differenziamento in vitro di cellule ossee

Transdifferenziazione
e
differenziazione di cellule ossee

Osteoclasti
Osteoblasti
Osteociti
Cellule ossee
Riassorbimento
osseo
Formazione di
matrice ossea
Meccanocettori, riparano
microdanneggiamenti
delle lacune osteocitarie

5%
94%
1%

Seeman E and Delmas P. N Engl J Med 2006;354:2250-2261

RIMODELLAMENTO OSSEO IN CONDIZIONI FISIOLOGICHE
Osteoclast
Precursors
Differentiated
Osteoclast
Riassorbimento
osseo
Osteoblasts
Osteodeposizione

Differenziamento da precursori
midollari
• Cellule osteoclastiche
e
• Cellule osteoblastiche

Skeletal Integrity in Oncology
clinicaloptions.com/oncology

M-CSF M-CSF
cellula
progenitrice
monocita
macrofagica
(GM-CFU)
OSTEOCLASTOGENESI
OPG OPG
RANKL RANKL
Preosteoclasto Cluster di
preosteoclasti
Osteoclasto
maturo
TRAP +
CTR +
αvβ3+

Pre OCs
c-FMS
c-FMS
RANK
RANK
of macrophage
of macrophage

NFAT2
Nature Reviews Immunology 7, 292-304 (April 2007)
DAP12 FcRγ
DC-STAMP
FUSIONE

© 2005 Rockefeller University Press
Potential mechanism of fusion of preosteoclasts and of macrophages.
Vignery A J Exp Med 2005;202:337-340

•Acidificazione
–Anidrasi carbonica
–Pompa H+/ATPasi
•Proteolisi
–Catepsina

Dopo 24 ore di coltura si recupera
la frazione cellulare non aderente
e si coltiva con
MCSF e RANKL

TRAP+ Osteoclasts in culture

Osteoclasta
coltivato su
fettina di osso
Lacuna di riassorbimento
scavata dalla cellula

Bone Marrow
Stromal Cells
AP1
Runx2
Fra-1
JunD
Wnt
Pre-Osteoblasts
ALP+
Coll I+
Early phase of osteoblastogenesis
Osteoblastogenesis
Late phase of osteoblastogenesis
Wnt
Runx2
Osterix
Fra-2
JunD
AP1
Osteoblasts
ALP++
Coll I++
Osteocalcin
BSP

DKK-1
kremen
LRP5/6
APC
Axin
Citoplasma
Sclerostin
LRP5/6
GSK-3
Nucleo
WNT WIF-1
Frizzled
P
β-Catenin
TCF/LEF
RUNX2, AP-1
Inhibition Activation of of canonical Wnt signaling
WNT sFRP
Axin
WNT
LRP5/6
Frat-1
Dsh
GSK-3
Frizzled
β-Catenin
β-Catenin
β-Catenin
β-Catenin

Cellule stromali
del midollo osseo
(BMSCs)
AP1
Runx2
Fra-1
JunD
Wnt
Pre-osteoblasti
ALP+
Coll I+
Wnt
Osterix
Fra-2
JunD
OSTEOBLASTOGENESI
AP1
OSTEOBLASTI OSTEOCITI
ALP++
Coll I++
Osteocalcin
BSP II

OSTEOBLASTI E ADIPOCITI HANNO UN PROGENITORE COMUNE
BMSC
C/EBPα
Wnt
Cbfa1/Runx2
PPARγ
Wnt
Cbfa1/Runx2
PPARγ
OSTEOBLAST
FORMATION
ADIPOCYTE
FORMATION

CELLULE MESENCHIMALI
STAMINALI
Sono conosciute anche come:
•colony forming fibroblastic cells
•stromal fibroblasts
•marrow stromal stem cells
•mesenchymal progenitor cells
Costituiscono una popolazione residente nel midollo osseo
capace di differenziare in cellule del tessuto adiposo,
del tessuto cartilagineo, del tessuto osseo e nello
stroma che supporta l’ematopoiesi.

