Mesenchymal stem cells of dental origin as promising tools for neuroregeneration

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1 Mesenchymal stem cells of dental origin as promising tools for neuroregeneration Gábor Varga Department of Oral Biology Faculty of Dentistry Semmelweis University, Budapest, Hungary

2 Sections of tooth undergoing development.

3 Definition of stem cell Unlimited self-renewal Capability to differentiate and form tissues - Embryonic stem cells - pluripotent - Adult/postnatal stem cells - multipotent

4 zygote Totipotent cells embryoblast Blastocyst Embryo Adult

5 zygote Totipotent cells embryoblast Blastocyst Embryo Stem cell culture Stem cells from differentiated tissues Adult

6 The plated periodontal ligamentum derived cells form colonies. This photo shows 14 days old colonies

7 hdpsc cell morphology during culture, and osteogenic differentiation A B C D E F Király et al., Neurochem Int, 2009

8 control (w/o primary antibody) anti-stro-1 anti-stro-1 anti-stro-1 STRO-1 immunostaining was observed in the primary DPSC cultures (5th passage, 5 week old) (400x magn.) Kádár et al., Fogorvosi Szle, 2009

9 Neuronal development: a link to tooth development Tucker, A., and Sharpe, P. The cutting-edge of mammalian development; how the embryo makes teeth. Nature reviews 5, 499, 2004.

10 Background Chronic neurodegenerative disorders such as Parkinson disease or Alzheimer disease are usually termed incurable. Acute traumatic brain and spinal cord injuries are also usually irreversible. Stem cell applications offer reasonable hope to treat these diseases. But neuronal stem cell sources are extremely limited in the human body.

11 Purpose of our studies Neuronal differentiation of dental pulp cells of human origin in vitro Application of these cells in vivo in a rat brain injury model

12 Neural differentiation by a new three-step protocol Neural induction was carried out first by demethylation, then by simultaneous PKC and cyclic AMP pathway activation, finally by a neurodifferentiation coctail.

13 Changes in DPSC cell morphology and cell viability during neuronal differentiation A D B C D E F Király et al., Neurochem Int, 2009

14 Differentiated cells: Complex neurite-like structures 10µm Cells after 10 days of differentiation display multi- and bipolar neuron-like structures

15 Changes in gene expression A B C D E F Vimentin (200 bp) Nestin (220 bp) NGN2 (196 bp) N-tubulin (243 bp) NSE (329 bp) NF-M (333 bp) GFAP (266 bp) β-actin (234 bp) 100,0 * * Relative normalized quantity 10,0 1,0 0,1 A B C D E F A B C D E F A B C D E F * * * * * * * * * * * * * * * * * * * * A B C D E F A B C D E F A B C D E F

16 Immuncytohemistry after neuronal differentiation (A) neuron specific microtubule marker N-tubulin, (B) neuronal NF-M and (C) glial GFAP intermediate filament (D) NeuN neuronal nuclei protein. Nuclei are visualized by DAPI A B C D Király et al., Neurochem Int, 2009

17 Patch clamp recordings to provide evidence for functional neurons A Trace (pa) Time (ms) V [mv] I [pa] B K DR current TEA C Na VD current sodium current TTX blocking Király et al., Neurochem Int, 2009

18

19 In vivo utilization of differentiated hdpscs in rat brain damage Vybrant DID+ labeling Király et al., 2011

20 Incorporation of pre-differentiated DPSCderived neural cells into the progenitor zones of the brain

21 Distribution of specific marker expression in engrafted Vybrant DiD pre-differentiated DPSCs in the rat brain

22 The patch clamp setup for brain slice studies

23 Phase-contrast Fluorescent Király et al., 2011

24 I-V relationship of voltage-dependent currents recorded from pre-differentiated engrafted DPSCs + TTX and TEA

25 Conclusions Our in vivo data demonstrate that at the single-cell level, grafted DPSC-derived neurons undergo morphological and functional integration into the host brain. Immunohistochemical and electrophysiological data both reveal that Vybrant DiD-labeled DPSCs can provide an appropriate model for studying neuro- and gliogenesis in vivo. These cells show progression in their neural specific marker expression and electrophysiological properties after brain injury, displaying a behavior very similar to that of the normal SVZ, SGZ and SCZ cells.

26 Stem Cell Clinical Trials are already on the way for Spinal Cord Injuries

27 Study of spinal cord injury in rat

28 Application of stem cells of dental origin based their antiinflammatory/immunomodulatory effect Acut pancreatitis (Peter Hegyi and Zoltan Rakonczay, Szeged Univ) IBD/colitis ulcerosa (Klara Gyires and Zoltan Zadori,Semmelweis Univ) Periodontal inflammation (Peter Windisch, Zsolt Lohinai, Janos Vag)

29

30 SU Oral Biology Ákos Nagy Orsolya Hegyesi Csilla Páska Gábor Rácz Irma Demeter Anna Földes József Blazsek Vanda Szlávik János Vág Erzsébet Bori Karola Kálló Kristóf Kádár Bálint Molnár Marianna Király Balázs Porcsalmy Gabriella Jobbágy-Óvári Perczel Kovács Katalin Borbély Zoltán Nagy Krisztina Fülöp Márta SU DCGI András Falus Éva Pálinger László Kőhidai SU Periodontology and Prostodontics István Gera Péter Hermann SU Maxillafacial Surgery József Barabás Sándor Bogdán SU Physiology Ákos Zsembery Lacza Zsombor SU Anatomy Imre Oláh Gábor Gerber HAS Inst. Exptl. Medicine Emília Madarász Márta Jelitai SU Oral Radiology Dobó Nagy Csaba Eotvos Univ., Biophysics Tamás Vécsek Bálint Szabó SU Nanotechnology Kellermayer Miklós Zrinyi Miklós NIH NIDCR Bruce Baum Songtao Shi Jay Chiorini Univ. Manchester Maynard Case Martin Steward Univ. Szeged Penke Botond Fülöp Lívia Univ Witten-Herdecke Wolf-Dieter Grimm Univ. Toronto Ben Ganss Eszter Somogyi-Ganss

31 Apical side Cl - ;HCO 3ˉ Na + Basolateral side Support: OTKA: NI69008, CK8092 TÁMOP-4.2.1/B-09/1/KMR TÁMOP-4.2.2/B-10/

32 Thank you for your attention

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