem cells (green) clustered near the base of the hippocampal DG granule cell layer (GCL) give rise to transit amplifying progenitors (light blue). These give rise to neuroblasts (blue), newborn neurons (purple), and eventually to immature (magenta) and mature (peach) granule cell neurons that primarily exist in the inner or hilar half of the GCL but extend their processes out to the molecular layer to receive cortical input. Note that SVZ neuroblasts migrate a relatively long distance to the OB to give rise to mature neurons, while SGZ progenitors move barely into the GCL to give rise to ma- ture neurons. NSCs TACs NBs Derived from VZ of cortex SVZ = Subventricular Zone, RMS = Rostral Migratory Stream, SGZ = Subgranular Zone of Dentate Gyrus OB = Olfactory bulb, NSC = neural stem cell, TAC = transient amplifying cells (progenitors), NB = neuroblast Modified from: Madeleine A. Johnson, Jessica L. Ables & Amelia J. EischCell-intrinsic signals that regulate adult neurogenesis.BMB Reports 2010 Mouse hippocampus development
FIGURE 1| Development of the mouse hippocampus. Schematic representation of the dorsal telencephalon at different embryonic (E )stages and at birth (P0). The indicated area in each picture corresponds to the hippocampal region and is magnified on its right hand side (bluesquares). (A) At E12.5 the presumptive DNE (dentate neuroepithelium) is located between the HNE (hippocampal meuroepithelium) and the CH (cortical hem), which produces Cajal-Retzius cells (orange), shown lining the pial side of the cortex. (B) At E14.5 dentate precursors of the primary matrix (dark blue circles) are located in the VZ, and precursor cells start to migrate towards the pial side of the cortex forming the secondary matrix. In the VZ of the HNE, radial glial precursors (depicted in dark blue and triangular body shape) will give rise to hippocampal neurons. (C) At E17.5 the hippocampal fissure is formed and dentate precursor cells migrate to and accumulate there, forming the tertiary matrix (light blue).Cajal-Retzius cells are also present and follow the hippocampal fissure. At this stage the glial scaffold (not shown) extends from the CH to the hippocampal fissure and pial surface, directing the migration of dentate precursor cells. From the HNE, hippocampal neurons (red triangles) are born and migrate along radial glial cells towards their location in the hippocampal fields (CA1 and CA3 are shown). (D) At birth the blades of the DG (dentate gyrus) start to form. Granule neurons in the DG (red triangles) appear first in the upper blade, below the hippocampal fissure. The continuous migration of Cajal-Retzius cells reaches the pial side and promotes the formation of the lower blade of the DG. Precursor cells in the primary and secondary matrix will soon disappear, but cells in the tertiary matrix continue actively dividing and producing granule neurons through postnatal DG development. HNE, hippocampal neuroepithelium; DNE, dentate neuroepithelium; CH, cortical hem; VZ, ventricular zone; 1ry, primary matrix; 2ry, secondary matrix;3ry, tertiary matrix; DG, dentate gyrus; D, dorsal; M, medial; V, ventral; L, lateral. HNE, hippocampal neuroepithelium DNE, dentate neuroepithelium CH, cortical hem VZ, ventricular zone 1ry, primary matrix 2ry, secondary matrix 3ry, tertiary matrix DG, dentate gyru D, dorsal; M, medial; V, ventral; L, lateral. Some differences between embryonic and adult neurogenesis Birthdating of progenitors
Inject retrovirus on specific day with GFP or other label (only labels dividing cells) Follow fate of labeled cells over time Can also use tamoxifen and Cre-ERT2 and a flox-stopped FP Can also use BrdU or EdU to label cell division. Can follow over days, weeks, months and then stain for "Birthdating marker" Adult neurogenesis Adult neurogenesis SVZ to OB Dentate Gyrus of hippocampus Summary of embryonic and adult neurogenesis
Protoplasmic or fibrous astrocytes DCX+ Nestin- GFAP- GLAST+ Nestin+ GFAP+ Pax6+ Tbr2+ Figure 1 | Patterns of gliogenesis in embryonic and adult progenitor zones. The progression from the embryo to the adult is shown from left to right (a to c). Black arrows indicate self-renewal or differentiation from one cell type to another. Markers of macroglia and their precursors are listed. a, Self-renewing neuroepithelial cells line the ventricles throughout the neuraxis at the stages of neural tube closure. These cells may generate some neurons. Neuroepithelial cells are transformed into radial glial cells as neurogenesis begins. b, Radial glia produce intermediate progenitor cells and oligodendrocyte precursor cells (OPCs), which in turn produce neurons and oligodendrocytes, respectively. Radial glia can also become astrocytes, as well as producing intermediate progenitors that expand in number before producing astrocytes. Protoplasmic astrocytes and fibrous astrocytes might arise from common or independent progenitors. Radial glia also produce ependymal cells. c, In adults, oligodendrocytes are produced by two independent pathways: type B cells in the cortical subventricular zone produce transit-amplifying cells (known as type C cells), which in turn produce OPCs as well as neurons. The OPCs subsequently generate oligodendrocytes, and OPCs that are already resident in the grey matter also produce oligodendrocytes. ALDH1L1, aldehyde dehydrogenase 1 family, member L1; APC, adenomatous polyposis coli; GFAP, glial fibrillary acidic protein; MBP, myelin basic protein; PDGFR-, platelet-derived growth factor receptor-; PLP, proteolipid protein 1. All green cells are intermediate progenitors, with type C cells being a subset of these, and all blue cells are neural stem cells (even though each blue cell is a different type). Subgranular zone hippocampus Nestin+, GFAP+ Pax6+ Blue cells - stem cellsGreen cells - intermediate progenitor cells Orange cells neuronal progenitors and neurons Modified from: Developmental genetics of vertebrate glial-cell specification.Rowitch DH, Kriegstein AR. Nature Nov 11;468(7321):214-22 Summary and ongoing questions
Symmetric vs. Asymmetric cell divisions Quiescence vs. proliferation Types of NSCs, SVZ vs. SGZ and others Regulation by Niche Regulation by hormones Regulation by exercise How do they mediate memory Why is there a decrease with aging Will they be useful for therapies For Thursday Read paper Do Figure Facts sheet
Be ready to discuss paper Let me know what paper you'll use for your term paper