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Le cellule staminali nella patologia degenerativa ossea
Maria Luisa BrandiDipartimento di Medicina Interna
Convegno UniSalute
Bologna, 30 settembre 2011
Dipartimento di Medicina Interna Azienda Ospedaliera Careggi di Firenze
TERMINOLOGY
Cell-Based Therapies
Stem Cells and Stem Cell Lines
Regenerative Medicine
Tissue EngineeringTissue Engineering
Human Therapeutic Cloning
Clear language and differentiation of respective ethical, legal,
and social issues are required to prevent inaccurate vernacular
usage, source of confused public perception of “cell therapies”
Cell-Based Therapies
Blood Cell Transplantation
……………………………………
……………………………………
Articular Cartilage CellsArticular Cartilage Cells
Bone Marrow Stromal Cells
the the futurefuture…………………………..
StemStem CellsCells
WHAT ARE STEM CELLS ?
STEM CELL: a cell with unique capacity to self-renew and
to generate differentiated progeny, existing at all stages of
development
EMBRYONIC STEM CELLS: totipotent giving rise to all types
of somatic and germ-line cellsof somatic and germ-line cells
FETAL STEM CELLS: multipotent contributing to all somatic
lineages, but not the germ line
ADULT STEM CELLS: do they exist ? Do all times express
the some plasticity ?
Stem Cells Develop and Maintain Their
Ability to Self-Renew within a Specific NicheAnagen Catagen TelogenStratum corneum
Granular layer
Spinous layer
Basal layer
Dermis
A. EPIDERMAL
Sebaceous gland
Bulge
Matrix
Dermal papilla
C. INTESTINAL D. NEURALB. FOLLICULAR
E. HEMATOPOIETIC
Goblet cellsAbsorptive enterocytes
V
Paneth cells
Crypt cells
Entero-endocrine cells
OB
SVCE
Cellule staminali adulte
� Localizzate in specifiche nicchie, provvedono al rinnovo fisiologico e alla riparazione dei tessuti
� Poco numerose
�� Velocità di duplicazione ridotta
� Si riteneva fossero specializzate nel generare cellule tipiche del tessuto di provenienza
� Studi recenti hanno dimostrato che possono dare origine a diversi tipi cellulari
ADULT STEM CELLS
TRANSDIFFERENTIATION
Physiologic homing of allogenic mesenchymal
stem cells to damaged myocardium
Can Adult Stem Cells Transdifferentiate ?
Defying scientific dogma adult stem cells can morph into
many types of cells
I this due to true “reprogramming” or to simple “fusion” ?
Science 295:1989, 2002
SCAFFOLDS AND TISSUE REPAIR
First generation biomaterials: a suitable
continuation of physical properties with a
minimal toxic response in the host
Second generation biomaterials: components
that could elicit a controlled action and that could elicit a controlled action and
reaction in the physiological environment
(either bioactive or resorbable)
Third generation biomaterials: designed to
stimulate specific cellular responses at the
molecular level (both bioactive and resorbable)
• Stroma di midollo osseo• Ependimo e zona subventricolare del sistema nervoso centrale• Strato basale dell’epidermide• Sangue di cordone ombelicale• Sangue periferico
Sorgenti di cellule staminali
• Sangue periferico• Placenta• Tessuto adiposo• Sinovia• Muscolo scheletrico• Follicolo pilifero• Cripta intestinale
�Metodica di prelievo invasiva
�Resa cellulare limitata (circa 1 MSC ogni 10.000 cellule
aderenti alla piastra di coltura)
�Necessità di una fase di espansione cellulare in vitro
(riduzione della capacità replicativa e della capacità di
differenziazione nel tempo, aumento di tempi, costi e rischi
Limiti delle BMSC
differenziazione nel tempo, aumento di tempi, costi e rischi
di contaminazione)
IL TESSUTO ADIPOSO RAPPRESENTA UNA FONTE ALTERNATIVA DI
CELLULE STAMINALI CON CARATTERISTICHE IDEALI
• Morfologicamente simili alle cellule stromali di midollo
• Facilmente ottenibili
• In vitro possono differenziarsi in:�Osso (Zuk PA et al. 2001. Tissue Eng. 7:211-28)
Cellule mesenchimali staminali da tessuto adiposoAdipose Tissue-Derived Stromal Cells (ADSC)
�Osso (Zuk PA et al. 2001. Tissue Eng. 7:211-28)
�Cartilagine (Zuk PA et al. 2001. Tissue Eng. 7:211-28)
�Muscolo scheletrico (Zuk PA et al. 2001. Tissue Eng. 7:211-28)
�Grasso (Zuk PA et al. 2001. Tissue Eng. 7:211-28)
�Tessuto nervoso (Yang LY et al. 2004. Chin Med J. 17:425-9)
Recenti studi confermano che le ADSC possono essere utilizzate come valida alternativa alle BMSC
nella rigenerazione del tessuto osseo
�ADSC are negative for immunologically relevant surface markers and inhibit proliferation of allogenic T cells in vitro
(Niemeyer P et al. 2007. Tissue Eng. 13:111-21)
�ADSC are capable of both adipogenic and osteogenic differentiation through 10 passages (34 population doublings)
ADSC
differentiation through 10 passages (34 population doublings)(Wall ME et al. 2007. Tissue Eng. 13:1291-8)
�The osteogenic potential of adipose-derived mesenchymal cells is maintained with aging(Shi YY et al. 2005. Plast. Reconstr. Surg. 116: 1686-96;
Weinzieri K et al. J Craniomaxillofac Surg. 34: 466-71)
ISOLATION AND ESTABLISHMENT OF
ADIPOSE TISSUE BONE MARROW
BONE MARROW CELLS
BMC
PREADIPOCYTES
PA
ISOLATION AND ESTABLISHMENT OF PRIMARY CELL CULTURES
PHENOTYPIC CHARACTERIZATION OF PRIMARY CELL CULTURES
1. MSC have to be plastic-adherent when maintained under 1. MSC have to be plastic-adherent when maintained under standard culture conditions.
