1. University of Plovdiv Department of Developmental Biology MSc Thesis: Neurogenic Potential of Bone-Marrow Derived Mesenchymal Stem Cells and Adult Neural Stem Cells by Tsvetelina Georgieva Batsalova Supervisor M. Draganov, PhD, Assoc. Prof Head of Department N.Popov, PhD, Assoc. Prof

2. 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 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

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

4. 1. Type of stem cells MSCsaNSCs 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).

5. 2. Sources in adult organism MSCsaNSCs 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)

6. 3. Isolation MSCsaNSCs 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).

7. 4. In vitro expansion MSCsaNSCs Basal medium with 10% FCS and FGF-2 Basal medium with 10% FCS and FGF-2 Feeder – independent growth Feeder – independent growth Adherent cultures Adherent cultures (Pittenger et al., 1999; Colter et al., 2001) Minimum essential medium with mitogens (EGF; FGF-2), without serum supplement Minimum essential medium with mitogens (EGF; FGF-2), without serum supplement Feeder – independent growth Feeder – independent growth Neurosphere forming suspension cultures Neurosphere forming suspension cultures (Galli et al., 2003)

8. 5. Multipotential MSCsaNSCs MSCs can give rise to: Osteoblasts Osteoblasts Chondrocytes Chondrocytes Adipocytes Adipocytes Tenocytes Tenocytes hematopoietic-supporting stroma 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. under appropriate culture conditions or after transplantation. NSCs can give rise to: Neurons Neurons Astrocytes Astrocytes Oligodendrocytes Oligodendrocytes (Gage et al., 2000; Temple et al., 2001; Ivanova et al., 2002; Galli et al., 2003) under appropriate culture conditions or after transplantation. under appropriate culture conditions or after transplantation.

9. 5. Multipotential MSCsaNSCs MSCs can also give rise to: cardiac and skeletal muscle (Peshle & Condorelli, 2005) cardiac and skeletal muscle (Peshle & Condorelli, 2005) hepatocytes (Theise et al., 2000) hepatocytes (Theise et al., 2000) neural cells (Kopen et al., 1999; Sanchez – Ramos 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 ) red and white blood cells (Bjornson et al., 1999 ) skeletal muscle (Galli et al., 2000) skeletal muscle (Galli et al., 2000) under appropriate culture conditions or after transplantation.

10. 6. Differentiation conditions for neural phenotype development in vitro 6. Differentiation conditions for neural phenotype development in vitro MSCsaNSCs Neuronal phenotype: β-mercaptoethanol, BHA (Woodbury et al., 2000) DMSO, BHA / β-mercaptoethanol (Hofstetter et al., 2002) Neuronal & astroglial phenotype: EGF, BDNF, β-mercaptoethanol, Retinoic acid (Sanchez – Ramos et al., 2000) NSCs express neural, astroglial or oligodendroglial phenotype after removing the mitogens (Galli et al., 2003) oligodendroglial phenotype after removing the mitogens (Galli et al., 2003)

11. 7. Typical markers expressed after neural induction MSCsaNSCs Early neural markers Nestin Nestin β -tubulin III β -tubulin III (Long et al., 2005) Early neural markers Nestin Nestin β -tubulin III β -tubulin III (Galli et al., 2005) Mature neural markers NF NF GABA GABA NeuN (atypical localisation) NeuN (atypical localisation) Tau (atypical localisation) Tau (atypical localisation) glial GFAP glial GFAP (Long et al., 2005) Mature neural markers NF NF GABA GABA NeuN NeuN Tau Tau glial GFAP glial GFAP glial GalC glial GalC (Galli et al., 2005)

12. 8. Electrophysiological properties in vitro MSCsaNSCs MSCs can generate: single action potential only after co-culturing with mice granular cerebelar neurons single action potential only after co-culturing with mice granular cerebelar neurons no synaptic activity and repetitive action potentials no synaptic activity and repetitive action potentials (Wislet-Gendebien et al., 2005). Generate single action potential Generate single action potential Repetitive action potentials Repetitive action potentials pre- and postsynaptic activity pre- and postsynaptic activity (Song et al., 2002; van Praag et al., 2002)

13. 9. Transplantation MSCsaNSCs Direct transplantation is possible but not necessary 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 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 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 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)

14. 10. Functional improvement in model organisms MSCsaNSCs 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)

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

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

17. Our preliminary results and conclusions Medium DMEM + 10% FCS + 5ng/ml FGF-2 is optimal for hMSCs XII in vitro expansion 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 supplement do not show good growth index Normal serum is appropriate supplement to the freezing medium according to the index cell survival after thawing 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 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 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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