Cardiac channelopathy and functional study

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Young at heart Evidence for cardiomyocyte
renewal in humans

• Carbon14-dated age of cardiomyocytes
• Cardiomyocytes renewed with a gradual decrease
  from 1 turning over annually at the age of 25,
  to 0.45 at the age of 75. Fewer than 50 of
  cardiomyocytes are exchanged during a normal life
  span

Bergmann et al. 2009, Science
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Nuclear weapons and neurogenesisno more cortical
neurons in normal adults
How about after stroke ?
Bhardwaj et al. 2006, PNAS
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Barriers restrain CNS regeneration

• Neuronal death
• Glial cells inhibit nerve growth
• Most neural stem cells are constrained
• Few exceptions in lower vertebrates and in
  olfactory pathway and hippocampus in mammals

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Plasticity of adult human brain
Ward NS et al., Brain, 2003
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Spontaneous changes in brain after stroke
Cramer, 08, Annals of Neurology
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Consequence of hypoxia-ischemia

• Features of energy metabolism in brain
• High metabolic rate
• Limited intrinsic energy stores
• Dependence on aerobic metabolism of glucose
• Within seconds, ATP level fall, lactic acidosis
• ? ion pumps dysfunction (Na/K ATPase)
• ? rundown of transmembrane ion gradients
• ? membrane depolarization
• ? voltage-sensitive ion channels open
• ? K efflux after a min, Na, Ca ion gradient lost
  and influx
• ? Ca overload enhance glutamate release
• ? cell death

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Ischemic apoptosis in parallel with necrosis

• Deprivation of growth factor support
• Addition of NGF or bFGF reduce injury
• Oxidative stress
• Antioxidants glutathione, superoxide dismutase
  (SOD)1
• Prolonged deficits in energy metabolism
• Increased inflammatory cytokines
• IL-1?, TNF?, TGF?
• Transgenic mice overexpressing bl2 had smaller
  infarcts

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Penumbra
Lo, 2008
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Recent therapeutic developments of stroke

• Prevention
• Acute treatment
• rt-PA thrombolysis
• even for minor stroke (Gonzales et al. 2006)
• Neuroprotection
• Ebselen, anti-inflammatory selenocomound mimic
  glutathione
• NXY-059, antioxidant nitrogen spin trap
• Stem cell transplantation
• G-CSF, endogenous stem cell-mobilizing agent
• phase I for acute MCA stroke by Shyu et al., 2006
• Rehabilitation
• Transcranial magnectic or direct current
  stimulation
• Hummel Cohen 2006 Review

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tPA for brain attack !

• In US, 50 pt arrive at ER lt 1h
• In Taiwan, 50 pt arrive at ER lt 4h
• NINCDS trial in 1990s 13 more favorable
• Symptomatic ICH rate
• (no protocol violation) 4 (violation) 8
• lt 3 of patients currently treated in US
• lt 2 of patients currently treated in Taiwan
• Increase likelihood of return to normal by 30

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Carotid endarterectomy or stent angioplasty

• Severe (gt 70) carotid stenosis in symptomatic
  patients
• Moderate (50-69) stenosis depend on age,
  comorbidities, symptoms
• For asymptomatic patients, only recommend for gt
  80 carotid stenosis

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Types of cell therapy for CNS diseases

• Peripheral nerve grafts (Schawann cells)
• Olfactory ensheathing glia
• Stem/ progenitors cells
• Adult bone marrow mesenchymal stem cells
• Induced pluripotent stem (iPS) cells from skin
  fibroblasts
• key pluripotency genes Oct-4, SOX2, c-Myc, Klf4
• Endogenous neural progenitor cells

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Critical issues for stem cell therapy

• What is a good source of stem cells for neural
  repair ? (effective obtainable)
• What factors stimulate stem cells to home
  (migrate) to sites of injury?
• What cues are needed for stem cells to
  differentiate into the desired cell type?
• Limited graft survival
• Avoid transplanting undifferentiated cells that
  cause tumors (purification)

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Astrocyte-like stem cells in subventricular zone
Ependymal cells
Slowly dividing GFAP
Johansson et al., Cell, 9625-34, 1999. Doetsch
et al., Cell, 97703-16, 1999.
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Endogenous neuronal replacement after stroke in
adult rats
No neurogenesis in the cortex! Arvidsson et
al., Nat Med 2002
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Expanding endogenous stem cells

• Adult NG2 oligodendrocyte precursors
• generate functional neurons and glia in vitro
• After selective elimination of cortical neurons,
  some endogenous neural precursors proliferated
  and incorporated BrdU, migrated to the injury
  site, most are glia
• Infusion of growth factors
• Epidermal growth factor
• Sonic hedgehog
• Antagonize inhibitory micro-environment

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De-differentiation reprogram to youth

