neuronal survival in vitro
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Human bone marrow-derived
mesenchymal stem cells secretebrain-derived neurotrophic
factor which promotesneuronal survival in vitro
ByPatel Devang V.
M.S.Pharm (Pharmaceutics)
NIPER-Ahmedabad
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STEM CELLS
Nonspecialized cells that have the capacity to selfrenew and to differentiate into specialized cells
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Stem cell type Description Examples
UnipotentStem cells can form only one type of
specialized cell type
Muscle stem
cells
MultipotentStem cells can form multiple types of
cells and tissue types
Fetal tissue,
Adult stem
cells
Pluripotent
Stem cells can form any adult cell type.However, they alone cannot develop into
adult animal because they lack the
potential to contribute to extraembryonic
tissue(such as the Placenta).
Blastocyst
(4 to 5 days
old embryo)
Totipotent
Stem cells can differentiate into
embryonic and extraembryonic cell
types (eg. Placenta). Such cells can
construct a complete, viable organism
Cells from
early (1-3
days)
embryo
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Embryonic Stem Cells can be obtained fromblastocysts and aborted fetuses.
Adult Stem Cells (Non-embryonic stem cells)
have been found in the blood, bone marrow,liver, kidney, cornea, dental pulp, brain, skin,
muscle, salivary gland etc.
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MESENCHYMAL STEM CELLS(MSCs)
Morphologically, mesenchymal stem cells (MSCs)have long and thin cell bodies with a largenucleus.
Mesenchymal stem cells are a distinct entity to the
mesenchyme (embryonic connective tissue whichis derived from the mesoderm).
MSCs are adult stem cells found in the bonemarrow, cord blood, peripheral blood, fallopian
tube, fetal liver and lung. MSCs have capacity to form multiple types of
tissue including adipocytes (fat cells),chondrocytes (cartilage cells), osteoblasts (bone
cells), tendons, muscle, skin, neurons.
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APPLICATIONS OF STEM CELLS
Stem cell therapy has the potential to treat manyhuman diseases like:
Brain damage
Leukemia Spinal cord injury
Heart damage
Muscle damage Parkinson's disease
Baldness
Missing teeth
Diabetes
Blindness and visionimpairment
Amyotrophic lateralsclerosis
Multiple sclerosis
Wound healing
Infertility
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STEM CELLS IN NEUROLOGICAL
DISEASESIn recent years,there has beenconsiderable
interest in thepotential of stemcells as therapeuticagents in
neurologicaldiseases includingstroke and spinalcord injury.
(A)Human ESCs
(A)Neuronsderived fromHuman ESCs
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In neurological diseases it has been postulated thatstem cell therapies may replace lost cells bydifferentiating into functional neural tissue; modulate
the immune system to prevent further
neurodegeneration and effect repair; or provide asource of trophic support for the diseased nervous
system.
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Human bone marrow-derived
mesenchymal stem cells secrete brain-derived neurotrophic factor whichpromotes neuronal survival in vitro
Published In
Stem Cell Research, 2009, 3, 6370By
Alastair Wilkins et al.,
Department of Neurology ,
University of Bristol,
UK
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AIM OF EXPERIMENT
To define mechanisms of neuronal celldeath under conditions of trophic
deprivation and exposure to nitric oxide To determine potential mechanism by
which human bone marrow-derived
mesenchymal stem cells (MSCs) mayprotect neurons from trophic deprivation or
NO-mediated damage
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MATERIALS USED Neuronal cultures prepared from cortices of E16 rat embryos
Bone marrow: Taken by the time of total hip replacementsurgery by orthopedic surgeons at the Avon Orthopedic
Centre, Bristol, UK
Dulbecco's modified Eagles medium (DMEM) supplemented
with 2% B27
MIN (DMEM supplemented with chemically defined mediumwith no serum)
CM (Mesenchymal stem cell-conditioned medium)
Neuronal marker bisbenzamide
DETANONOate (NO donor)
LY290042 (PI3kinase/Akt inhibitor)
Neutralising antibodies to BDNF
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EXPERIMENT AND RESULT[A] Determination of the influence of MSC-
conditioned medium on signaling changesoccurring during trophic factor withdrawal
Cortical neurons (1.4103 cells/mm2) were
maintained in B27-supplemented Dulbecco'smodified Eagles medium (DMEM).
This was taken as Control throughoutexperiment and other values expressed as a
percentage of this control.
For determination of neuronal survival, cultureswere fixed and stained by the nuclear marker
bisbenzamide.
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MIN: Chemically defined medium with no serum
CM: MSC-conditioned medium
CM/LY: MSC-conditioned medium plus LY290042
FIG : MSC-conditionedmedium increases
survival of neuronsexposed to trophicdeprivation
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FIG : MSC-conditionedmedium increasessurvival of neurons
exposed to trophicdeprivation
Neurons exposed to MIC (Chemically defined medium with no
serum) showed decreased survival compared to control.
Neurons exposed to CM (MSC-conditioned medium showedincreased survival compared to those exposed to MIC (Chemically
defined medium with no serum).
The PI3 Kinase / Akt inhibitor LY290042 inhibits the survival effect ofMSC-conditioned medium.
