Laboratorio di ricerca sulle cellule staminali ( www.stemcellreslab-verona.it ) Dr. Mauro Sezione di Ematologia, Dipartimento di Medicina Università degli Studi di Verona Azienda Ospedaliera Universitaria Integrata Verona mauro.krampera@univr.it
Laboratorio di ricerca sulle cellule staminali Tappe principali 2-21: stage del Dr. come Academic Visitor e Research Associate presso il Dept. of Haematology e il Dept. of Immunology, Imperial College School of Medicine, London, U.K., con progetto sull effetto immunomodulatorio delle cellule staminali mesenchimali nel trapianto di midollo osseo Dal 21: creazione di una rete di collaborazioni locali, nazionali ed internazionali 25: creazione dell indirizzo di Dottorato in Biologia ed Applicazioni Cliniche delle Cellule Staminali e del Laboratorio di Ricerca sulle Cellule Staminali (www.stemcellreslab-verona.it) Dal 25: stage di collaboratori presso: Cardiovascular Research Institute - New York Medical College, USA (Responsabile: Prof. Piero Anversa - Dr. Federico Mosna, 25-27); Stem Cell Institute Leuven, Katholieke Universiteit Leuven, Belgio (Responsabile: Prof. Catherine Verfaillie D.ssa Elda Mimiola, 28-29); Center for Regenerative Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA (Responsabile: Prof. Piero Anversa - Dr. Mario Ricciardi, 211-212); Laboratory of Angiogenesis and Neurovascular Link, Vesalius Research Center, Leuven, Belgium (Responsabile: Prof. Peter Carmeliet - Dr. Francesco Bifari, 212-presente); Dept. of Clinical and Experimental Medicine, Pneumology Unit, Katholieke Universiteit Leuven, Belgium (Prof. Bart Vanaudenaerde Dr. Mario Ricciardi) 26: creazione del gruppo europeo CASCADE (Cultivated Adult Stem Cells for Damaged Epithelia) FP7 (Dr. Luc Sensebé, EFS, Tours! Toulouse, France) 28 novembre 27: inaugurazione Cell Factory Verona (DIMT Policlinico) 28: creazione del gruppo europeo REBORNE (Regenerating Bone defects using New biomedical Engineering approaches) FP7 (Dr. Pierre Layrolle, INSERM, Nantes, France) Dal 211: coinvolgimento nella Mesenchymal Stromal Cell (MSC) Committee of the International Society for Cellular Therapy (ISCT), presieduta dal Dr. Luc Sensebé (EFS, Toulouse, France) Dal 213: coinvolgimento nel Mesenchymal Stromal Cell (MSC) Ruler Working Group, coordinato dal Prof. Armand Keating (University of Toronto, Canada).
Produzione in GMP di MSC per uso clinico Cell Factory - Verona Dipartimento Interaziendale di Medicina Trasfusionale (Dr. Giuseppe Aprili) Policlinico G.B. Rossi - Verona, AOUI Verona
Cell Factory Verona SIT Policlinico (dr. G. Aprili)
Laboratorio di ricerca sulle cellule staminali Progetti finanziati
CASCADE - 7 th FP EU 28 (Cultivated Adult Stem Cells as Alternative for Damaged tissue) Coordinator: Dr. Luc Sensebé, EFS, Tours! Toulouse, France
CASCADE
REBORNE - 7 th FP EU 28 (Regenerating Bone defects using New biomedical Engineering approaches) Coordinator: Dr. Pierre Layrolle, INSERM, Nantes, France
REBORNE
Laboratorio di ricerca sulle cellule staminali Produzione scientifica 23 213: 58 lavori pubblicati o in press (I.F. globale 334,122 - SJIF 212), riguardanti: - biologia delle cellule staminali - immunologia delle cellule staminali - applicazione clinica delle cellule staminali - cellule staminali leucemiche e supporto stromale alla crescita tumorale 3 lavori attualmente sottomessi a riviste internazionali e in corso di valutazione 4 lavori in attesa di completamento per sottomissione 2 capitoli di libro (Springer Ed.) su metodiche di coltura e analisi delle cellule staminali 1 brevetto (WO21114612, 9/1/211 - Device for cell culture and analysis )
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Cellule Staminali da Leptomeningi (LeSC) F. Bifari, I. Decimo, C. Chiamulera, E. Bersan, G. Malpeli, J. Johanson, V. Lisi, B. Bonetti, G. Fumagalli, G. Pizzolo, M.. Novel stem/progenitor cells with neuronal differentiation potential reside in the leptomeningeal niche. J Cell Mol Medicine 29;13(9B): 3195-328.
Cellule perivascolari CD146+ (precursori MSC) Polmone umano M. Ricciardi, G. Malpeli, F. Bifari, G. Bassi, L. Pacelli, A.H. Nwabo Kamdje, M. Chilosi, M.. Comparison of epithelial differentiation and immune regulatory properties of mesenchymal stromal cells derived from human lung and bone marrow. PLoS One 212;7 (5):e35639.
