Modulation of StemProgenitor Cells in CNS Pathology: Current and Future Project Overview

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Modulation of Stem/Progenitor Cells in CNS
Pathology Current and Future Project Overview

• Heather H Ross, PhD, MPT
• Department of Physical Therapy
• K12 Annual Advisory Meeting
• May 19, 2009

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Professional Background
East Carolina University
1998-2000 Department of Physical
Therapy Virginia Commonwealth University
2000-2006 Mentor Helen L. Fillmore,
Ph.D. Dissertation Title Identification of a
novel human MT5-MMP transcript in multipotent NT2
cells University of Florida
2006-2009 Postdoctoral Supervisor Eric D.
Laywell, Ph.D. Area of Study Role of thymidine
analogs in stem/progenitor cells/cancer
cells K12 Scholar January
2009-Present Mentors Drs. Behrman, Laywell and
Steindler
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Agenda

• Thymidine Analogs as Anti-proliferative Agents
  in Neural and Cancer Stem/Progenitor Cells
• Background
• Data Summary
• Future Directions
• Hypoxia-Driven Modulation of Neural
  Stem/Progenitor Cell Transplant Following
  Cervical Spinal Cord Injury
• Background
• Proposed Specific Aims

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Death of a Dogma
The adult mammalian brain harbors multipotent
stem cells, which reside and participate in
specialized niches that support self-renewal and
differentiation. -Doetsch, 2003
In adult centers the nerve paths are something
fixed, ended, immutable. Everything may die,
nothing may be regenerated. It is for the science
of the future to change, if possible, this harsh
decree. -Santiago Ramón y Cajal
Gould et al., 1992 Cameron et at., 1993 Gould et
al., 1997 Kornack Rakic, 1999
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Basic Definition of a Stem Cell

• Stem Cell A single cell that possesses two key
  features
• Self-renewal ability to maintain division over
  time to retain a stem cell pool
• Multipotency ability to generate multiple cell
  types

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The Neurogenic Niche SVZ and SGZ
Vescovi et al., 2006
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Generation of Multipotent SVZ Neural Progenitor
Cells Neurospheres
BL6 neonates (P4-8)
GFAP ß-III Tubulin DAPI
Plate Neurosphere Forming Cells (NFC)
Neurosphere Assays
Count and Measure Floating NS
Stain Attached for Markers of terminal differentia
tion
BrdU
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Summary Project Overview
Modulation of Stem Cell Biology
Hypoxia
Thymidine Analogs
Target Neural Stem Cell Transplant Population
Target Cancer Stem Cell
Optimize Transplant Environment in
Combination With Rehab Strategies
Translational Benefit of Stem Cell Therapeutics
Ameliorate Primary and Recurrent Brain CA
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Thymidine Analogs - BrdU and EdU
Thymidine
BrdU
Br
EdU (5-ethynyl-2'-deoxyuridine)
Adapted Slide courtesy of Lindsay Levkoff
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TA Treatment Yields Neurospheres with Altered
Morphology and Multipotency
Ross et al., 2008
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TA Treatment Yields Neurospheres with Reduced
Self-Renewal
Ross et al., 2008
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Brain Cancer and the Cancer Stem Cell

• 52,000 new primary brain tumors
• diagnosed each year
• Multiple types of brain tumors
• Primary tumors and their recurrence
• Metastatic disease
• Benign vs. Malignant
• High grade gliomas (glioblastoma, GBM) account
  for 52 of all primary cases
• Mean Survival lt2 years.
• Cancer Stem Cell May represent a therapeutic
  target

www.frommedskool.com
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Generation of Human GBM-derived Neurospheres
www.frommedskool.com
Tumor Resection and Collection from O.R.
Plate sphere Forming Cells
Neurosphere Assays
Count and Measure Floating NS
Stain Attached for Markers of terminal differentia
tion
Nestin CD133
BrdU
Singh, et al., 2003
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TA Treatment Results in Decreased Tumor Mass and
Enhanced Animal Survival
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Ongoing and Future Studies

• Mechanism of action how do TA alter cell cycle
  dynamics?
• In vivo studies optimization of treatment
  paradigm for primary and recurrent brain tumor.

