A Brief History of Neural Stem Cells

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INT 94JY Getting New Brain Cells Neurogenesis in
the Adult Brain Seminar 5 Adult Neurogenesis.
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Blood (hematopoietic) Stem Cells
Individual cells in bone marrow can repopulate
blood system. -proliferate, -multipotential,
-last lifetime. -Unique gene expression profile
Proof of Principle Cancer (leukemia) treament
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Early/Basic Ideas of Brain Repair

• Structures of the brain are determined shortly
  after birth.
• Damage to the brain produces irreversible
  deficits.
• Post-morten analyses shows that neurons do not
  grow back into damaged areas get glial scarring.
  
• Thus, there is likely no neurons produced in the
  adult brain.

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Cajals Nervous System circa 1900 Neuron
Doctrine -Individual discrete cells -Cells
communicate by contact -No new neurons after birth
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Adult Neurogenesis Discovered?

• 1962-, Joseph Altman publishes a series of
  reports that regions of the adult mammalian brain
  incorporated tritiated thymidine.

Process Tissue and Film.
Inject Thy-H3
Labeled-Cells in Olfactory Bulb Hippocampus Cereb
ellum
-gtReceive little attention due to technical
limitations (Dogma withstands)
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Reasons for Altmans research being ignored.

• Relied on morphological analyses to determine if
  neurons or glia had incorporated thymidine.
• Limited microscopic techniques.
• Dogma was supported by developmental studies.

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Adult Neurogenesis Re-Discovered!

• In the 1990s a number of studies established the
  validity of Altmans early findings.
• Importantly, these newer studies were able to
  conclusively show that new neurons were being
  formed in the adult mammalian brain.
• Required sophisticated technical developments
  that were not previously available.

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Improved Methods Verified Adult Neurogenesis

• Immunocytochemstry and bromo-deo-xyuridine
  labeling
• Viral-labeling of proliferating cells
• Combine with neuron specific markers
• Confocal microscopy colocalization in 3-D.

Neurogenesis confirmed in adult brain of rats,
primates, and humans.
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Detection of in vivo proliferation via DNA
Labelling
1 hr
Inject thymidine analog BrdU
Stain for BrdU in Tissue Sample
Fluorescent Microscopy
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Survival of BrdU-labeled Cells
30 d
BrdU NeuN
Inject thymidine analog BrdU
Stain for BrdU in Tissue Sample
Fluorescent Microscopy
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Parallel Paths in Olfactory Bulb
Sensory Input

• Distinct Glomeruli have parallel projections to
  the higher areas of the brain (ie piriform
  cortex, amygdala).
• Granule cells serve to produce lateral inhibition.

Neurogenesis in these populations
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Adult-born neurons in OB Periglomerular cells
GABA marker
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Adult-born neurons in OB Granule Cells
Dopamine marker
TH
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Neurogenesis in the Olfactory Bulb

• Olfactory bulb neurogenesis path is well
  established
• Proliferation in subventricular zone
• Migration in rostal migratory stream
• Differentiation into interneurons in olfactory
  bulb.

Where would you expect a neural stem cell?
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Stem cells in the adult brain
Mouse
The Neurosphere Assay for Neural Stem Cells
Reynolds Weiss, 1992
Proof of Principle Transplant repopulation
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Neural Stem Cell Reside Only in the SE
Seaberg van der Kooy. J Neurosci. 2002
22(5)1784-93.
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Location of Neural Stem Cells
-Brain is hollow with the center filled with
fluid. Ventricles -Stem cells reside in
lining of the ventricles subventricular zone
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SVZ NSC Morphology

• EM microscopy to identify all cells types in the
  subventricular zone
• Three of which incorporated H3-thymidineA,
  B, C type.
• Doetsch et al., 1997 1999

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Organization of the Adult Germinal Zone Doetsch
et al., 1997 and 1999
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Identity of NSCs - Astroglia?

