Che cosa sono le cellule staminali e come si differenziano

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1 MSOffice1 Convegno UniSalute Le Cellule Staminali e il loro impiego attuale nella clinica Bologna, 30 Settembre 2011 Che cosa sono le cellule staminali e come si differenziano Roberto M. Lemoli Dipartimento di Ematologia e Scienze Oncologiche, Istituto di Ematologia L. e A. Seràgnoli, Università di Bologna, Italia

2 Diapositiva 1 MSOffice1 ; 01/11/2009

3 Definition of a Stem Cell Long-term Stem Cells Short-term Stem Cells Multipotent Progenitors Oligolineage Progenitors Terminally Differentiated Cells clonogenic self-renewing differentiating into multiple lineages regenerates the organ or tissue of origin after transplantation high degree of plasticity among these options a morphological entity

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5 Cell type Hepatocytes Oval cells Cardiac stem cells Endothelial stem cells Neuronal stem cells Ductal and islet pancreatic cells Pulmonary epithelial cells Glomerular mesangial cells; Renal tubular epithelial cells Keratinocytes; Buccal cells Satellite cells Model Experimental Experimental/clinical Experimental/clinical Experimental/clinical Experimental Experimental/clinical experimental Experimental/clinical Experimental/clinical Disease category that benefits Metabolic liver disease; Hepatic fibrosis Myocardial infarction Ischemic heart disease; Ischemic vascular disease; Limb ischemia; Ischemic retinopathy Spinal cord injury; Parkinson s disease; Alzheimer s disease Diabetes Treatment related pulmonary toxicity Acute tubular necrosis; Glomerulonephritis; Diabetic nephropathy Skin injury (wound healing); Treatment related mucosa injury Muscular dystrophy

6 CFU-GEMM MYELOID PROGENITOR CELL PLURIPOTENT STEM CELL LYMPHOID PROGENITOR CELL CD34 + / CD34 - CD38 - CD133+/Lin - c-kit low Thy-1 + HLA-DR - BFU-E BFU-MK SCF CFU-Eo CFU-Bas IL-3 IL-3 IL-3 FLT-3L GM-CSF GM-CSF GM-CSF GM-CSF EPO IL-6 G-CSF TNF-α SCF IL-9 IL-11 CSF IL-1 IL-2 IL-3 IL-4 IL-5 IL-6 IL-11 THYMUS IL-1 IL-2 IL-3 IL-4 IL-5 IL-6 IL-7 IL-12 CD34 + CD38 + c-kit + HLA-DR + CFU-E IL-3 GM-CSF EPO EPO TPO CFU-MK IL-3 GM-CSF GM-CSF M-CSF CFU-M CFU-GM IL-3 GM-CSF G-CSF CFU-C IL-3 GM-CSF G-CSF CFU-DC IL-3 GM-CSF IL-3 IL-4 CD34 - CD38 + Lin + ERYTHROCYTE PLATELETS EOSINOPHIL BASOPHIL B MONOCYTE DC NEUTROPHIL LYMPHOCYTE T LYMPHOCYTE CD3 CD4 CD8 CD11b CD14 CD15 CD19 CD20 CD64 MACROPHAGE

7 The Hematopoietic System CLP Common Lymphoid Progenitor Pro-T Pro-B T B NK LT - HSC ST - HSC MPP GMP Granulocytes Monocytes Long-term Short-term Multipotent Progenitor CMP Common Myeloid Progenitor Granulocyte- Monocyte Progenitor MEP Megakaryocyte/ Erythrocyte Progenitor Dendritic Cells Erythrocytes Platelets Manz MG, Akashi K, Weissman IL. Biology of Hematopoietic Stem and Progentior Cells. In: Blume KG, Forman SJ, Applebaum FR et al. Thomas Hematopoietic Cell Transplantation. 3 rd Ed. Malden, MA: Blackwell; 2004:69-95.

8 CD34 EXPRESSION CD34 - CD34 + Culture 5-FU Transplantation CD34 - CD34 + CD34 + RESTING SELF-RENEWING ACTIVATED SELF-RENEWING DIFFERENTIATING NOT SELF-RENEWING Goodell M.A., Blood, 1999

9 HEMATOPOIETIC CD34 + STEM CELLS AND BONE MARROW MICROENVIROMENT CD34+ CD34 +

10 Moore KA Science 2006

11 Wilson A and Trumpp A Nature Reviews 2006

12 APOPTOSIS SUPPRESSORS MCL1 TRANSCRIPTION FACTORS TEL/ETV6 HOXB4 JAK/STAT (STAT5) PTEN BMI1 SELF-RENEWAL INTRACYTOPLASMIC SIGNALING TIE2/ANG1 NOTCH WNT

13 hmsc SEPARATION FROM BONE MARROW 2nd passage MSC in culture MNC + MSC precursors plating 7-10 days adherent MSC

14 DIFFERENTIATION POTENTIAL OF hmsc IN VITRO 7-day culture 14-day culture Osteoblastic AP+ cells Cartilagine col II+ cells Adipogenic oil red-o+ cells

15 Multi-Organ, Multi-Lineage Engraftment by a Single Bone Marrow-Derived Stem Cell HOMED CD34 + SINGLE CELL LUNG ESOPHAGUS STOMACH INTESTINE LIVER Krause DS et al, Cell 2001.