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[B] Determination of the influence of MSC-conditionedmedium on signaling changes occurring during NOexposure
FIG: MSC-conditioned mediumincreases survival of neuronsexposed to nitric oxide
MIN: Chemically definedmedium with no serum
NO: DETANONOate
NO/CM: DETANONOate plusMSC-conditioned medium
NO/CM/LY: DETANONOate
plus MSC-conditioned medium
plus LY290042
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Neurons exposed to the DETANONOate (nitric oxide
donor) showed decreased survival compared tocontrol, a process which was attenuated by MSC-
conditioned medium.
The PI3 Kinase / Akt inhibitor LY290042 inhibits thesurvival effect of MSC-conditioned medium.
FIG: MSC-conditioned mediumincreases survival of neuronsexposed to nitric oxide
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[C] Determination of the influence of MSC-conditionedmedium on neuronal survival via PI3kinase/Akt-dependent pathways
FIG: MSC-conditionedmedium activates Akt inneurons exposed to
trophic deprivation
MIN: Chemically defined
medium with no serum
CM: MSC-conditioned
medium
CM/LY: MSC-conditionedmedium plus LY290042
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Exposure of neurons to CM (MSC-
conditioned medium) increased activation ofAkt compared to those exposed to MIN
(Chemically defined medium with no serum).
Furthermore, addition of the PI3kinase/Aktinhibitor LY290042 inhibited CM (MSC
conditioned medium)-induced survival of
cortical neurons exposed to trophic factor
withdrawal.
FIG: MSC-conditionedmedium activates Akt inneurons exposed to
trophic deprivation
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FIG: MSC-conditioned medium activates Akt in neuronsexposed to DETANONOate
B27: Neurons exposed to 2% B27
MIN: Chemically defined medium with no serum
NO: DETANONOate
NO/CM: DETANONOate plus MSC-conditioned medium
NO/CM/LY: DETANONOate plus MSC-conditioned
medium plus LY290042
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FIG: MSC-conditioned medium activates Akt in neuronsexposed to DETANONOate
Akt activation was seen in neurons exposed toCM (MSC-conditioned medium) in the presence
of DETANONOate, compared to neurons
exposed to DETANONOate alone. Furthermore, addition of the PI3kinase/Akt
inhibitor LY290042 inhibited CM (MSC
conditioned medium)-induced survival of cortical
neurons exposed to NO exposure.
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FIG: MSC-conditioned medium reduces p38 activation in
neurons exposed to DETANONOate
MIN: Chemically defined medium with no serum
NO: DETANONOate
NO/CM: DETANONOate plus MSC-conditionedmedium
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Furthermore exposure of neurons to MIN(Chemically defined medium with no serum) alone
did not lead to activation of p38 MAPkinase, whichoccurred on exposure to DETANONOate.
CM (MSC-conditioned medium) attenuatedDETANONOate-induced p38 activation within
cortical neurons.
FIG: MSC-conditioned medium reduces p38 activation in
neurons exposed to DETANONOate
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[D] Determination whether BDNF is important inmediating the MSC effects on neuronal survival
FIG: Human MSCs
produce BDNF
MIN: Chemically defined medium
with no serum
MSC16: Different MSCpopulations (Derived from different
patients)
NeuronNO: Neurons exposed to
DETANONOate
BDNF ELISA demonstratedsignificant amounts of BDNF
secreted from MSCs.
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FIG: Neutralisingantibodies to BDNFattenuate MSC-conditioned mediumsurvival effects underconditions of trophicdeprivation
CM/aBDNF: MSCconditioned medium
plus neutralising
antibodies to BDNF
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FIG: Neutralising antibodiesto BDNF attenuate MSC
conditioned mediumsurvival effects underconditions ofDETANONOate exposure
NO/CM: DETANONOateplus MSC-conditioned
medium
NO/CM/aBDNF:DETANONOate plus MSC-
conditioned medium plus
neutralizing antibodies to
BDNF
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CONCLUSION
Human bone marrow derivedmesenchymal stem cells secrete factorswhich protect rodent neurons from trophic
deprivation and nitric oxide-induced death.
Therefore human MSC transplantation hasbeen shown to improve the outcome in a
variety of neurological diseases including
stroke and spinal cord injury.
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REFERENCES
Alastair Wilkins, Kevin Kemp et al., Human bonemarrow-derived mesenchymal stem cells secrete
brain-derived neurotrophic factor which promotes
neuronal survival in vitro, Stem Cell Research, 2009,
3, 6370. Hokari M., Kuroda S., Shichinohe H. et al., Bone
marrow stromal cells protect and repair damaged
neurons through multiple mechanisms,
Neuroscience, 2008, 1024-1035.
Rice C.M., Scolding N.J., Autologous bone marrowstem cells-properties and advantages, Neurological
Science, 2008, 265, 59-62.
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Parr A.M. Tator C.H., Bone marrow-derivedmesenchymal stromal cells for the repair of central
nervous system injury, Bone Marrow
Transplantation,2008, 40, 609619.
Rosser A.E., Zietlow R., Dunnett S.B., Stem cell
transplantation for neurodegenerative diseases,Curr. Opin. Neurol., 2007, 20, 688-692.
http://www.nature.com/bmt/journal/v45/n8
http://www.ncbi.nlm.nih.gov/pubmed/20028455#
http://www.journal-inflammation.com/content/2/1/8
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