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

Convegno UniSalute“Le Cellule Staminali e il loro impiego attuale nella clinica”

Bologna, 30 Settembre 2011

di Bologna, Italia

MSOffice1

Diapositiva 1

MSOffice1 ; 01/11/2009

Definition of a Stem CellDefinition of a Stem Cell

• clonogenic • self-renewing• differentiating into

multiple lineages• regenerates the

• clonogenic • self-renewing• differentiating into

multiple lineages• regenerates the

Long-term Stem Cells

Short-term Stem Cells• regenerates the

organ or tissue of origin after transplantation

• high degree of “plasticity” among these options

• a morphological entity

• regenerates the organ or tissue of origin after transplantation

• high degree of “plasticity” among these options

• a morphological entity

Multipotent Progenitors

Oligolineage Progenitors

Terminally DifferentiatedCells

Spinal cord injury; Parkinson’s disease; Alzheimer’s disease

Experimental/clinicalNeuronal stem cells

Ischemic heart disease;

Ischemic vascular disease; Limb ischemia;

Ischemic retinopathy

Experimental/clinicalEndothelial stem cells

Myocardial infarctionExperimental/clinicalCardiac stem cells

Metabolic liver disease;

Hepatic fibrosis

ExperimentalHepatocytes

Oval cells

Disease category that benefitsModelCell type

Acute tubular necrosis; Glomerulonephritis; Diabetic nephropathy

experimentalGlomerular mesangial cells;

Renal tubular epithelial cells

DiabetesExperimentalDuctal and islet pancreatic cells

Treatment related pulmonary toxicity

Experimental/clinicalPulmonary epithelial cells

Skin injury (wound healing); Treatment related mucosa injury

Experimental/clinicalKeratinocytes;

Buccal cells

Muscular dystrophyExperimental/clinicalSatellite cells

disease; Alzheimer’s disease

CD34+ / CD34-

CD38-

CD133+/Lin-

c-kit low

Thy-1+

HLA-DR -

CD34+

CD38+

c-kit+

HLA-DR +

THYMUS

PLURIPOTENT STEM CELL

LYMPHOID PROGENITOR

CELL

MYELOID PROGENITOR

CELL

CFU-GEMM

CFU-GMCFU-MK

BFU-E BFU-MK CFU-Eo CFU-Bas

IL-3 GM-CSF

EPO SCF IL-9

IL-3 GM-CSF

IL-6 IL-11 CSF

IL-3 GM-CSF G-CSF

SCF FLT-3L GM-CSF TNF-αααα

IL-3 GM-CSF

IL-3 IL -4

IL-1 IL-2 IL-3 IL-4 IL-5 IL-6 IL-11

IL-1 IL-2 IL-3 IL-4 IL-5 IL-6 IL-7 IL-12

CD34-

CD38+

Lin +

CD3CD4 CD8 CD11b CD14

CD15CD19 CD20 CD64 …

CFU-GMCFU-MKCFU-E CFU-DC

CFU-M CFU-C

ERYTHROCYTE

PLATELETS

MACROPHAGE

MONOCYTE NEUTROPHILEOSINOPHIL BASOPHIL B

LYMPHOCYTE T LYMPHOCYTE

EPO TPO GM-CSF M-CSF

IL-3 GM-CSF G-CSF

IL-3 GM-CSF

EPO

IL-3 GM-CSF G-CSF

IL-3 GM-CSF

GM-CSF IL -4

DC

The Hematopoietic System

LT - HSC ST - HSC MPP

Common Lymphoid Progenitor

CLP Pro-T

Pro-B

NK

B

T

GMP

Monocytes

Granulocytes

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 . 3rd Ed. Malden, MA: Blackwell; 2004:69-95.

Long-term Short-term Multipotent Progenitor

Common Myeloid Progenitor

CMPMEP

Megakaryocyte/ Erythrocyte Progenitor

Erythrocytes

Platelets

Monocytes

Dendritic CellsGranulocyte-Monocyte Progenitor

CD34- CD34+

Transplantation

5-FUCulture

CD34 EXPRESSION

CD34-CD34- CD34+CD34+ CD34+CD34+

RESTING SELF-RENEWING

ACTIVATED SELF-RENEWING

DIFFERENTIATING NOT SELF-RENEWING

Goodell M.A., Blood, 1999

HEMATOPOIETIC CD34+

STEM CELLS AND BONE MARROW

MICROENVIROMENT

CD34+CD34+

Moore KA Science 2006

Wilson A and Trumpp A Nature Reviews 2006

SELF-RENEWAL

APOPTOSISSUPPRESSORS

MCL1

TRANSCRIPTION FACTORSTEL/ETV6

HOXB4JAK/STAT (STAT5)

PTENBMI1

SELF-RENEWAL

INTRACYTOPLASMIC SIGNALINGTIE2/ANG1

NOTCHWNT

hMSC SEPARATION FROM BONE MARROW

2nd passage MSC in culture

MNC + MSC precursors adherent MSCplating

7-10 days

7-day culture 14-day culture

DIFFERENTIATION POTENTIAL OF hMSC IN VITRO

OsteoblasticAP+ cells

Adipogenicoil red-O+ cells

Cartilagine col II+ cells

Multi-Organ, Multi-Lineage Engraftment by

a Single Bone Marrow-Derived Stem Cell

Multi-Organ, Multi-Lineage Engraftment by

a Single Bone Marrow-Derived Stem Cell

“HOMED”CD34+

SINGLE CELL

Krause DS et al, Cell 2001.