16 Pluripotency of mesenchymal stem cells derived from adult marrow MSCs LUNG SPLEEN BLOOD CELLS INTESTINE LIVER Jiang Y et al, Nature 2002

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18 CABG +Intramyocardial Stem Cell Injection up to 8 x 10 6 CD34+/CD133+ BMSC 10 x 0.2 ml injections

19 Rosenzweig A. New Engl J Med, 2006

20 ACTIVE CLINICAL TRIALS ON HSCs AND REGENERATIVE MEDICINE TISSUE / ORGAN HEART NEO- ANGIOGENESIS* PANCREAS CNS** LUNG N. TRIAL PURIFIED HSCs (CD34+, CD133+) G-CSF RECRUITING TERMINATED OR NOT YET RECRUITING * Arterial occlusive disease ** Ischemic Stroke Pulmonary artery idiophatic ipertension revascularization 31/08/2010

21 BONE MARROW-DERIVED STEM CELLS POTENTIAL ROLE IN LIVER INJURY MAPC Transdifferentiation MSC Paracrine effect Anti-fibrotic Pro-angiogenic HSC Multipotent stem cells EPC Pluripotent stem cells Effects within the liver Houlihan & Newsome, Gastroenterology 2008

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23 Phase I Patient G-CSF (5-7 days) Apheresis (day+4; CD133 + >8/µL) Immunomagnetic isolation CD133 + Sterility Test CD133 + Cryopreservation Viability Biological assays Phenotype Clonogenic assay Molecular evaluation Hemopoietic Endothelial

24 Phase II (at least 4 weeks after apheresis) 40 ml CD133 + (5x10 4 /kg 1x10 6 /kg) Hepatic artery infusion (Followed by G-CSF x 3 days) BIOLOGICAL STUDIES FOLLOW-UP (12 months) Stem cell Mobilization: T0, T+5,T+14 Stem cell Infusion: R-2, R+1, R+3, R+7, R+15 1 week: daily 1, 2, 3, 6, 9, 12 months

25 -CD133 + cell production -

26 STEM CELL INFUSION IN CIRRHOTIC PATIENTS Hepato-mesentheric trunk Hepatic artery dx

27 Patient characteristics Patient Hepatic cirrhosis (viral/ alcholic) Mobilization with G-CSF Apheresis Total number of isolated CD133 + stem cells Intrahepatic reinfusion Cohort (number of reinfused CD133 + stem cells) 01 viral yes yes yes I (50,000 CD133 + /Kg) 02 alcholic yes yes yes I (50,000 CD133 + /Kg) 03 viral yes yes yes I (50,000 CD133 + /Kg) 04 viral no no / / II (150,000 CD133 + /Kg) 05 viral yes yes no II (150,000 CD133 + /Kg) 06 viral yes yes yes II (150,000 CD133 + /Kg) 07 viral yes yes yes II (150,000 CD133 + /Kg) 08 viral yes yes no II (150,000 CD133 + /Kg) 09 viral yes yes yes III (400,000 CD133 + /Kg)

28 Phenotype of isolated CD133 + stem cells %CD133 + %CD34 + %CD90 + %CD117 + %CD184 + %CD105 + %C-MET + 86±6 93±5 19±17 26±18 8±7 9± ±0.03

29 Dept. of Medicine University of Bologna Stefania Lorenzini Paolo Caraceni Lucia Brodosi Massimo Domenicali Stefano Gitto Pietro Andreone Mauro Bernardi Francesco Losinno Cristina Rossi Andrea Casadei Romeo Canini Dept. Of Radiology University of Bologna MRC, Centre for Inflammation Research The Queen s Medical Research Institute University of Edinburgh, UK Stuart J Forbes John P Iredale Institute of Hematology L & A Seràgnoli University of Bologna Maria Rosa Motta Lucia Catani Michele Baccarani Immunohematology and Blood Bank Azienda Ospedaliero-Universitaria di Bologna Valeria Giudice Pasquale Paolo Pagliaro Cell Factory, Fondazione IRCCS Ca Granda Ospedale Maggiore, Policlinico Milano Rosaria Giordano Tiziana Montemurro Lorenza Lazzari

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