Transcript Neural Stem Cell Biology

Neural Stem Cell Biology
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Postnatal neurogenesis discovery
Neural stem cell discovery
Embryonic NSCs
Adult NSCs
BCH/GGB512 Richard Gronostajski
History of postnatal neurogenesis
discovery
http://www.nature.com/nrn/journal/v1/n1/pdf/nrn1000_067a.pdf
History of postnatal neurogenesis
discovery
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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
http://braindevelopmentmaps.org
History of postnatal neurogenesis
discovery
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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 celltype markers
1999 Rakic showed neurogenesis with BrdU incorporation
and cell-type specific markers.
Postnatal neurogenesis
Neurobiology: Kornack and Rakic Proc. Natl. Acad. Sci. USA 96 (1999) 5771
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 TuJ1positive cell (green, arrow) with a BrdU-positive nucleus
(orange) has an immature migratory appearance. Note the
thin trailing process (arrowheads) and a nearby BrdUnegative 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.]
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 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. Neurospheres
(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 idenitify
the 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.
Neural Stem Cells in vitro
FIG. 1. Morphology of neurons
generated by culturing adult
brain cells with bFGF and then
with medium conditioned by Ast1 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. (af 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.
What is the evidence?
A
Summary of embryonic and adult
neurogenesis
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Protoplasmic or fibrous astrocytes
DCX+
Tbr2+
Nestin+, GFAP+
Pax6+
NestinGFAPGLAST+
Nestin+
GFAP+
Pax6+
Gliogenic
Switch
Subgranular
zone
hippocampus
Blue cells - stem cells Green cells - intermediate progenitor cells
Orange cells – neuronal progenitors and neurons
Modified from: Developmental genetics of vertebrate glial-cell specification. Rowitch DH, Kriegstein AR.
Nature. 2010 Nov 11;468(7321):214-22
Evidence
GFAP-GFP transgene expression
E16
E14
GFAP-GFP transgene is
expressed in GLAST+ cells
that form radial pattern
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 material
Types of colonies made in vitro
Filled arrowheads indicate double-labeled cells,
open arrowheads indicate single-labeled cells in
corresponding micrographs. Note that GLASTpositive precursor cells generate neurons and
astrocytes in two separate lineages. Scale bars:
25um.
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 mousespecific 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
~E14-E15
Neural tube E11-12
~E14-E15
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!
Neural progenitor cell
not Neural Stem cell
Niches of adult neurogenesis
Newly generated
NSCs
TACs
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. Eisch Cell-intrinsic signals that regulate adult neurogenesis.
BMB Reports 2010
Mouse hippocampus development
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
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
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B
C
Protoplasmic or fibrous astrocytes
DCX+
Tbr2+
NestinGFAPGLAST+
Nestin+
GFAP+
Pax6+
Nestin+, GFAP+
Pax6+
Subgranular
zone
hippocampus
Blue cells - stem cells Green cells - intermediate progenitor cells
Orange cells – neuronal progenitors and neurons
Modified from: Developmental genetics of vertebrate glial-cell specification. Rowitch DH, Kriegstein AR.
Nature. 2010 Nov 11;468(7321):214-22
Quiescent vs. active Neural Stem Cells
Disposable Hippocampal Neural Stem Cells
Multiple Neural Stem Cell models
Summary and ongoing questions
1. Symmetric vs. Asymmetric cell divisions
2. Quiescence vs. proliferation
3. Gliogeneic Switch and "Disposable SCs"
4. Types of NSCs, SVZ vs. SGZ and others
5. Regulation by Niche
6. Regulation by hormones
7. Regulation by exercise
8. How do they mediate memory?
9. Why is there a decrease with aging?
10. Will they be useful for therapies?
For Next Tuesday
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Read paper
Do Figure Facts sheet
Be ready to discuss paper
Let me know what paper you'll
use for your term paper
• Next Thursday, we'll go from
1-3PM, 10 minutes each
paper, 8 presentation, 2 for
discussion, unless.....