Postnatal neurogenesis discovery Neural stem cell discovery Embryonic NSCs Adult NSCs BCHGGB512 Richard Gronostajski History of postnatal neurogenesis discovery http wwwnaturecom nrn ID: 497146
Download Presentation The PPT/PDF document "Neural Stem Cell Biology" is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
Slide1
Neural Stem Cell Biology
Postnatal neurogenesis discoveryNeural stem cell discoveryEmbryonic NSCsAdult NSCs
BCH/GGB512 Richard GronostajskiSlide2
History of postnatal neurogenesis discovery
http://
www.nature.com
/
nrn
/journal/v1/n1/
pdf
/nrn1000_067a.pdfSlide3
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)Slide4
History of postnatal neurogenesis discovery
Injected P20+21, harvested P60
http://
braindevelopmentmaps.orgSlide5
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.
1985-88
Pasko
Rakic's
papers found "little or no" adult neurogenesis.
1988 Stanfield and Trice showed adult neurogenesis with fluorescent tracer + 3H
TdR
1997-99 Fred Gage and coworkers used BrdU and cell-type markers
1999
Rakic
showed neurogenesis with BrdU incorporation and cell-type specific markers.Slide6
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. (a–d) 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 (arrow–cross). (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 (arrow–cross). [Bar (a–l) 5 10 mm.]
Neurobiology:
Kornack
and
Rakic
Proc. Natl. Acad. Sci. USA 96 (1999) 5771Slide7
Major reasons for 40 year delay
Lack of good markers for both proliferation and cell types.Bias against the idea.Slide8
Neural Stem Cells in vitroSlide9
Neural Stem Cells in vitro
Fig. 1. EGF-induced proliferation of cells isolated from the adult mouse striatum.
(A)
After 2 DIV, cells that had undergone 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 8 DIV, spheres of cells lifted off the substrate and floated in suspension. Line in substrate (A through C) serves to identify 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 were
immunoreactive
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 cell after 2DIV
(G)
began to divide and form cluster of cells during the subsequent
3
(H)
, 4
(I)
, and 6
(J)
DIV.
Scratches
in
substrate serve to idenitifythe field. Scale bars: (A through D) bar in (D) denotes 50 um (E), 50 um; (F), 25 um; (G through J) bar in (J), 50 um.
NeurospheresSlide10
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 (arrows in 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.)Slide11
Neural Stem Cells in vitroSlide12
Neurosphere assay
Primary neurospheres may measure stem and progenitor cells. Initial passage.Secondary neurospheres may measure stem cells. Second passage.Assay controversial, spheres split or merge, best to make at limiting dilution.Slide13
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. What is the evidence?Slide14
Modified from:
Developmental genetics of vertebrate glial-cell specification. Rowitch DH, Kriegstein
AR. Nature. 2010 Nov 11;468(7321):214-22
Blue cells
- stem cells
Green cells
- intermediate progenitor cells
Orange cells
– neuronal progenitors and neurons
Nestin
-
GFAP
-
GLAST
+
Nestin
+
, GFAP
+
Pax6
+
Tbr2
+
Nestin
+
GFAP
+
Pax6
+
DCX
+
Subgranular zone
hippocampus
Summary of embryonic and adult neurogenesis
Protoplasmic or fibrous astrocytes
A
B
C
Gliogenic
SwitchSlide15
EvidenceSlide16
GFAP-GFP transgene expression
E16
E14
GFAP-GFP transgene is expressed in GLAST+ cells that form radial patternSlide17
Sorted GFP+ and put in culture
GFAP-GFP+ cells made neurons, glia and mixed colonies when put into culture of 5-7 days. Some contaminating neurons present in starting materialSlide18
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.
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.
Types of colonies made
in vitroSlide19
Patterns of embryonic neurogenesis
Neural tube E11-12
Neural tube E11-12
~E14-E15
~E14-E15Slide20
Multiple types of embryonic neural progenitors
Similar to what you saw in the retina lecture,
Interkinetic
nuclear migration
Symmetric vs. Asymmetric cell divisions!
Neural progenitor cell
not Neural Stem cell
Neural progenitor cell
not Neural Stem cellSlide21
Niches of adult neurogenesis
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.
Eisch
Cell-intrinsic signals that regulate adult neurogenesis.
BMB Reports 2010
NSCs
TACs
NBs
NSCs
TACs
Derived from VZ of cortex
Newly generated Slide22
Mouse hippocampus development
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.Slide23Slide24
Some differences between embryonic and adult neurogenesisSlide25
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"Slide26
Adult neurogenesisSlide27
Adult neurogenesis
SVZ to OB
Dentate Gyrus of hippocampusSlide28
Modified from:
Developmental genetics of vertebrate glial-cell specification. Rowitch DH, Kriegstein
AR. Nature. 2010 Nov 11;468(7321):214-22
Blue cells
- stem cells
Green cells
- intermediate progenitor cells
Orange cells
– neuronal progenitors and neurons
Nestin
-
GFAP
-
GLAST
+
Nestin
+
, GFAP
+
Pax6
+
Tbr2
+
Nestin
+
GFAP
+
Pax6
+
DCX
+
Subgranular zone
hippocampus
Summary of embryonic and adult neurogenesis
Protoplasmic or fibrous astrocytes
A
B
CSlide29
Quiescent vs. active Neural Stem CellsSlide30
Disposable Hippocampal Neural Stem CellsSlide31
Multiple Neural Stem Cell modelsSlide32
Summary and ongoing questions
Symmetric vs. Asymmetric cell divisionsQuiescence vs. proliferationGliogeneic Switch and "Disposable SCs"
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?Slide33
For Next Tuesday
Read paperDo Figure Facts sheetBe ready to discuss paperLet me know what paper you'll use for your term paperNext Tuesday and Thursday, we'll go from 1-2PM, 20 minutes each paper, 15 presentation, 5 for discussion.