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Human bone marrow-derivedmesenchymal stem cells secrete brain-derived neurotrophic factor which promotes neuronal survival in vitro By Patel Devang V. M.S.Pharm (Pharmaceutics) NIPER-Ahmedabad

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STEM CELLS“Nonspecialized cells that have the capacity to self renew and to differentiate into specialized cells”

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Stem cell type Description Examples Stem cells can form only one type of Muscle stem Unipotent specialized cell type cells Fetal tissue, Stem cells can form multiple types ofMultipotent Adult stem cells and tissue types cells Stem cells can form any adult cell type. However, they alone cannot develop into Blastocyst Pluripotent adult animal because they lack the (4 to 5 days potential to contribute to extraembryonic old embryo) tissue(such as the Placenta). Stem cells can differentiate into Cells from embryonic and extraembryonic cell early (1-3 Totipotent types (eg. Placenta). Such cells can days) construct a complete, viable organism embryo

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• Embryonic Stem Cells can be obtained from blastocysts 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 large nucleus. • Mesenchymal stem cells are a distinct entity to the mesenchyme (embryonic connective tissue which is derived from the mesoderm). • MSCs are adult stem cells found in the bone marrow, 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 CELLSStem cell therapy has the potential to treat many human diseases like:• Brain damage • Diabetes• Leukemia • Blindness and vision• Spinal cord injury impairment• Heart damage • Amyotrophic lateral• Muscle damage sclerosis• Parkinsons disease • Multiple sclerosis• Baldness • Wound healing• Missing teeth • Infertility

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STEM CELLS IN NEUROLOGICAL DISEASES In recent years, there has been (A) Human ESCs considerable interest in the potential of stem cells as therapeutic agents in (A) Neurons neurological derived from diseases including Human ESCs stroke and spinal cord injury.

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• In neurological diseases it has been postulated that stem cell therapies may replace lost cells by differentiating into functional neural tissue; modulate the immune system to prevent further neurodegeneration and effect repair; or provide a source of trophic support for the diseased nervous system.

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Human bone marrow-derivedmesenchymal stem cells secrete brain- derived neurotrophic factor which promotes neuronal survival in vitro Published In Stem Cell Research, 2009, 3, 63–70 By Alastair Wilkins et al., Department of Neurology , University of Bristol, UK

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AIM OF EXPERIMENT• To define mechanisms of neuronal cell death under conditions of trophic deprivation and exposure to nitric oxide• To determine potential mechanism by which human bone marrow-derived mesenchymal stem cells (MSCs) may protect 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 replacement surgery by orthopedic surgeons at the Avon Orthopedic Centre, Bristol, UK• Dulbeccos modified Eagles medium (DMEM) supplemented with 2% B27• MIN (DMEM supplemented with chemically defined medium with 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 changes occurring during trophic factor withdrawal• Cortical neurons (1.4 103 cells/mm2) were maintained in B27-supplemented Dulbeccos modified Eagles medium (DMEM).• This was taken as Control throughout experiment and other values expressed as a percentage of this control.• For determination of neuronal survival, cultures were fixed and stained by the nuclear marker bisbenzamide.

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FIG : MSC-conditioned medium increases survival of neurons exposed to trophic deprivation• MIN: Chemically defined medium with no serum• CM: MSC-conditioned medium• CM/LY: MSC-conditioned medium plus LY290042

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FIG : MSC-conditioned medium increases survival of neurons exposed to trophic deprivation• Neurons exposed to MIC (Chemically defined medium with no serum) showed decreased survival compared to control.• Neurons exposed to CM (MSC-conditioned medium showed increased survival compared to those exposed to MIC (Chemically defined medium with no serum).• The PI3 Kinase / Akt inhibitor LY290042 inhibits the survival effect of MSC-conditioned medium.

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[B] Determination of the influence of MSC-conditioned medium on signaling changes occurring during NO exposure FIG: MSC-conditioned medium increases survival of neurons exposed to nitric oxide 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 increases survival of neurons exposed to nitric oxide• Neurons exposed to the DETANONOate (nitric oxide donor) showed decreased survival compared to control, a process which was attenuated by MSC- conditioned medium.• The PI3 Kinase / Akt inhibitor LY290042 inhibits the survival effect of MSC-conditioned medium.

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[C] Determination of the influence of MSC-conditioned medium on neuronal survival via PI3kinase/Akt- dependent pathways FIG: MSC-conditioned medium activates Akt in neurons exposed to trophic deprivation MIN: Chemically defined medium with no serum CM: MSC-conditioned medium CM/LY: MSC-conditioned medium plus LY290042

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FIG: MSC-conditioned medium activates Akt in neurons exposed to trophic deprivation• Exposure of neurons to CM (MSC- conditioned medium) increased activation of Akt compared to those exposed to MIN (Chemically defined medium with no serum).• Furthermore, addition of the PI3kinase/Akt inhibitor LY290042 inhibited CM (MSC conditioned medium)-induced survival of cortical neurons exposed to trophic factor withdrawal.

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FIG: MSC-conditioned medium activates Akt in neurons exposed 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 neurons exposed to DETANONOate• Akt activation was seen in neurons exposed to CM (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-conditioned medium

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FIG: MSC-conditioned medium reduces p38 activation in neurons exposed to DETANONOate• Furthermore exposure of neurons to MIN (Chemically defined medium with no serum) alone did not lead to activation of p38 MAPkinase, which occurred on exposure to DETANONOate.• CM (MSC-conditioned medium) attenuated DETANONOate-induced p38 activation within cortical neurons.

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[D] Determination whether BDNF is important in mediating the MSC effects on neuronal survival MIN: Chemically defined medium with no serum MSC1–6: Different MSC populations (Derived from different patients) NeuronNO: Neurons exposed to DETANONOate BDNF ELISA demonstratedFIG: Human MSCs significant amounts of BDNF secreted from MSCs. produce BDNF

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FIG: Neutralising antibodies to BDNF attenuate MSC- conditioned medium survival effects under conditions of trophic deprivationCM/aBDNF: MSC conditioned medium plus neutralising antibodies to BDNF

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FIG: Neutralising antibodies to BDNF attenuate MSC conditioned medium survival effects under conditions of DETANONOate exposure• NO/CM: DETANONOate plus MSC-conditioned medium• NO/CM/aBDNF: DETANONOate plus MSC- conditioned medium plus neutralizing antibodies to BDNF

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CONCLUSION• Human bone marrow derived mesenchymal stem cells secrete factors which protect rodent neurons from trophic deprivation and nitric oxide-induced death.• Therefore human MSC transplantation has been 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 bone marrow-derived mesenchymal stem cells secrete brain-derived neurotrophic factor which promotes neuronal survival in vitro, Stem Cell Research, 2009, 3, 63–70.• 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 marrow stem cells-properties and advantages, Neurological Science, 2008, 265, 59-62.

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• Parr A.M. Tator C.H., Bone marrow-derived mesenchymal stromal cells for the repair of central nervous system injury, Bone Marrow Transplantation, 2008, 40, 609–619.• 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