University%20of%20Plovdiv%20Department%20of%20Developmental%20Biology%20MSc%20Thesis:%20Neurogenic%20Potential%20of%20Bone-Marrow%20Derived%20Mesenchymal%20Stem%20Cells%20and%20Adult%20Neural%20Stem%20Cells%20by%20Tsvetelina%20Georgieva%20Batsalova - PowerPoint PPT Presentation

Title: University%20of%20Plovdiv%20Department%20of%20Developmental%20Biology%20MSc%20Thesis:%20Neurogenic%20Potential%20of%20Bone-Marrow%20Derived%20Mesenchymal%20Stem%20Cells%20and%20Adult%20Neural%20Stem%20Cells%20by%20Tsvetelina%20Georgieva%20Batsalova


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University of PlovdivDepartment of
Developmental BiologyMSc Thesis Neurogenic
Potential of Bone-Marrow Derived Mesenchymal Stem
Cells and Adult Neural Stem Cellsby Tsvetelina
Georgieva Batsalova

• Supervisor
• M. Draganov, PhD, Assoc. Prof
• Head of Department
• N.Popov, PhD, Assoc. Prof

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Aim

• theoretical examination of adult neural stem
  cells (aNSCs) and bone marrow-derived mesenchymal
  stem cells (MSCs) potential for generating
  functional neural cells in vitro and in vivo

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Introduction

• Classical vision - neural tissue has fixed number
  of neural cells
• Neural tissue recovery by stem cells
• endogenous NSCs
• exogenous stem cells
• Embryonic and fetal stem cells
• Adult stem cells
• MSCs
• aNSCs