ISOLAMENTO DELLE MSC DA
MIDOLLO OSSEO UMANO
1. Centrifugazione su gradiente di Ficoll
2. Coltura su piastre alla densità di 10000 cell/cm 2
3. Rimozione cellule non aderenti

2. Coltura su piastre di polistirene non rivestite alla
densità di 10000 cell/cm 2
Dopo 24-48 ore
3. Rimozione cellule non aderenti
Dopo 10-14 giorni
Cellule subconfluenti
Le cellule possono duplicarsi ed essere
espanse per circa 20 passaggi mantenendo
le caratteristiche di multipotenza senza
ridurre il tasso di crescita. Le cellule così
ottenute non hanno proprietà di cellule
immortalizzate e hanno un tempo di
crescita definita.

Differenziamento osteogenico
Il differenziamento delle MSCs in osteoblasti non è sorprendente; il
midollo osseo è contenuto all’interno del canale diafisario delle ossa lunghe
dove la struttura dell’osso è estremamente simile a quella dell’osso
spugnoso che si rimodella continuamente, perciò non sorprende che
campioni di midollo prelevati dall’osso possano contenere precursori di
osteoblasti.
Acido ascorbico
Desametasone
Β Glicerofosfato
MSCs Osteoblasti

Differenziamento osteogenico
Acido ascorbico (vitamina C): funziona come cofattore
nella idrossilazione dei residui di prolina e lisina nelle
molecole di collageno, promuovendo la formazione della
matrice extracellulare, la maturazione e la deposizione di
collagene; induce l’attività della fosfatasi alcalina della
membrana plasmatica degli osteoprogenitori.
Β glicerofosfato: I fosfati organici promuovono la
mineralizzazione dal momento che il fosfato viene
incorporato nei cristalli di idrossiapatite della matrice.
Desametasone: promuove il differenziamento, agisce sui
promotori responsivi dei fattori di trascrizione necessari
per il committment delle MSCs nel lineage osteogenico;
promuove la calcificazione in vitro.

Conoscere i processi
differenziativi è importante per
• Studiare i meccanismi responsabili
dell’alterazione del rimodellamento
osseo in condizioni patologiche
•Sviluppare nuovi farmaci

Bone Is Removed by Osteoclast Activity
Skeletal Integrity in Oncology
Bone Remodeling is Altered in
clinicaloptions.com/oncology
and Replaced by Osteoblast Activity
Bone
Resorption
RANK/RANKL/OPG
pathological conditions
Normal Bone Remodeling
Courtesy of Dr. G R Mundy, Vanderbilt University.
Bone
Wnt
Formation
signaling

Incidence of Bone Metastases in Cancers
• Myeloma 95-100
• Breast 65-75
• Prostate 65-75
• Thyroid 60
• Bladder 40
• Lung 30-40
• Renal 20-25
• Melanoma 14-45
Incidence of
Bone Metastases (%)

OSTEOLISI MASSIVA

Bone Is Removed by Osteoclast Activity
Skeletal Integrity in Oncology
clinicaloptions.com/oncology
Bone Remodeling is Uncoupled in MM
and Replaced by Osteoblast Activity
RANKL/OPG
Normal Bone Remodeling
Courtesy of Dr. G R Mundy, Vanderbilt University.
Wnt signaling

Cellular mechanisms of myeloma bone disease
The original relationship between
myeloma cells and osteoclasts
Current understanding of mechanisms of
myeloma bone disease

Pathogenesis of Multiple Myeloma Bone Disease
Alteration of
RANK/RANKL/OPG
Inhibition of
Wnt signaling

SCOPO DEL LAVORO
Studiare, nel MM, ulteriori meccanismi responsabili:
1
Dell’incrementata formazione
e sopravvivenza degli
osteoclasti
OSTEOLISI
2
Della ridotta formazione e
attivazione degli osteoblasti

Formazione di OCs da PBMCs di soggetti affetti da
Mieloma Multiplo
30 days
T cells support osteoclastogenesis in
an in vitro model derived from human
multiple myeloma bone disease through
RANKL production
Colucci S et al. Blood 2004
60 days

-
+
Formazione di OCs da PBMCs di soggetti affetti da
Mieloma Multiplo
MCSF
RANKL
MCSF
RANKL
Colucci S et al. Blood 2004

-
+
T cells depleted MM PBMC cultures
MCSF
MCSF
RANKL RANKL
RANKL
MCSF
MCSF
RANKL RANKL
RANKL
T cells support osteoclastogenesis