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Summary Project Overview
Modulation of Stem Cell Biology
Hypoxia
Thymidine Analogs
Target Neural Stem Cell Transplant Population
Target Cancer Stem Cell
Optimize Transplant Environment in
Combination With Rehab Strategies
Translational Benefit of Stem Cell Therapeutics
Ameliorate Primary and Recurrent Brain CA
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Cervical Spinal Cord Injury

• Approximately 10,000 new SCI cases per year
• gt50 of these are cervical-SCI
• Cervical injuries involve
• Upper extremity involvement
• Lower extremity involvement
• Respiratory compromise (high cervical injury)

www.frommedskool.com
Approach Pair stem/progenitor cell transplant
with priming modalities
followed by targeted rehabilitation strategies
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Transplant of Neural Stem/Progenitor Cells into
SCI
Stem cells have been transplanted into acute and
sub-acute SCI Lesion Thoracic/lumbargtgtcervical
Cell Source ES cellsgtfetalgtadult Result Glial
CellsgtNeuronal Cells
Karimi-Abdolrezaee et al., 2006
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Hypoxic Conditioning as a Priming Modality
Baker-Herman et al., 2004
Studer et al., 2000
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Specific Aim I
Specific Aim I To determine if hypoxic exposure
increases SVZ neural progenitor cell (NPC)
survival, self-renewal and neuronal
differentiation. Hypothesis Both in vitro
hypoxic pre-conditioning and systemic in vivo
intermittent hypoxia will increase expression of
hypoxia-driven genes, NPC viability,
proliferation, self-renewal and neuronal
differentiation as compared to control
cells.
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Specific Aim I
Experimental Design
Endpoint Analyses -mRNA and protein levels of
hypoxia-driven genes -Neurosphere (NS) assay
assess neurosphere forming cell (NFC) viability,
proliferation and self-renewal -Cell fate
choice when plated under permissive conditions
for terminal differentiation.
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Specific Aim II
Specific Aim II To determine if a combined
approach of hypoxic conditioning plus
rehabilitation strategies relevant to
respiratory recovery will improve functional
integration of NPC transplants following
cervical SCI. Hypothesis The addition
of rehabilitative strategies will result in a
further improved NPC engraftment and
survival, as well as respiratory recovery as
compared to animals that receive no further
rehabilitative activity.
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Specific Aim II

• Experimental Design
• -All animals will receive cervical hemisection
• -NPC transplant one week following injury
• -Analysis from Aim I will dictate which hypoxic
  conditioning will be utilized to
• generate enriched NPC cells
• Experimental Groups
• Enriched NPC cell transplant with no further
  intervention
• Enriched NPC cell transplant plus tracheal
  occluder training
• Enriched NPC cell transplant plus voluntary
  running wheel
• Enriched NPC cell transplant plus treadmill
  locomotor training

Endpoint Analyses -Graft histology and
stereology -Electrophysiology -Barometric
plethysmography
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Future Direction
To pursue study of the combinatorial approach of
neural stem cell therapies and rehabilitation
strategies following neural insult. Long-term
goals -To further the basic understanding of
mechanisms behind the modulation of neural stem
cells. -To use this information to optimize the
translational benefit of neural cell replacement
therapies and subsequent patient outcomes.
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ACKNOWLEDGEMENTS
RRCD Program Mentorship Team Andrea Behrman, PhD,
PT Eric Laywell, PhD Dennis Steindler,
PhD Department of Anatomy and Cell
Biology Department of Physical Therapy Laywell
Lab Greg Marshall II, PhD Meryem
Demir John Aris, PhD Tom Foster, PhD David
Fuller, PhD Brendan Dougherty Milapjit
Sandhu Ashok Kumar, PhD Brandi Ormerod, PhD Brent
Reynolds, PhD Lindsay Levkoff, PhD Kyle Roux,
PhD Dan Silver Florian Siebzehnrubl, PhD
Financial Support RRCD Program Florida Center for
Brain Tumor Research