LUNG ESOPHAGUS STOMACH INTESTINE LIVER

MSCs

Pluripotency of mesenchymal stem cells

derived from adult marrow

Pluripotency of mesenchymal stem cells

derived from adult marrow

LUNG SPLEEN BLOOD CELLS LIVER

Jiang Y et al, Nature 2002

INTESTINE

CABG +Intramyocardial Stem

Cell Injection

CABG +Intramyocardial Stem

Cell Injection

• up to 8 x 106 CD34+/CD133+ BMSC• 10 x 0.2 ml injections

Rosenzweig A. New Engl J Med, 2006

-3-510G-CSF

-2-1011PURIFIED HSCs (CD34+, CD133+)

41033560N. TRIAL

LUNG§CNS**PANCREASNEO-

ANGIOGENESIS*HEART°

TISSUE / ORGAN

ACTIVE CLINICAL TRIALS ON HSCs AND REGENERATIVE MEDICINE

* Arterial occlusive disease

** Ischemic Stroke

§ Pulmonary artery idiophatic ipertension° revascularization

http://www.clinicaltrials.gov 31/08/2010

241929TERMINATED OR NOT YET RECRUITING

2622431RECRUITING

BONE MARROWBONE MARROW--DERIVED STEM CELLSDERIVED STEM CELLSPOTENTIAL ROLE IN LIVER INJURYPOTENTIAL ROLE IN LIVER INJURY

MAPC

MSC Paracrine effect

Anti-fibrotic

Transdifferentiation

Houlihan & Newsome, Gastroenterology 2008

Multipotentstem cells

HSC

EPC

Pluripotentstem cells

Effectswithin the liver

Pro-angiogenic

Anti-fibrotic

Phase I

Patient

G-CSF (5-7 days)

Apheresis (day+4; CD133+ >8/µL)

Immunomagnetic isolation CD133+

CD133+

Cryopreservation

Sterility Test

Viability

Biological assays

Phenotype

Clonogenic assay

Molecular evaluation

Hemopoietic Endothelial

Phase II(at least 4 weeks after apheresis)

≤ 40 ml CD133+(5x104/kg – 1x106/kg)

Hepatic artery infusion (Followed by G-CSF x 3 days)

BIOLOGICAL STUDIES

FOLLOW-UP (12 months)

– 1° week: daily

– 1, 2, 3, 6, 9, 12 months

Stem cell Mobilization: T0, T+5,T+14

Stem cell Infusion: R-2, R+1, R+3,R+7, R+15

- CD133+ cell production -

S TEM CELL I NFUS I ON I N CIRRHOTIC PATIENTS

Hepato-mesentheric trunk

Hepatic artery dx

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 63.000.000 yes I (50,000 CD133+/Kg)

02 alcholic yes yes 53.660.000 yes I (50,000 CD133+/Kg)

03 viral yes yes 54.530.000 yes I (50,000 CD133+/Kg)

Patient characteristics

04 viral no no / / II (150,000 CD133+/Kg)

05 viral yes yes 164.551.000 no II (150,000 CD133+/Kg)

06 viral yes yes 69.193.000 yes II (150,000 CD133+/Kg)

07 viral yes yes 106.897.000 yes II (150,000 CD133+/Kg)

08 viral yes yes 49.842.000 no II (150,000 CD133+/Kg)

09 viral yes yes 36.449.600 yes III (400,000 CD133+/Kg)

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±12 0.06±0.03

Dept. of Medicine

University of Bologna

• Stefania Lorenzini Paolo Caraceni

• Lucia Brodosi Massimo Domenicali

• Stefano Gitto Pietro Andreone

• Mauro Bernardi

Institute of Hematology “L & A Seràgnoli”

University of Bologna

•Maria Rosa Motta

• Lucia Catani

• Michele Baccarani

Dept. Of Radiology Immunohematology and Blood Bank Dept. Of Radiology

University of Bologna

• Francesco Losinno

• Cristina Rossi

• Andrea Casadei

• Romeo Canini

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

MRC, Centre for Inflammation Research

The Queen’s Medical Research Institute

University of Edinburgh, UK

•Stuart J Forbes

• John P Iredale