• Irreversible differentiation and germ layer
  restriction can be broken by extracellular cues,
  especially following injury and in cell culture
• First evidence of de-differentiation in
  vertebrate cells came from studies of urodeles
  and avians
• Regenerated limb following limb amputation in
  urodeles (Brockes 1997, Brockes Kumar 2002)
• Newly formed lens after resection in salamander
  (Eguchi Kodama 1993, Stone 1967)

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De-differentiation in mammals

• Schwann cells de-differentiated into precursors,
  proliferate, and redifferentiate after peripheral
  nerve injury (Brockes Kumar 2002)
• Oligodendrocyte precursor cells can produce
  neurons, astrocytes, and oligodendrocytes in
  vitro (Kondo Raff 2000)
• Precursor-to-stem cell conversion
• In intestinal crypt (Marshman et al. 2002)
• EGF convert transit-amplifying precursors in the
  adult brain into stem cells (Doetsch et al. 2002)

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Bone marrow stem cells not restricted to the
blood cell lineage

• Adult bone marrow stem cells (BMSC) give rise to
  cells of all three germ layers, including neural
  cells in vitro
• Some transplanted BMSC in the brain had
  characteristics of macrophage/microglia,
  astrocytes, neurons?
• Tissue injury may recruit BMSC to additional cell
  types
• mdx muscular dystrophy mice BMSC? myocytes

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Immunosuppressive properties of mesenchymal stem
cells
Nauta and Fibbe. 2007, Blood
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CNS entry of peripherally injected umbilical cord
blood cells is not required for neuroprotection
in stroke
Borlongan et al., 2004, Stroke
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Brain cells may be derived from non-neural stem
cells ?

• Transdifferentiated cells from one primary germ
  lineage to another ?
• Bone marrow SC give rise to astrocytes
• Umbilical cord SC form neurons and glia
• Adult NSC reconstitute hematopoietic system
• Controversies concerning plasticity of bone
  marrow and hematopoietic SC (Castro et al., 2002
  Wagers et al., 2002)
• Few differentiated cells present in the graft,
  most derived from cell fusion most suggest
  trophic supply that facilitate endogenous repair
  process

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Neural stem cells differ from those in other
systems

• Unlike hematopoietic system, the nervous system
  is largely formed during early development
• Unlike epithelial system, neurons form intricate
  connections over long distances

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Neuron-like morphology?

• Reevaluation of in vitro differentiation of bone
  marrow stromal cells Disruption of actin
  cytoskeleton induces rapid morphological changes
  and mimics neuronal phenotype (Neuhuber et al.,
  2004)
• Its unclear if neurons can be differentiated
  from any source other than neural stem cells and
  ES cells in vivo

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Grafted neural stem cells develop into functional
pyramidal neurons
Englund et al., 2002
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Induced Pluripotent Stem Cells (iPS)

• Promising alternative of embryonic stem cells
  (ES), Overexpression of OCT4, SOX2, KLF4 and
  C-MYC, or Nanog and Lin 28 (Takahashi and
  Yamanaka, 2006 Okita et al., 2007 Wernig et
  al., 2007 Yu et al., 2007)
• Chimera or differentiate into 3 germ layers (Park
  et al., 2007 Takahashi et al., 2007)
• Disease-specific iPS provide resources for
  disease investigation and drug development (Park
  et al., 2008)
• iPS cells from patients with ALS can be
  differentiated into motor neurons (Dimos et al.,
  2008)
• Transplantation of neural precursor cells derived
  from iPS cells functional integration in the
  fetal rat brain, enhance functional recovery in
  Parkinsonism rats (Wernig et al., 2008)

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A fresh look of iPS
Yamanaka, 2009, Cell
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Mechanisms of cell therapy-mediated recovery

• Trophic factors and reduce death of host cells
• VEGF, GDNF, FGF, BDNF
• Increased neovascularization
• Attenuation of inflammation
• T- cells inhibition
• Induce host plasticity
• Neurogenesis
• Recruitment of endogenous progenitors
• Neuronal replacement and functional integration

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Adverse effects of cell therapy

• Cell-related
• Tumorigenic
• Graft rejection
• Allodynia (Hofstetter et al. 2005)
• Procedure-related
• Intracranial hemorrhage during stereotactic
  transplantation without general anesthesia
• Infection

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Post-stroke neuroplasticity

• Corticospinal excitability decreased at the
  lesion
• Weakened intracortical inhibition and
  facilitation in the ipsilesional hemisphere

Swayne et al., Cerebral cortex, 2008
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Strategies of brain stimulation after stroke
Hummel and Cohen, 2006, Lancet Neurology
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After-effects of repetitive transcranial
stimulation

• Short-term effects (minutes)
• Shifting ionic balance
• Short-term synaptic plasticity
• Neurotransmitter release
• Long-lasting effects (hours)
• Long-term depression (LTD)
• and potentiation (LTP)
• Induced gene expression
• Regulation of postsynaptic receptors (ex AMPA,
  NMDA, GABA)

Ridding Rothwell, Nature Rev, 2007