Antonio Uccelli Neuroimmunology Unit Dept. Of Neurosciences University of Genoa, Italy Workshop on Transplant and Cellular Therapy for Autoimmune Diseases CIBMTR, April 19-20 2013, Milwaukee, WI
Stem Cells Portfolio for the treatment of MS Immune reconstitution Eradication of autoimmunity Reset of the immune system HSC HSC Mobilization Chemotherapy HSC Infusion Induction of immune tolerance MSC Neuroprotection Induction of oligodendrogenesis NPC Transdifferentiation and neuroprotection Induction of neurogenesis Uccelli and Mancardi, Curr Opin Neurol 2010 Immunomodulation
Uccelli A, et al, Nat Rev Immunol 2008 The hematopoietic stem cell niche is the site where HSCs home in the BM in close contact with osteoblasts, endothelial and stromal cells, a rare cell population of non-heamatopoietic origin(representing < 0.01% of total BM cells) named mesenchymal stem cells (MSC)
Why should we use MSC for the treatment of brain repair?
In vitro neuronal (trans)differentiation of MSC: is this biologically relevant in vivo? Neural differentiation: prolonged (30 days) and specific nestin GFAP Anghileri et al, 2008 MAP2 GABA-A
Mesenchymal stem cells protect CNS neurons against glutamate excitotoxicity Voulgari-Kokota et al Exp Neurol 2012
Mesenchymal stem cells instruct oligodendrogenic fate decision on adult neural stem cells. Control MSC-CM GalC Rivera et al., Stem Cells, 2006 GFAP MBP
MSC promote in vivo neurogenesis MSCs promote proliferation, migration, and survival of mouse NSCs Implantation of human MSCs increased synthesis of trophic factors Munoz et al, PNAS 2006
MSC inhibit T cell proliferation arresting cell division Benvenuto et al, Stem Cells 2007
MSC inhibit B cell functions 80000 B cells cpm 60000 40000 20000 ** * B cells + MSC 1:1 B cells + MSC 1:2 MSC 0 ng/ml 350 300 250 200 150 MSC inhibit B cell proliferation * ** ** B cells B cells + MSC 1:1 100 50 0 IgM IgG IgA MSC inhibit immunoglobulins production IgM IgG IgA B cells B cells + MSC 1:1 B cells B cells + MSC 1:1 B cells B cells + MSC 1:1 MSC inhibit B cells differentiation to antibody secreting cells In collaboration with V. Pistoia Corcione et al, Blood 2005
MSC A impair LPS-induced maturation of DC 50 CD11c CD86 78% vs 51% 72% vs 59 % 0 50 CD80 45% vs 16% CD40 41% vs 20% 0 10 0 10 1 10 2 10 3 10 10 0 10 1 10 2 10 3 10 FL1-H 4 FL1-H 4 MHC cl.i MHC cl.ii 50 55% vs 22% 48% vs 19% 0 10 0 10 1 10 2 10 3 10 10 0 10 1 10 2 10 3 10 FL2-H 4 FL2-H 4 CCR7 CD49d 1 50 68% vs 32% 89% vs 75% 0 10 0 10 1 10 2 10 FL3-H 3 10 4 Control DC MSC treated DC Chiesa et al, PNAS 2011
MSC impair DC antigen presentation to DO11.10 CD4 + T cells and cross-presentation to OT.1 CD8 + T cells A 500 DC + CD4 + T MSC exposed DC + CD4 + T 67755 57210 B DC + CD8 + T MSC exposed DC + CD8 + T 2 M OVA CD69 48h 0 500 69885 16035 CFSE Cell number 0 0.2 M OVA 400 DC + CD8 + T MSC exposed DC + CD8 + T 20800 2226 48h 50 4500 2610 M OVA Cell number 0 400 78110 2428 0 72h CFSE 0 CFSE Chiesa et al, PNAS 2011
The complex interplay between MSCs and the immune system Uccelli et al, Nat Rev Immunol 2008
Do all these results translate into relevant in vivo biological effects in experimental models of neurological diseases?
MSCs ameliorate EAE inducing in vivo tolerance to myelin antigens inside secondary lymphoid organs (Zappia et al 2005; Gerdoni et al 2007) Lymph nodes engraftment MSC Demyelination T cell 50 m T cells 8000 7000 Amelioration of EAE Macrophages T cells proliferation (CPM) 6000 5000 4000 3000 2000 1000 0 Mog 1 M Mog 3 M Mog 10 M Inhibition of Ag-specific T and B cell response B cells A B Axonal loss Neuroprotection without transdifferentiation O4 EA GFP B Inhibition of T cell encephalitogenic potential GFAP Zappia et al, Blood NeuN2005 C D
Is MSCs therapeutic effect based also on long term engraftment and cell replacement?