Cellule perivascolari CD146+ (precursori MSC) Immune modulatory properties M. Ricciardi, G. Malpeli, F. Bifari, G. Bassi, L. Pacelli, A.H. Nwabo Kamdje, M. Chilosi, M.. Comparison of epithelial differentiation and immune regulatory properties of mesenchymal stromal cells derived from human lung and bone marrow. PLoS One 212;7(5):e35639.
Cellule perivascolari CD146+ (precursori MSC) Epithelial differentiation Dept. of Clinical and Experimental Medicine Pneumology Unit Katholieke Universiteit Leuven, Belgium (Prof. Bart Vanaudenaerde) M. Ricciardi, G. Malpeli, F. Bifari, G. Bassi, L. Pacelli, A.H. Nwabo Kamdje, M. Chilosi, M.. Comparison of epithelial differentiation and immune regulatory properties of mesenchymal stromal cells derived from human lung and bone marrow. PLoS One 212;7(5):e35639.
Menard C, Pacelli L, Bassi G, Dulong J, Bifari F, Bezier I, Zanoncello J, Ricciardi M, Latour M, Bourin P, Schrezenmeier H, Sensebé L, Tarte K, M. Clinical-grade mesenchymal stromal cells produced under various GMP processes differ in their immunomodulatory properties: Standardization of immune quality controls Stem Cells and Development 213;22(12):1789-81 &;3-4'%)*()<-*(,(4#=)>4(?-&.('@)*-8()2'/*()*()A-&%4#) G&#.#6(%4#-=)
)9(:#&;3-4'%)*()<-*(,(4#=)>4(?-&.('@)*-8()2'/*()*()A-&%4#) G&#.#6(%4#-=) Cryopreserved ex-vivo expanded GMP-grade human MSCs at passage 1 (P1) ADSC-PL from French Blood Bank of Toulouse (France) (5 samples) BMMSC-FCS + bfgf from French Blood Bank of Tours (France) (5 samples) BMMSC-PL from the Institute of Clinical Transfusion Medicine and Immunogenetics in Ulm (Germany) (5 samples) Two Immunological laboratories (Verona - Italy, and Rennes - France) performing the same tests in parallel on the same samples, by using the same batches of reagents Immunophenotyping Gene expression by RT-PCR (INDO, PTGS2/Cox2, NOS2, and TNFAIP6/TSG6 mrna expression) Functional tests (purified immune effector cell proliferation and vitality, neutralization experiments with specific inhibitors, MSC immunogenicity, IDO activity expressed as kynurenine/tryptophan ratio by high-performance liquid chromatography) MSCs were thawed, seeded at 1, cells/cm 2 and expanded, in the same culture medium used for production, until almost confluence to avoid any bias associated with the use of freshly thawed MSCs. All phenotypic and functional experiments were then performed at the end of P2. Menard C et al. Clinical-grade mesenchymal stromal cells produced under various GMP processes differ in their immunomodulatory properties: Standardization of immune quality controls. Stem Cells and Development 213;22(12):1789-81.
Primed ADSC-PL HLA-ABC high / CD4 high / inos2 high / TSG6 high primed BMMSC-FCS HLA-ABC low / PD-L1 high / CD2 dim / NK ligands high primed BMMSC-PL HLA-ABC high DR high / PD-L1 high / CD2 dim / CD4 high / NK ligands high all the 3 types main role for IFN-g-induced IDO activity
SC Types/immunological behaviors Immunosuppression behavior Mechanism Immunogenicity Immune Effector Cells T NK B T NK BM-MSC + + IDO *** pbm-msc ++ + ++ IDO * OE-MSC ++ +++ - // *** poe-msc ++ +++ +++ IDO * LeSC +++ - IDO / COX-2 *** plesc +++ +++ +++ IDO * AFS + IDO / IFN-γ *** pafs ++ + IDO / IFN-γ * CSC +++ + - IDO / HO-1 *** pcsc +++ + ++ IDO ** LSC +++ + - IDO *** plsc +++ + +++ IDO ** Inhibition range (%) Symbol supportive effect - - 2 2-5 + 5-75 ++ > 75 +++ Susceptibility to NKmediated lysis (%) Symbol - 3 3-5 > 5 * ** *** Comparison of immune regulatory properties of stem cells from different tissues. Di Trapani M., Bassi G. et al. Stem Cells and Development 213 (in press) BM-MSC: bone marrow mesenchymal stromal cells OE-MSC: olfactory ecto-mesenchymal stem cells LeSC: leptomeningeal stem cells AFS: c-kit+ amniotic fluid stem cells CSC: c-kit+ cardiac stem cells LSC: c-kit+ lung stem cells p: priming with IFN-γ + TNF-α
Cytotherapy. 213 Sep;15(9):154-61. doi: 1.116/j.jcyt.213.2.1. Epub 213 Apr 17.
