neuronal survival in vitro

7/29/2019 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


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


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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neuronal survival in vitro

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