MSC are effective across MHC constraints are they immunoprivileged? MSC T cell inhibition overcome inter and intraspecies barriers Syngeneic, allogeneic and xenogeneic MSC can inhibit EAE Rafei et al, Mol Ther 2010 Zappia and Casazza, unpublished results
Allogeneic marrow stromal cells are immune rejected by MHC class I and class II mismatched recipient mice Eliopoulos et al, Blood 2005
MSCs are killed by cells of the innate immunity NK cells Spaggiari et al, Blood 2006 γδ cells Prigione et al, Stem Cells 2009
Bone Marrow-Derived Mesenchymal Stem Cells after Infusion principally distribute to the lungs Gao et al, 2001 Barbash et al, 2003 Lee et al, 2009
Identification of the optimal route of administration 3 2.5 2 1.5 1 ctrl iv ctrl ic msc iv msc ic 0.5 0 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 MSC I.V. MSC I.C. CTRL I:V: 10X CTRL I.C. 10X 10X 10X *P < 0.05: **P < 0.01 Demyelination areas (%) 60 50 40 30 20 10 0 * * ctrl iv msc iv ctrl ic msc ic Morando et al., Stem Cell Res & Ther 2012
Demyelination T cells MSCs do not transdifferentiate but protect neurons in vivo Gerdoni et al, Ann Neurol 2007 MAP2 MSC 40x Macrophages O4 A E GFP B MAP2 CTRL 40x B cells GFAP Axonal loss C NeuN D
Conditioned growth medium from human MSCs biases the development of neurosphere derived cells toward oligodendrocytes and neurons, and promotes functional recovery in EAE Bai et al Nat Neurosci 2012
MSC reduce the EAE-dependent oxidative stress in the CNS Lanza et al, JNC 2009
MSCs reduces astrogliosis and microglia activation in EAE SPINAL CORD CTR 40X SPINAL CORD MSC 40X GFAP IB4 Casazza and Principato unpublished results
Microglia dynamically changes phenotype and functions depending on environmental cues Perry, V. H. et al. (2010) Nat. Rev. Neurol.
Microglial plays a key role in CNS homeostasis Neumann H et al. Brain 2009;132:288-295
MSC inhibit microglia production of proinflammatory molecules and upregulation of stress associated proteins following LPS activation inos TNF IL1β * p<0.05 * p<0.01 * p<0.001 N9 +LPS +LPS+MSC N9 +LPS +LPS+MSC N9 +LPS +LPS+MSC Metallothioneins * p<0.001 HOX1 * p<0.001 PARP * p<0.001 N9 +LPS +LPS+MSC N9 +LPS +LPS+MSC N9 +LPS +LPS+MSC Giunti et al, Stem Cells 2012
MSC reverse and enhance the LPS-mediated decrease of molecules associated with neuroprotection CX3CR1 * p<0.05 NURR1 * p<0.05 CD200R * p<0.05 N9 +LPS +LPS+MSC N9 +LPS +LPS+MSC N9 +LPS +LPS+MSC IGF1 * p<0.05 EP2 * p<0.05 N9 +LPS +LPS+MSC N9 +LPS +LPS+MSC Giunti et al, Stem Cells 2012
MSC induce functional changes on microglia Intracellular Ca 2+ * p<0.001 Phagocytosis * p<0.001 TREM2 * p<0.001 N9 +LPS +LPS+MSC N9 +LPS +LPS+MSC N9 +LPS +LPS+MSC N9 +LPS +LPS+MSC Giunti et al, Stem Cells 2012
CX3CL1 expressed by neurons modulates the activity of CX3CR1-positive microglia The disruption of CX3CL1-CX3CR1 signaling induces neurotoxicity in mouse models of neurodegeneration (Cardona et al, Nat Neurosci, 2006)
MSC express and produce fractalkine under inflammatory conditions CX3CL1 production * p<0,05 CX3CL1 expression * p<0,01 MSC +IFN +LPS +IFN MSC +IFN +LPS +IFN +LPS +LPS Giunti et al, Stem Cells 2012
CX3CL1 silencing reverts MSC effect on the microglia expression of inflammatory and neuroprotective genes TNF * p<0.01 ** p<0.01 IL1β * p<0.001 ** p<0.05 N9 +LPS +LPS +MSC +LPS+MSC CX3CL1si N9 +LPS +LPS +MSC +LPS+MSC CX3CL1si CX3CR1 * p<0.01 ** p<0.01 NURR1 * p<0.05 ** p<0.05 EP2 * p<0.01 ** p<0.01 N9 +LPS +LPS +MSC +LPS+MSC CX3CL1si N9 +LPS +LPS +MSC +LPS+MSC CX3CL1si N9 +LPS +LPS +MSC +LPS+MSC CX3CL1si Giunti et al, Stem Cells 2012
Silencing of fractalkine in reverts functional changes exerted by MSCs on microglia Intracellular Ca 2+ * p<0.001 ** p<0.001 Phagocytosis * p<0.001 ** p<0.001 TREM2 * p<0.001 ** p<0.001 N9 +LPS +LPS +MSC +LPS+MSC +acx3cl1 N9 +LPS +LPS +MSC +LPS+MSC +acx3cl1 N9 +LPS +LPS +MSC +LPS+MSC sicx3cl1 N9 +LPS +LPS+MSC +LPS+MSC+ acx3cl1 Giunti et al, Stem Cells 2012