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Microvescicole da MSC Di Trapani M., Bassi G
Shwachman-Diamond Syndrome (SDS) Cr. 7 SBDS gene mutation ribosomal biogenesis mitotic-spindle stabilization Anemia Neutropenia Thrombocytopenia bone homeostasis cell survival chemiotaxis Pancreatic disfunction Growth retardation bone marrow failure myelodysplasia Skeletal defects Cognitive defects Hematologic abnormalities acute myeloid leukemia
Shwachman-Diamond Syndrome (SDS) BM immunophenotype Hematopoietic-lineage maturation a) e) 1 2K 3 1K 1K 5K 5K 2K 15K 5K 5K 1 3 1 4 1 5 CD34+ cells 1 3 1 4 1 3 1 4 1 5 1 3 CD45 CD 1 4 1 5 CD19 19 1 5 CD34 1 5 =TGG8' 5 relative % of cells 1 5 ** 4 1 4 1 3 1 5 Granulocytes 1 1 4 1 1 3 1 3 1 3 1 4 1 1 5 5 1 3 1 1 4 3 4 1 1 3 1 4 1 5 1 3 1 4 1 5 =TEF' =TGFR' ** 8 3 5 =TIK' 1 1 =TGI' B cells 4 Monocytes 2 5 Erythrocytes =TGGP' 1 5 1 4 CD33 =TGK' 1 4 1 3 CD45 1 3 1K CD34 1 3 15K HSC 1 4 =TGF' 1 SSC CD38 1K 25K 5 2K 15K CD71 Hematopoietic Precursors 1 4 15K g) 25K SSC 1 5 2K SSC-A c) 25K 25K SSC CD34+ cells CD34+ relative % of cells 6 ns HSC granulo. Hematopoietic Precursors CD34+CD19+ mono. erythro. 1 6 lympho. ** 2 1 ns 8 CD15+ ns 15 4 2 ns * *** 6 ns ns 4 2 1 5 ** CD117+ 2 15 *** * 3 relative % of cells CD34+CD117+ ns ** relative % of cells *** 2 promyelo. myelo. metamyelo. neutrophils * promono. imm. mono. mat. mono. relative % of cells 1 ns *** 4 4 relative % of cells 2 MIF on CD34+CD38- relative % of cells 3 ** 1 ns 5 * pro-erythro. bas. erythro. polyc. erythro. pro-b ly. pre-b ly. B lympho. plasmacells
Leukemia Stem Cells (LSC) Acute Leukemia CD38!"##$%&'(!)*+(,--.( LSC in Acute Myeloid Leukemia (AML) in CD34+/CD38-? CD34
Systemic Mastocytosis (SM) Abnormal MC tissue infiltration Clonal MC 'U&5,%'?.((",(' Abnormal Morphology Skin BM - MC-mediators release symptoms: flushing, orticaria CD25/CD2 expression - MC-infiltration symptoms: liver defects, BM failure - Anaphylactic allergic reactions: hymenoptera, drugs - Osteoporosis KIT mutation (codon 816) Biological studies on SM
Produzione di MSC per uso clinico Medicina Tradizionale (farmaco ad effetto sistemico o locale per alleviare i sintomi) Advanced Therapies (uso di cellule per risolvere la patologia) - Trapianti - Radiofarmaci - Sangue ed emoderivati - Cell-Based Medicinal Products (CBMP) Terapia cellulare Terapia genica (cellule modificate o espanse) (materiale genetico + cellule) Ingegneria tissutale (cellule + scaffold per riparare tessuti)
Advanced Therapies Impianto Prelievo (Assemblaggio) Isolamento Espansione ex-vivo (+ fattori di crescita) Approccio farmaceutico in GMP
Produzione di MSC per uso clinico - Che tipo di trattamento è?! Advanced Therapy (AT) - Che tipo di regolamentazione ha?! EU + ITA (GMP, AIFA, ISS, EU Pharmacopeia, EMA) - In quale struttura è possibile farla?! Cell Factory accreditata AIFA - Che tipo di progetto si deve fare?! Dossier per sperimentazione di fase I con studi in vitro e pre-clinici (autorizzazione ISS) - Quali controlli si devono fare?! Contaminazione + stabilità genetica + reattività immunologica - Come standardizzare la produzione?! Studi cooperativi
Rigenerazione ossea mediante MSC Dossier per sperimentazione clinica di fase I Eudract 212-2424-33 - Ricostruzione di deficit dell osso scafoide mediante l uso di MSC autologhe espanse ex-vivo Vantaggi 1) riduzione di tempi e rischi chirurgici (unico intervento); 2) no anestesia generale per espianto osseo dalla cresta iliaca per innesto; 3) no dolore da espianto da cresta iliaca per innesto; 4) possible miglioramento della guarigione della pseudoartrosi dello scafoide. Selezione dei pazienti (U.O. di Chirurgia della Mano - AOUI Verona) Espianto di midollo osseo Espansione ex-vivo di MSC caratterizzazione (QC) e criopreservazione (Cell Factory - AOUI Verona) Preparazione trapianto composito Intervento chirurgico Follow-up (6 mesi per efficacia / 2 anni per sicurezza) (U.O. di Chirurgia della Mano - AOUI Verona)
Laboratorio di ricerca sulle cellule staminali www.stemcellreslab-verona.it