• Morshead et al 2003.
• Transgenic mice expressing thymidine kinase under
  GFAP promotor ganciclovir (GCV) kills
  proliferating GFAP cells
• What about neurospheres?
• Does GFAP astrocyte NSC?

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Elimination of proliferating GFAP cells
eliminates Dg and OB neurogenesis.
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Identification of Neural Stem Cells
NSC Markers based of FACS Nestin Mushashi GFAP
LEX ABC Transporter (Hoetsch exclusion) -only
enrichment none purify (i.e. 100 NSCs)
therefore no definitive markers
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Repopulation of SE by neurosphere-forming cells

• Morshead et al 1994
• Mice are treated with high dose of Thy-H3 that
  is lethal to proliferating cells either once or
  twice (2 or 4 days apart).

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Regulation of NSCs by Growth Factors in vivo

• Craig et al., 1996.
• Direct infusion of epidermal growth factor (EGF)
  or basic fibroblast growth factor (bFGF) into
  lateral ventricle.

BrdU- labeling
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Life as a Neural Stem Cell
-Initially derived directly from toti-potent ES
cells by default lineage restriction at embryonic
day 5-7 (Tropepe et al. 2001 Hitoshi et al.,
2004). -Expansion of population until birth
(Martens et al. 2000). -Population remains
constant from birth until mid-adulthood (Kippin
et al., 2004 2005 Seaberg et al. 2005).
-Population declines in late adulthood (Kippin
et al. 2004 2005 Maslov et al. 2004 Enwere et
al. 2004). -Population is multipotential but
relatively quiescent cell cycle time of about 2
weeks (Morshead et al. 1994).
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Neural Stem Cell are Derived from ES cells via
Default Pathway.
What about life as an adult neural stem cell
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Cell Cycle Control and p21
cdc2
CDK2
Cyclin A
Cyclin A
G2
cdc2
S
Cyclin B
M
CDK2
Cyclin E
G1
G0
p21 p27 p57
p15, p16 p18, p19
CDK4-6
Cyclin D
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p21 -/- and / NSCs Burn out or fade away
Kippin et al. (2005) Genes Development
19756-767.
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Exhaustion of NSC proliferative capacity in
vitro in the absence of p21.
NSCs that live fast die young.
Kippin et al. (2005) Genes Development
19756-767.
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Neurogenesis in the Dentate Gyrus of the
hippocampus.

• Dentate gyrus neurogenesis
• is well established
• Proliferation in subgranular zone
• Migration into dentate gyrus
• Differentiation into neurons with inputs from
  entorhinal cortex and outputs to CA3.

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Developmental Profile of DG neurogenesis
DG
SGZ
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DG NG time course
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Adult-born neurons in DG are (electrically)
functional!
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Immature and mature neurons in DG

• Immature neurons are more responsive than older
  neurons.
• Lower threshold.
• Higher max firing.
• Excited by GABA

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Neural Stem Cell Reside Only in the SE
Seaberg van der Kooy. J Neurosci. 2002
22(5)1784-93.

• Interpretations
• No NSC (precursors migrate)
• Different NSC

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Cerebellum
Limited evidence for neurogenesis in adult
cerebellum
Thus, although Altman turned out to be correct
about adult neurogenesis, his critics were also
correctneed for scientific skepticism!!!
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Neocortex (Gould et al 1999 2001)
Reported co-localization of BRDU and neural
markers in primate cortex.
NeuN BrdU DAPI
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Neocortex (Kornack Rakic, 2001)
Failed to find any co-localization of BRDU and
neural markers (some co-localization with glial
markers).
NeuN BrdU GFAP
Controversy of these data continues but mostly
there is limited support for adult neurogenesis
in cortex.
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Neo- Paleo-Cortex
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The extent/limits of neurogenesis

• Field is still defining where in the adult brain
  new neurons are produced and their function

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Limbic System
Limbic system is a series of forebrain structures
that are critically involved in learning and
memory and emotional responses.
Includes structures with the highest amount of
adult neurogenesis the hippocampus and olfactory
bulbs.