Alterazione dell’asse RANKL/OPG nel Mieloma Multiplo
Linfociti T
di MM
TRAIL
Apoptosi
Colucci S et al. Blood 2004
OPG
RANKL
Inibizione OCs
RANK RANK
RANK
Plasmacellule
Mielomatose
Osteolisi
Precursori
osteoclastici

Alterazione dell’asse RANKL/OPG e TNFα nel Mieloma Multiplo
apoptosis
Fas-L
T cells
Colucci S et al Leukemia 2009
DcR3 Plasmacells
TNF-α
RANKL
TNF-α
CD14+
preosteoclasts
Multiple Myeloma osteolysis

RANK/RANKL/OPG
NEL RIMODELLAMENTO OSSEO
IN CONDIZIONI FISIOLOGICHE E
PATOLOGICHE: NUOVE PROSPETTIVE
TERAPEUTICHE

Il RANKL è implicato nella
perdita di massa ossea in un’ampia
Perdita ossea patologica
Osteoporosi
postmenopausale
Osteoporosi
maschile
gamma di patologie
Artrite
reumatoide
Perdita ossea
da trattamento
farmacologico
Glucocorticoide
Osteoporosi
indotta da
glucocorticoidi
Inibitori
aromatasi
Terapia di
deprivazione
androgenica
Perdita ossea
collegata alla
terapia
Distruzione
ossea
indotta da
tumore
Metastasi ossea/
Mieloma multiplo

Unopposed RANK Ligand Activity
Causes Long Bone Fragility Fractures
OPG knockout
mouse model
Radiograph of 1-month-old OPG knockout
mouse with spontaneous fragility fractures
Bucay N, et al. Genes Dev 1998;12:1260-1268. Reprinted with permission.
X

Role of OPG in the Regulation of
Bone Mineral Density
X
Normal OPG absent OPG excess
No BMD Change
Decreased BMD
Increased BMD
Bolon B, et al. Arthritis Rheum. 2002; 46: 3121-3135. Reprinted with permission of Wiley-Liss, Inc., a
subsidiary of John Wiley & Sons, Inc.

Inhibition of RANK Ligand – a Potential Future
CFU-GM
Mimic the activity
and binding
specificity of OPG
for RANK-L
Therapeutic Option
Pre-fusion
Osteoclast
Bone Formation
Osteoblasts
Osteoclast
Formation
Inhibited
RANKL
RANK
OPG
Bone Resorption
Inhibited
denosumab
Osteoclast
Function and Survival
Inhibited
CFU-GM = colony forming unit granulocyte-macrophage; M-CSF = macrophage colony stimulating factor.
Boyle WJ, et al. Nature 2003;423:337-342.

Denosumab Is the First Fully Human
Monoclonal Antibody Targeting RANK Ligand
1997
in Clinical Development
Present
Fc-OPG OPG-Fc RANK-Fc Denosumab
OPG = osteoprotegerin.
Simonet WS, et al. Cell. 1997;89:309-319.
Data on file, Amgen.
Fusion proteins
Fc OPG
RANK
Fully human
monoclonal
antibody

Denosumab Is a Fully Human
Murine
100%
mouse protein
Example:
Orthoclone OKT ® 3
(muromonab-CD3)
Monoclonal Antibody
Chimeric
34%
mouse protein
Example:
ReoPro ®
(abciximab)
Humanized
5%–10%
mouse protein
Example:
Herceptin ®
(trastuzumab)
Orthoclone OKT ® 3 is a registered trademark of Johnson & Johnson; ReoPro ® is a registered trademark of
Eli Lilly and Company; Herceptin ® is a registered trademark of Genentech, Inc.
Bekker PJ, et al. J Bone Miner Res. 2004;19:1059-1066; Lonberg N. Nat Biotechnol. 2005;23:1117-1125;
Ternant D, et al. Expert Opin Biol Ther. 2005;5(suppl 1):S37-S47; Weiner LM. J Immunother . 2006;29:1-9;
Yang XD, et al. Crit Rev Oncol Hematol. 2001;38:17-23.
Denosumab
Fully Human
100%
human protein

