1. Proliferation and Differentiation of
Human Neural Stem Cells via Selective
Agonism of AT1 & AT2 Receptors
Research performed at Nova Southeastern University
Brigitte Blanco
Pine Crest School
2. What is the Renin-Angiotensin-System (RAS)?
Endocrine system that regulates cardiovascular homeostasis,
and fluid balance
RAS enzymes and proteins found in almost every tissue
RAS is found in areas of the brain and neurons involved
cognitive and motor function
RAS is responsible for proliferation and differentiation of
neural stem cells
4. •
•
AT1 and AT2 counterbalance each other by mediating invariably opposite functions in
order to maintain cardiovascular and endocrine balance or homeostasis
The RAS promotes proliferation and differentiation of neural stem cells via the protein
AngII which mediates its function via its two receptors AT1 and AT2
• AT1 induces Proliferation and AT2 induces Differentiation
RAS pathway
AT1 receptor
AT2 receptor
Vasoconstriction
Proliferation
Hypertrophy
Aldosterone
release
Oxidative stress
Baroreflex
Vasodilation
Neurite outgrowth
Cerebral blood flow
Spatial memory
Differentiation
Antiremodeling
Neuronal
excitability
Guimond MO, Gallo-Payet N (2012) The Angiotensin II
Type 2 Receptor in Brain Functions: An Update.
International journal of hypertension 2012: 351758
5. Application
Selective agonism of AT1 and AT2 receptors could possibly induce
proliferation and differentiation in damaged areas of the brain
Therapy for traumatic brain injuries, post-stroke ischemic damage,
and chemotherapy
Neurodegenerative diseases and cognitive deficits are associated
with neuronal and synaptic dysfunctions
Alzheimer’s, Parkinson’s, Huntington’s, mental retardation
Therapeutic treatments for non-proliferating and non-functioning
cells associated with neurodegenerative diseases, traumatic brain
injuries, and more to proliferate and function again could become a
reality
6. Purpose:
To determine if selective agonism of both the AT1 and AT2 receptor
in proliferating and differentiating conditions could induce
proliferation and differentiation of Human Neural Stem Cells
Proliferation
AT1 receptor
AT2 receptor
Vasoconstriction
Proliferation
Hypertrophy
Aldosterone release
Oxidative stress
Baroreflex
Vasodilation
Neurite outgrowth
Cerebral blood flow
Spatial memory
Differentiation
Antiremodeling
Neuronal
excitability
Differentiation
7. Methods:
Human Neural Stem Cell Culture:
StemPro® NSC SFM (Serum-Free Human Neural Stem Cell Culture Medium) kit from
Life TechnologiesTM was used to make proliferation and differentiation media
Proliferation media was made with Recombinant Human Epidermal and Fibroblast Growth Factors, whereas
Differentiation media was not
N7800-200 Gibco H-9 derived, Human Neural Stem Cells (hNSC) from Life
TechnologiesTM were seeded at 25,000 cells/cm2 and cultured at 37˚C in a
humidified incubator with 5% CO2
half-media changes were performed every other day
After reaching 70-90% confluency, hNSC were expanded in adherent monolayer
cultures in 8 or 4 well chamber slides using CELLSTARTTM (Gibco), in D-PBS with
calcium and magnesium
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do.com
Usascientific.com
8. Methods:
Selective Agonist Drug Treatments
Selective Agonists (below) were administered daily to their respective chamber slides at 10µM
AT1 selective agonist (sar1 AngII)
AT2R agonist (CGP42112)
AT2 Selective Antagonist administered with AT1 selective agonist
AT2 antagonism control (PD123319)
The relationship between these two receptors is unidirectional, meaning that if
the AT1 receptor is stimulated, the AT2 receptor will also produce a response.
HOWEVER, if the AT2 receptor is stimulated, the AT1 receptor will NOT respond
For this reason, only and AT2 antagonist was added in addition to the AT1
agonist
9. Methods:
Addition of Primary and Secondary Antibodies
hNSC were fixed and treated with antibodies after selective agonist
drug treatments
Tagged Primary and Secondary Antibodies form a fluorescent
complex to visualize proliferating or differentiating cells
Proliferation Antibodies
mPCNA (proliferation marker)
Differentiation Antibodies
mHuCD (neuronal differentiation)
rNestin (neural stem/progenitor cell marker) rGFAP (astrocyte marker)
mNeuN (proliferation marker)
mOligo (oligodendrocyte marker)
rs100β (proliferation marker)
rDCX (migrating neuroblast marker)
Data in Results only related to mPCNA and mHuCD
10. Methods:
Click-iT ApoTag TUNEL Assay
Degraded DNA fragments will be analyzed by terminal transferase
nick-end-labeling (TUNEL) to detect and quantify the percentage
of cells undergoing apoptosis through immunofluorescence
Slide Preparation
DAPI DNA counterstain was added to each well to visualize cell
nuclei
17. Conclusions:
AT1 Receptor Agonism INCREASED PROLIFERATION
In proliferating conditions:
Increased the percentage of proliferating cells by 4.84%
AT2 Receptor Agonism: INCREASED DIFFERENTIATION
In proliferating conditions:
Increased the percentage of differentiating cells by 58.02%
In differentiating conditions:
Increased the percentage of differentiating cells by 78.80%
ApoTag TUNEL Assay:
Levels of cellular apoptosis were not consequential to overall cellular
viability
18. Future Research:
Repeat these methods with increased drug concentrations
and exposure time to increase the statistical power of our
analysis and confirm hypotheses
Introduce EdU, a thymidine analog, which incorporates into
dividing cells during S-phase of cell division
To better understand the cell cycle kinetics, EdU will allow
visualization of cells before and after cells enter and leave cellcycle and the timespan involved in these processes
19. Bibliography
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Guimond MO, Gallo-Payet N (2012) The Angiotensin II Type 2 Receptor in Brain Functions: An Update.
International journal of hypertension 2012: 351758
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M. Shum, S. Pinard, M. O. Guimond et al., “Angiotensin II Type 2 receptor promotes adipocyte
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8) Guimond MO, Gallo-Payet N (2012) The Angiotensin II Type 2 Receptor in Brain Functions:
An Update. International journal of hypertension 2012: 351758
9) Usascientific.com
10) International Rules for Pre-college Science Research: Guidelines for Science and Engineering Fairs 20122013 http://www.societyforscience.org/document.doc?id=398