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1. Type of stem cells
MSCs aNSCs
Adult stem cells -patients own stem cells can be used avoiding ethical questions, immune rejection and tumor formation (Pagano et al., 2006). Adult stem cells -patients own stem cells can be used avoiding ethical questions, immune rejection and tumor formation (Pagano et al., 2006).
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2. Sources in adult organism
MSCs aNSCs
Bone marrow (Pittenger et al., 1999 Colter et al., 2001) Central Nervous System main sources are the subventricular zone and the hippocampus (Gage, 2000 Galli et al., 2003)
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3. Isolation
MSCs aNSCs
Enough amount of autologous MSCs can be isolated from small bone marrow aspirates (10-20ml) without risk for patients health (Pittenger et al., 1999 Colter et al., 2001). NSC can be obtained from patients undergoing invasive neurosurgery (difficult and risky isolation of autologous aNSCs) (Pagano et al., 2006).
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4. In vitro expansion
MSCs aNSCs
Basal medium with 10 FCS and FGF-2 Feeder independent growth Adherent cultures (Pittenger et al., 1999 Colter et al., 2001) Minimum essential medium with mitogens (EGF FGF-2), without serum supplement Feeder independent growth Neurosphere forming suspension cultures (Galli et al., 2003)
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5. Multipotential
MSCs aNSCs
MSCs can give rise to Osteoblasts Chondrocytes Adipocytes Tenocytes hematopoietic-supporting stroma (Pittenger et al., 1999 Muraglia et al., 2000 Colter et al., 2001 Sekiya et al., 2002) under appropriate culture conditions or after transplantation. NSCs can give rise to Neurons Astrocytes Oligodendrocytes (Gage et al., 2000 Temple et al., 2001 Ivanova et al., 2002 Galli et al., 2003) under appropriate culture conditions or after transplantation.
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5. Multipotential
MSCs aNSCs
MSCs can also give rise to cardiac and skeletal muscle (Peshle Condorelli, 2005) hepatocytes (Theise et al., 2000) neural cells (Kopen et al., 1999 Sanchez Ramos et al., 2000) under appropriate culture conditions or after transplantation. NSCs can also give rise to red and white blood cells (Bjornson et al., 1999) skeletal muscle (Galli et al., 2000) under appropriate culture conditions or after transplantation.
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6. Differentiation conditions for neural
phenotype development in vitro
MSCs aNSCs
Neuronal phenotype ß-mercaptoethanol, BHA (Woodbury et al., 2000) DMSO, BHA / ß-mercaptoethanol (Hofstetter et al., 2002) Neuronal astroglial phenotype EGF, BDNF, ß-mercaptoethanol, Retinoic acid (Sanchez Ramos et al., 2000) NSCs express neural, astroglial or oligodendroglial phenotype after removing the mitogens (Galli et al., 2003)
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7. Typical markers expressed after neural
induction
MSCs aNSCs
Early neural markers Nestin ß-tubulin III (Long et al., 2005) Early neural markers Nestin ß-tubulin III (Galli et al., 2005)
Mature neural markers NF GABA NeuN (atypical localisation) Tau (atypical localisation) glial GFAP (Long et al., 2005) Mature neural markers NF GABA NeuN Tau glial GFAP glial GalC (Galli et al., 2005)
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8. Electrophysiological properties in vitro
MSCs aNSCs
MSCs can generate single action potential only after co-culturing with mice granular cerebelar neurons no synaptic activity and repetitive action potentials (Wislet-Gendebien et al., 2005). Generate single action potential Repetitive action potentials pre- and postsynaptic activity (Song et al., 2002 van Praag et al., 2002)
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9. Transplantation
MSCs aNSCs
Direct transplantation is possible but not necessary Intravenous transplantation MSCs can be transplanted more easily intravenously because of their capability to migrate and home selectively in the injured tissue (Krause et al., 2003) Direct transplantation is possible but not necessary Intravenous transplantation NSCs can be transplanted more easily intravenously because of their capability to migrate and home selectively in the injured tissue (Martino Pluchino, 2006)
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10. Functional improvement in model organisms
MSCs aNSCs
In some experiments has been shown that transplanted MSCs might acquire neuronal- or astroglial-like phenotype (Kopen et al., 1999 Hofstetter et al., 2002). Although, the functional improvement observed after MSC transplantation in model organisms with different CNS disorders was mainly a result of neurotrophic factors secretion (NGF, VEGF, CNTF,FGF-2) that activate nearby astrocytes. Activation of astrocytes results in increase in neurogenesis by NSCs located in close proximity (Munoz et al., 2005). Functional recovery obtained by aNSC transplantation scarcely correlates with the number of transplant-derived, newly generated, terminally differentiated neural cells. NSC might be therapeutically efficacious by secreting neurotrophines NGF, BDNF, CNTF, GDNF (activating astrocytes and microglia) (Martino Pluchino, 2006). NSC also might promote improvement in neuroinflammatory models by inducing apoptosis of inflammatory T lymphocytes (Pluchino et al., 2005)
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Conclusions

• Therapeutic efficacy of MSC transplantation is
  equivalent to aNSCs transplantation
• MSCs can be preferred for CNS disorders therapy
  because of their easier isolation in bigger
  amount and easier in vitro expansion

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Our experiment

• Culturing of hMSCs XII (derived from 18 years old
  donor Cambrex)
• Tested growth media
• DMEM (Cambrex)
• DMEM/Hams F-12 (Sigma)
• Serum supplement
• FBS (Gibco)
• FCS (Cambrex)
• BS (BulBio)
• BS (our own production)
• Different serum concentration
• 20, 15, 10, 5, 2, 0
• Culturing without FGF-2

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Our preliminary results and conclusions

• Medium DMEM 10 FCS 5ng/ml FGF-2 is optimal
  for hMSCs XII in vitro expansion
• Normal serum supplement do not show good growth
  index
• Normal serum is appropriate supplement to the
  freezing medium according to the index cell
  survival after thawing
• Lower FCS concentrations decrease the hMSCs XII
  proliferation in vitro
• In conditions of FGF-2 absence and serum absence
  hMSCs XII do not proliferate but stay vital for a
  long period

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• Thank You for Your Attention!