Pharmacologic Properties of
• Fully human monoclonal
antibody - IgG 2 isotype
• High affinity for human
RANK ligand
Denosumab
• High specificity for RANK ligand
– No detectable binding to TNF-α,
TNF-β, TRAIL, or CD40L
• No neutralizing antibodies
detected in clinical trials to date
Ig = immunoglobulin; TNF = tumor necrosis factor;
TRAIL = TNF-α–related apoptosis-inducing ligand.
Bekker PJ, et al. J Bone Miner Res. 2004;19:1059-1066.
Elliott R, et al. Osteoporos Int. 2007;18:S54. Abstract P149.
McClung MR, et al. N Engl J Med. 2006;354:821-831.
Data on file, Amgen.
Model of Denosumab
This molecule is investigational and is not
approved by the FDA and EMEA

Hofbauer LC et al. Bone 1999
Sherman ML et al. J Clin Invest 1990
Cenci S et al J Clin Invest 2000
Lam J et al. J Clin Invest 2000
Zhang YH et al J Bol Chem 2001
Fuller K et al Endocrinology 2002
D Aeschlimann and BAJ Evans.
The vital osteoclast: how is it regulated?
Cell Death and Differentiation 2004
RANKL
Stromali cellule T
MCSF
(p55/60)
attivate
Pre OC
TNFα
Formazione di
OCs attivi
RANK

Pathogenesis of Multiple Myeloma Bone Disease
Alteration of
RANK/RANKL/OPG
Inhibition of
Wnt signaling

DKK
LRP5/6
Sclerostina
LRP5/6
WNT WIF-1 WNT sFRP
osteoblast
differentiation
WNT
LRP5/6
Frizzled

Osteoblast suppression in MM bone disease
has been related to the inhibition
of the canonical Wnt signaling
Tian E. et al. The role of the Wnt-signaling antagonist DKK1 in the
development of osteolytic lesions in multiple myeloma. N Engl J Med
349(26):2483-94; 2003.
DKK1
Oshima T et al. Myeloma cells suppress bone formation by secreting a
soluble Wnt inhibitor, sFRP-2. Blood 106(9):3160-5; 2005.
sFRP2
Giuliani N. et al. Production of Wnt inhibitors by myeloma cells: potential
effects on canonical Wnt pathway in the bone microenvironment. Cancer
Res. 67(16):7665-74; 2007.
DKK1, sFRP3

What is known about sclerostin
involvement in MM bone disease?
Terpos E et al., High Serum Sclerostin Correlates with Advanced Stage,
Increased Bone Resorption, Reduced Osteoblast Function, and Poor Survival
in Newly-Diagnosed Patients with Multiple Myeloma. (ASH Annual Meeting
Abstracts) Blood 2009; 114: (Abstract 425).
Terpos E et al., Circulating levels of the Wnt inhibitors Dickkopf-1 and
sclerostin in different phases of multiple myeloma: alterations post-therapy
with lenalidomide and dexamethasone with or without bortezomib. (ASH
Annual Meeting Abstracts) Blood 2010; 116: (Abstract 2963).

•Sclerostin, encoded by SOST gene, is the most important
negative regulator of bone formation and its production is
restricted to bone tissue
•Mutations in the SOST gene cause sclerosing bone
dysplasia, such as Sclerosteosis and Van Buchen disease
van Bezooijen et al Cytokine & Growth factor Rev 2005

Sclerostin represents a negative regulator
of osteoblast differentiation
Proliferation
(osteoblast
Progenitors) Matrix formation
(early osteoblasts)
Benzooijen et al Cytokine & Growth factor Rev 2005
Matrix maturation
& mineralization
(mature osteoblasts)
Sclerostin
Lining cells
Apoptosis
Osteocytes
Sclerostin alteration could be involved in bone diseases including MM

Aim of the work
Investigate the expression of
Sclerostin by myeloma cells
Study the Sclerostin involvement in
osteoblast differentiation in a co-colture
system between Bone Marrow Stromal
Cells and myeloma cells

Sclerostin
Sclerostin expression by CD138+ cells from MM patients
and HMCLs
Total ERK
OD RATIO
Sclerostin/Total ERK
CD138+
1,6
1,2
0,8
0,4
0
MGUS
MM10
no osteolysis osteolysis
MM11
MM12
MM17
MM19
MM7
MM8
MM4
MM9
MM20
0,4
0,3
0,2
0,1
0
U266
H929
Karpas 909
RPMI-8226