2. MSC must have the ability for osteogenic and adipogenic.
3. MSC must express CD71, CD90, CD144 and CD105
4. MSC must lack expression of the hematopoietic lineage markers CD34 and CD45
Vitamin C
B -Glycerophosphate
Dexamethasone
OSTEOGENIC MEDIUM
HAM F12 COON’S MODIFICATION + 10% FCS +1% Ab +
• Vitamin C: essential for the biosynthesis of collagen and extracellular matrix
• β-glycerophosphate: glycerophosphate acts as an exogenous source of phosphate groups
• Dexamethasone: promotes the differentiation of MSCs into mature osteoblasts with the ability to deposit mineralized matrix in vitro.
� CYTOFLUORIMETRY (?)
� Qualitative and quantitative analysis of osteogenic markers gene expression (RT-PCR and RT-PCR)
� Evaluation of ALP activity by cytochemical staining and fluorometric assay
� Analysis of expression of OCN and OPN expression using LASER SCANNING
OSTEOGENIC DIFFERENTIATION ANALYSIS
� Analysis of expression of OCN and OPN expression using LASER SCANNING CONFOCAL MICROSCOPY (LSCM)
� Qualitative and quantitave evaluation of mineralization using cytochemical staining (ALIZARINA RED S e CALCEIN) and ALIZARIN RED S ASSAY
Osteoblast-like cell line SaOS-2 as control
The aim of the investigation was to study the in vitroosteoblastic differentiation of human adipose tissue- and bonemarrow-derived stem cells (PA and BMC finite lines), and tocompare their in vitro osteogenic response to Ti6Al4V alloy.
ACTIVITY OF ALP
CYTOCHEMICAL STAINING
FLUOROMETRIC ASSAY1 day
40 days
Espressione di ALP come attività enzimatica/unità di superficie (µU/cm2)*
Tognarini I et al. 2008. Biomaterials. 29:809-24
SaOS-2 BMC PA
50 µm
1 day
OCN EXPRESSION(LSCM)
40 days
Tognarini I et al. 2008. Biomaterials. 29:809-24
MINERALIZATION ANALYSIS:Alizarina Red S
SaOS-2 BMC1 PA1
1 day 1 day 1 day
F
200 µm
20 days 40 days 40 days
Tognarini I et al. 2008. Biomaterials. 29:809-24
CONCLUSIONS
The study showed that ADSC and BMSC have similar ability todifferentiate in mature osteoblasts, confirming that the adiposetissue represents an abundant reservoir of stromal cells for bonetissue engineering.
The Ti6Al4V alloy has an in vitro preferential osteoinductive actionThe Ti6Al4V alloy has an in vitro preferential osteoinductive actionof on ADSC.
The novel description of an opens future avenues for investigationin the development of cell-bioengineered titanium alloys.
EVALUATION OF THE EFFECTS OF Sr 2+ ON PREOSTOBLATS FROM ADIPOSE TISSUE
•Proliferation ([3H] Thymidine incorporation assay)
•Osteogenic differentiation (quantitative RT-PCR)
•Mineralization (Alizarina S Red Staining, Alizarina Red assay)
RUNX2 ALP
0
2
4
6
8
10
12
14
0 15 30
days from induction
Exp
ress
ion
ratio
of R
UN
X2/
RP
S18
Control
Sr 1 ug/ml
Sr 10 ug/ml
Sr 100 ug/ml
0
5
10
15
20
25
0 15 30
days from induction
Exp
ress
ion
ratio
of A
LP/R
PS
18
Control
Sr 1ug/ml
Sr 10 ug/ml
Sr 100 ug/ml
The ion Sr2+increases the expression in time-dependent manner of ALP, RUNX2 and OCN during induction
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