A new role for meninges as a niche for stem/precursor cells with neural differentiation potential during development up to adulthood
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1 A new role for meninges as a niche for stem/precursor cells with neural differentiation potential during development up to adulthood Francesco Bifari, MD, PhD Mauro Krampera, MD, PhD Ilaria Decimo, PhD Guido Fumagalli, MD Stem Cell Research Laboratory Section of Hematology Dpt. of Medicine University of Verona, Italy mauro.krampera@univr.it Molecular Pharmacology Laboratory Section of Pharmacology Dpt. of Public Health and Community Medicine University of Verona, Italy guido.fumagalli@univr.it ilaria.decimo@univr.it francesco.bifari@univr.it
2 Background Neural Stem Cells (NSCs) have been found - in the main neurogenic regions of the brain, i.e. hippocampus, subventricular zone (SVZ), olfactory bulb, dentate gyrus - in some non-neurogenic regions, i.e.spinal cord and possess neural differentiation potential into neurons and glial cells Zhao C Cell. 2008, 132, neurospheres neurons glia
3 Background MENINGES Dura Mater Leptomeninges Arachnoid Pia mater role during corticogenesis anatomical peculiarities
4 Nestin + cells are present in leptomeninges of rat brain and spinal cord Bifari F, Decimo I, et al. J Cell Mol Med 2009, 13(9B): Decimo I, Bifari F, et al. Stem Cells 2011; 29(12): Nestin/Laminin/Dapi
5 Nestin + cells in meninges have specific stem cell features (Meningeal stem cells) Self-renewal Proliferating cells (rarely) doublecortin (DCX)- positive cells (neuroblast marker) at perivascular level, but NG2-negative (pericytes)
6 nestin/ki67/to-pro3 40um 180um Nestin + cells can be extracted and expanded and have NSC properties. They can be induced to differentiate into neurons in vitro and in vivo Intra-brain injection (hippocampus) MAP2/EGFP Nestin + cells are activated by injury and participate to CNS parenchymal reaction (Decimo et al. Stem Cells 2011, Nakagomi et al. Stem Cells & Development 2012). CTRL 7 days post-injury GFP-transduction (lentivirus) of meningeal cells prior to injury 7 days post-injury
7 Nestin + meningeal stem cells with neural differentiation potential Are they conserved during development from embryo to adulthood?
8 ADULT P15 P0 E20 E14 Nestin-laminin-TOPRO3 Nestin + cells are present in rat leptomeninges during embryonic stages up to adulthood E20 P1 P8 Nestin (ICH) A P15 B Adult C SVZ Nestin (ICH) top view D E F Choroi d plexus Total number of Nestin+ cells significally decreases during development (n = 9)
9 ADULT P15 P0 E20 E14 ADULT P15 E14 Nestin-Ki67-TOPRO3 Nestin + proliferating cells decrease during development but persist up to adulthood (n = 3)
10 ADULT P15 P0 E20 E14 P15 Nestin-Oct4-TOPRO 3 Nestin+ cells expressing embryonic, self renewal-related markers decrease during development but persist up to adulthood (n = 3)
11 NSC-related marker expression in meninges during development Vimentin-nestin-TOPRO3 DCX- laminin SOX2-laminin ADULT P15 P0 E20 E14
12 Expression pattern of NSC-related genes by meninges: laser capture microscopy and qrt-pcr before after
13 ADULT P15 P0 E20 Nestin + cells are in contact with different cell types pericytes endothelial cells neural crest cells nestin-ng2-topro3 nestin-cd31-topro3 nestin- p75 ADULT P15 E14
14 Nestin + cells are in contact with several extracellular matrix components E20 P0 P15 Nestin-laminin G H Nestin-fibronectin Heparan sulphate-laminin ADULT P15 E14 ADULT P0 E20
15 n of CFU / 10 5 cells of tissue extract Nestin + cells can be extracted and expanded ex-vivo as homogeneous adherent cells from embryo up to adulthood NESTIN NEUROSPHERE CULTURE E P0 P E20 P0 P15 adult Adult 50µm scale bar
16 ADULT P15 P0 Ex-vivo-expanded Nestin + cells can be induced to differentiate into neural cells MAP2/GFAP MAP2/BrdU Ca2+ imaging: response to KCl 50µm scale bar E20
17 Meningeal Niche in development EMBRYO ADULT Nestin Vimentin GFAP NG2 Ki67 Sox2 CD31 p75 Oct4 Laminin
18 CONCLUSIONS AND FUTURE DEVELOPMENTS Meningeal Nestin+ cells are present in rat leptomeninges during embryonic stages up to adulthood in a decreasing number, but with similar features: they express NSC-related, embryonic and selfrenewal-related markers, are capable of proliferating and differentiating into different neural cells, and may be expanded ex-vivo During development, meningeal Nestin+ cells are in direct contact with resident niche cells and extracellular matrix components, which are known to play a role in stem cell quiescence, self-renewal, and proliferation/differentiation in response to stimuli Further characterization of meningeal Nestin + cells in normal and pathological conditions may help to better understand their involvement in the pathogenesis of neurological diseases and their therapeutic potential
19 ACKNOWLEDGEMENTS Molecular Pharmacology Lab Ilaria Decimo Marjiana Kusalo Valeria Berton Vincenzo Tedesco Alessia Auber Thomas Zandonai Nazeema Sherin Marzia Di Chio Cristiano Chiamulera Stem Cell Research Laboratory Francesco Bifari Mauro Krampera Section of Pathological Anatomy Giorgio Malpeli Aldo Scarpa Eliana Amato Guido Fumagalli
20 Thank you for your attention!
21
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