…Co-coltures between
bone marrow stromal cells (BMSCs)
and H929/CD138+ cells
anti-Sclerostin
mAb
H929/CD138+
BMSCs

Formation of CFU-F and CFU-OB
in BMSCs co-coltured with H929 or CD138+ cells from the patients
H929
anti-Sclerostin
(ng/ml)
CD138+ cells
anti-Sclerostin
(ng/ml)
H929
anti-Sclerostin
(ng/ml)
CD138+ cells
anti-Sclerostin
(ng/ml)
CFU-F CFU-F
- + + + - + + +
- - 50 500 16
12
- - 50 500
CFU-F/well
(Mean ± SE)
0
- + + + - + + +
- - 50 500 16 - - 50 500
CFU-F/well
(Mean ± SE)
8
4
12
CFU-OB CFU-OB
- + + + - + + +
- - 50 500 20 - - 50 500
CFU-OB/well
(Mean ± SE)
- + + + - + + +
- - 50 500 20 - - 50 500
CFU-OB/well
(Mean ± SE)
15
10
5
0
15
10
5
0
8
4
0
*
*
*
*
*
*
*
*

H929
anti-Sclerostin
(ng/ml)
Effect of myeloma cells on
bone matrix protein expression by BMSCs
BSP II
β-Actina
H929
anti-Sclerostin (ng/ml)
Coll I
β-Actina
BMSCs
- + + +
- - 50 500
BMSCs
- + + +
- - 50 500
Osteocalcin mRNA relative
fold change (Mean ± SE)
1,2
0,8
0,4
0
H929
anti-Sclerostina
(ng/ml)
- + + +
- - 50 500
BMSCs
*
*

Effect of myeloma cells on transcription factor
expression by BMSCs
H929
anti-Sclerostin
(ng/ml)
Fra-2
Lamin B1
H929
anti-Sclerostin (ng/ml)
Fra-1
Lamin B1
BMSCs
- + + +
- - 50 500
BMSCs
- + + +
- - 50 500
H929
anti-Sclerostin
(ng/ml)
JunD
Lamin B1
BMSCs
- + + +
- - 50 500

WNT signaling regulates RANKL/OPG axes
Wnt3a over-expression
up-regulated OPG
Osteoclasts
Osteoblasts
Wnt inhibitors
down-regulated OPG expression
Osteoclasts
Osteoblasts

Effect of human myeloma cells on RANKL and OPG
expression by BMSCs
H929
anti-Sclerostina (ng/ml)
RANKL
β-Actin
H929
anti-Sclerostin (ng/ml)
OPG
β-Actin
BMSCs
- + + +
- - 50 500
BMSCs
- + + +
- - 50 500
OD RATIO
RANKL/ β-actin
OD RATIO
OPG/ β-actin
0,6
0,4
0,2
0
1,8
1,5
1,2
0,9
0,6
0,3
0
BMSCs
- + + +
- - 50 500
BMSCs
*
*
- + + +
- - 50 500
*
*

Sclerostin
Myeloma cells through sclerostin
secretion contribute to
MM Cells
1) Inhibit OB formation
and activity directly
OBs
Sclerostin
OPG
OBs
MM Cells
RANKL
2) Induce OC formation
and resorption indirectly
Sclerostin can be an attractive candidate for developing novel
targeted therapies for this disease

Stabilizzazione della frattura con placca
e viti ed esposizione del focolaio
psudoartrosico
estrazione delle
prelievo cellule dalla staminali: cresta iliaca
di metodo aspirato con midollare filtri
scaffold di tessuto
osseo
demineralizzato, (solo
collagene o
substrati sintetici di
idrossiapatite
o calcio fosfato)
RX postoperatoria con placca e
riempimento del difetto osseo con
lo scaffold arriccito di cellule
staminali

Riassorbimento dell’osso alveolare
Cellule Staminali
presenti nella Polpa Dentale

scaffold a base di collagene arricchito di
cellule staminali, prelevate dalla polpa
dentale (di terzi molari estratti).