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

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 


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 


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 


Inhibition of 


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


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% 


Example: 

ReoPro ® 

(abciximab) 

Humanized 

5%–10% 


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 


Inhibition of 


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) 


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 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 


(ng/ml) 

CD138+ cells 


(ng/ml) 

H929 


(ng/ml) 

CD138+ cells 


(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 


(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 


(ng/ml) 

Fra-2 

Lamin B1 

H929 


Fra-1 


BMSCs 

- + + + 

- - 50 500 

BMSCs 

- + + + 

- - 50 500 

H929 


(ng/ml) 

JunD 


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 


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 

* 

*


Myeloma cells through sclerostin 

secretion contribute to 

MM Cells 

1) Inhibit OB formation 

and activity directly 

OBs 


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).

differenziamento in vitro di cellule ossee

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