CONCLUSIONS MSC microglia interactions lead to the inhibition of the production of proinflammatory molecules and enhances molecules associated to neuroprotection. MSC promote microglia s functions involved in CNS homeostasis CX3CL1 produced by MSC plays a major role in the induction of a functional switch of microglia from a detrimental one to a beneficial one further pointing out a major role for molecules released by MSC upon interaction with the inflammatory environment.
MSCs improve survival and motor functions in SOD1/G93A(+) mice through inhibition of glutamate driven excitotoxicity Uccelli et al, Mol Med 2012
Mesenchymal stem cells improves experimental epilepsy rescuing hippocampal neurons from apoptosis Voulgari-Kokota et al, Exp Neurol 2012
Systemic administration of MSC increases neuronal survival in global cerebral ischemia Perasso et al 2010
MSC inhibit the autoimmune attack to the CNS and promote neural cell survival Neuron + Astrocytes TNF- - IFN- T demyelinated axon B - - + Oligodendrocytes + NO + ROI Microglia Neurons Neural precursor cells Adapted from Uccelli et al Trends Immunol 2007
MoA of MSC for brain repair: take home messeage Transdifferentiation potential Engraftment/cell contact mechanisms Direct inhibition of the effector functions of cells of the immune system By-stander effect through the release of: Anti-apoptotic and anti-oxidant molecules Trophic factors Factors inducing the mobilization and induction of proliferation of local precursors (committed) cells Non-cell autonomous mechanisms mediated by: Dendritic cells Monocytes/macrophages microglia
"Stemness" Does Not Explain the Repair of Many Tissues by Mesenchymal Stem/Multipotent Stromal Cells (MSCs) There has recently been an explosion of interest in adult stem/progenitor cells that have the potential to repair tissues, with over 3,000 citations to publications (PubMed) and numerous announcements of clinical trials in which the cells are used to treat individuals with a broad range of diseases. At the same time, the data present a paradox as the cells originally attracting attention because of their stem-cell like properties, frequently repair injured tissues without much evidence of either engraftment or differentiation. Darwin Prockop 2007
Neuroimmunology Unit University of Genoa F. Benvenuto S. Casazza D. Giunti L. Lovato F. Ivaldi S. Morando B. Parodi L. Serpero T. Vigo A. Uccelli Dept. Of Neurosciences,- University of Genoa GL. Mancardi ISPE San Raffaele Hospital G. Martino Department of Biology University of Genoa L. Vergani Dept. Of Nuclear Medicine University of Genoa G. Sambuceti Institute of Anatomy University of Bari D. Virgintino Dept of biomedical sciences - University of Teramo M. Maccarrone Hematology S. Martino Hospital, Genoa F. Gualandi University of California San Francisco J. Oksenberg S. Baranzini Gaslini Insitute, Genoa F. Frassoni S. Chiesa G. Candiano E. Traggiai L. Moretta V. Pistoia Dept. of Biochemistry University of Genoa A. De Flora Dept. Neuropharmacology, University Genoa GB. Bonanno CNR Genoa Cesare Usai Tor Vergata University Rome D. Centonze A. Urbani University of Verona Italy B. Bonetti Ospedali Riuniti Bergamo Italy M. Introna The studies on MSC have been supported by grants of the Italian Foundation for Multiple Sclerosis (FISM), Istituto Superiore di Sanità (ISS) National Program on Stem Cells, by the Ministero della Salute, Ricerca Finalizzata, by the Ministero dell Università e della Ricerca, Progetti PRIN, by the Fondazione CARIGE, by the Fondazione CARIPLO and the Regione LIGURIA
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