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

Body Building: the Bionic Human

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

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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 ?

WHAT ABOUT ADULT WHAT ABOUT ADULT

STEM CELLS ?

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.

Identificazione di un microRNA come regolatore della differenziazione osteogenica nelle ADSC

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

This is an exciting time for competent cell biologists and competent clinicians.

The two can work together