Biology of Stem Cells

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Biology of Stem Cells
Scott Zeitlin, Ph.D. Department of
Neuroscience soz4n_at_virginia.edu 924-5011
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Neuronal Stem Cells

• Multipotent self-renewing progenitor cells
• CNS neurons, astrocytes, oligodendrocytes
• PNS neurons, Schwann cells, neural crest
  derivatives (smooth muscle cells)

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Gage review, Science 2000
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Mouse ES cells growing on a layer of primary
fibroblasts
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ES cells maintenance of the undifferentiated
phenotype
JAK kinase
Y
Y
Y
STAT3
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Neuronal differentiation of ES cells

• Remove LIF, culture as embryoid bodies, then
  treat with RA
• Grow in defined medium supplemented with FGF2,
  followed by withdrawal from FGF2 and plating on a
  laminin-coated substrate
• Grow in the presence of Shh and FGF8 (generates
  predominantly dopaminergic neurons)

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ES cells grown in suspension form embryoid bodies
that recapitulate early embryonic development
Day 4
Day 10
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Neuronal differentiation of ES cells

• Grow ES cells at low density in defined medium on
  a non-adhering substrate
• Grow ES cells at low density in defined medium in
  the presence of LIF

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Neuronal differentiation a default state for ES
cells?
Tropepe et al. Neuron 3065-78 (2001)
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ES cell derived neural sphere colonies
differentiate into neurons and glia and can
contribute extensively in mouse chimeras
Tropepe et al. Neuron 3065-78 (2001)
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Neurogenesis in the adult songbird
brain(Goldman, S.A. and Nottebohm, F. PNAS
802390-94 1983)

• Occurs within an area of the dorsomediocaudal
  neostriatum involved in song learning the HVC
  (Higher Vocal Center)
• New neurons are generated in the
  ventricular/subventricular zone and migrate into
  the forebrain where they differentiate into
  functional neurons (both projection and
  interneurons)
• The adult canary HVC recruits over 1.4 of its
  neurons daily. Hormones regulate this process
• Avian radial glial cells may be the progenitor
  cells for both radial glia and neurons

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Goldman S.A., J. Neurobiol. 1998 267-86.
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Garcia-Verdugo et al., J. Neurobiol. 36 234-48
(1998)
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SVZ markers

• Musashi 1 (RNA binding protein)
• Nestin (intermediate filament protein)
• PSA-NCAM (cell adhesion)
• Vimentin (intermediate filament protein)
• Tenascin (extracellular matrix protein)
• GFAP (glial fibrillary acidic protein)
• TGFa?(growth factor)
• IGF-1 (growth factor)
• EGF-R (growth factor receptor)
• TrkB (growth factor receptor)
• TuJ1 (neuronal marker)

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IHC with anti-PSA-NCAM Ab
Garcia-Verdugo et al., J. Neurobiol. 36 234-48
(1998)
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Differentiated outgrowths from a neurosphere
RedTuJ1
GreenGFAP
Svendsen et al., J. Neurosci. Meth. 85 (1998)
141-52
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Svendsen et al., J. Neurosci. Meth. 85 (1998)
141-52
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Identity of the stem cell in the adult SVZ
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Stem cells observed in situ in the adult rodent
brain
F. Gage, Science 287, 2000
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Ependymal cells are stem cells?
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GFAP
bIII-tub
neurosphere
O4
ciliated ependymal cell
BrdU
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Transplantation into hippocampus
Transplantation into the rostral migratory stream
From Suhonen et al., Nature 383, 624 (1996)
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Isolation of endogenously proliferating cells
from the adult rat brain Palmer et al., J.
Neurosci. 198487-8497 1999
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The influence of FGF-2 on adult NSCs
Palmer et al. J. Neurosci.198487-97 (1999)
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Notch/delta signaling in NSCs
Notch 2 and DG
Notch 1 and 2 mRNAs are present in the adult
germinal zones Notch 1 is also expressed in
neurospheres Irvin, D.K. et al. J. Comp. Neurol.
436167-181 (2001)
Notch 1 and the SVZ
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NSCs express Notch receptors and variable levels
of delta ligand
Undiff.
Undiff.
Undiff.
delta ligand
Notch receptor
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Activation of Notch receptors in neighboring
cells induces a signal transduction casacade
either maintaining the cells in the
undifferentiated state or inducing gliogenesis
Undiff.
Undiff. or glial
neuronal
delta ligand
Notch receptor
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FGFs (directly or indirectly) active Notch signal
transduction in neurosphere cultures resulting in
maintenance of the undifferentiated state
Undiff.
Undiff.
delta ligand
Notch receptor
FGFR
FGF
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Noggin expression in the subependymal cells may
help maintain a population of neuronal stem cells
in the SVZ
Lim et al. (2000) Neuron 28 713-726
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PNAloHSAhi adult NSCs expanded in vitro can give
rise to neurons, astrocytes, oligodendrocytes,
and muscle cells upon differentiation. Rietze et
al., Nature 412736-9 2001
neurosphere
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Prospective isolation of human NSCs

• Prepare a single cell suspension of human fetal
  spinal cord or brain tissue
• Stain cells with an antibody to CD133, a
  transmembrane protein that is found on human
  hematopoietic stem cells
• Enrich for CD133 cells by FACS
• Expand in neurosphere culture

Uchida, N. et al. PNAS 9714720-14725 (2000)
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Uchida, N. et al. PNAS 9714720-14725 (2000)
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Transplantation of purified HNSCs into the
lateral ventricles of SCID mice (analyzed 7
months after engraftment)
HNA
Ki-67
HNAKi-67GFAP
HNATH
H-NCAM
HNA?uJ1
Uchida, N. et al. PNAS 9714720-14725 (2000)
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hNSCs injected into the embryonic monkey brain
LVs migrate from the VZ into the developing
neocortex. Brownvimentin (radial
glia) BlackBrdU labeled nuclei of hNSC-derived
cells Ourednik V et al. Science 2931820-24
(2001)
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hNSCs injected into the lateral ventricles of the
developing monkey brain segregate into two
populations (1) differentiated cells and (2)
cells colonizing adult germinal zones that retain
progenitor cell characteristics
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• Papers for discussion
• E. Gould, N. Vail, M. Wagers, and C.G. Gross
  (2001) Adult-generated hippocampal and
  neocortical neurons in macaques have a transient
  existence. PNAS 9810910-10917.
• D.R. Kornack and P. Rakic (2001) Cell
  proliferation without neurogenesis in adult
  primate neocortex. Science 294 2127-2130.
• P. Rakic (2002) Adult neurogenesis in mammals an
  identity crisis. J. Neurosci. 22 614-618.
• E. Gould and C.G. Gross (2002) Neurogenesis in
  adult mammals some progress and problems. J.
  Neurosci. 22 619-623.

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