Table of Contents I. INTRODUCTION .............................................................................................................. 3 1. Intercellular signaling through extracellular nucleotides ...............................................

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I. INTRODUCTION ... 3

1. Intercellular signaling through extracellular nucleotides ... 3

1.1. History ... 3

1.2. The nucleotides as extracellular signaling molecules ... 3

1.2.1. Nucleotides release ... 4

1.2.1.1. ATP release from dying cells ... 4

1.2.1.2. Regulated ATP release ... 4

• Secretory pathway ... 4

• Conductive release ... 4

o Anion channel ... 4

o Hemichannel : ... 5

o Transporter: ... 5

1.2.2. Extracellular metabolism ... 5

1.2.2.1. Ectonucleoside triphosphate diphosphohydrolase (E-‐NTPDase) family ... 5

1.2.2.2.Ecto-‐nucleotide pyrophosphatase/phosphodiesterase (E-‐NPP) family ... 6

1.2.2.3. 5’-‐Nucleotidase ... 6

1.3. Extracellular nucleotides receptor ... 6

1.3.1. P2 receptor family ... 7

1.3.1.1. P2X receptor family ... 7

1.3.1.2. P2Y receptor family ... 7

• Structure of P2Y receptors ... 7

• Coupling of P2Y receptors to G proteins ... 9

• Responsiveness to different nucleotides ... 11

o P2Y13 receptor (P2Y13R) ... 11

o P2Y12 receptor (P2Y12R) ... 12

1.3.2. Purinergic signaling as a complex network ... 13

2. Mesenchymal stem cell ... 14

2.1. Definition ... 14

2.1.1. Self-‐renewal of MSC: ... 15

2.1.2. Multipotent differential potential ... 16

2.1.3. Specific surface antigen expression ... 16

2.1.4. Adherence to plastic in standard culture condition ... 17

2.2. Function ... 17

2.2.1. Bone tissues ... 17

2.2.1.1. Bone and bone marrow: structure and composition ... 19

2.2.1.2. Bone remodeling ... 19

2.2.1.3. Differentiation of MSCs ... 20

2.2.1.3.1.

Osteogenesis ... 20

• Commitment of MSC into pre-osteoblast ... 20

• Maturation of pre-osteoblasts into osteoblasts ... 22

• Differentiation of osteoblast into osteocytes ... 22

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1

• Commitment of MSC into pre-adipocyte ... 24

• Maturation of pre-adipocyte into adipocyte ... 24

2.2.1.3.3.

Balance between adipogenesis and osteogenesis ... 25

2.2.1.3.4.

Transdifferentiation ... 25

2.3. Support of hematopoiesis ... 26

2.3.1. Niche components ... 26

• MSCs _ ... 26

• Osteoblast ... 27

• Endothelial cells ... 27

• Nervous system ... 27

2.3.2.Stem cell niche as a unit ... 29

2.4. Role of OsteoMacs in MSCs physiology ... 30

2.5. MSCs and cell therapy/tissue engineering ... 30

3. EXTRACELLULAR NUCLEOTIDES SIGNALING IN BONE PHYSIOLOGY ... 31

3.1. Role of extracellular nucleotides signaling in human MSC ... 31

3.1.1. Expression of P2 receptor in MSC ... 31

3.1.2. ATP release and extracellular metabolism in MSC ... 31

3.1.3. Effect of extracellular nucleotides in MSC physiology ... 32

3.2. Bone phenotype of P2Y

13

knockout mice ... 33

II. AIM OF THE WORK ... 34

III. RESULTS ... 35

1. Development of experimental protocols ... 35

1.1. Phenotype and properties of bone marrow stromal cells (BMSC) ... 35

1.1.1. Culture of BMSCs ... 35

1.1.2. Immunophenotyping of BMSC primary culture ... 35

1.2. Differentiation potential of BMSCs ... 37

1.2.1. Osteogenic potential of BMSCs ... 37

1.2.1.1. CFU assay ... 37

1.2.1.2. Expression kinetic of osteogenic gene expresion ... 39

1.2.2. Adipogenic potential of BMSCs ... 39

1.2.2.1. Oil Red O and Nile Red assay ... 39

1.2.2.2.Expression kinetic of adipogenic gene expression ... 39

2. Role of the P2Y

13

R in the differentiation of BMSCs into osteoblasts and adipocytes . 41

2.1. P2Y

13

mRNA expression in undifferentiated and differentiated BMSCs ... 41

2.2. Effect of ADPβS on osteoblast differentiation ... 41

2.2.1. Effect of ADPβS on osteoblast formation ... 41

2.2.2. Effect of ADPβs on osteogenic gene expression ... 41

2.3. Effect of P2Y

13

deletion on osteoblast differentiation ... 42

2.3.1. Effect of P2Y

13

deletion on osteoblast formation ... 46

2.3.2. Effect of P2Y

13

deletion on osteogenic genes expression ... 46

2.4. Effect of P2Y

13

deletion on adipogenic differentiation ... 46

2.4.1. Effect of P2Y

13

deletion on adipocyte formation ... 46

2.4.2. Effect of P2Y

13

deletion on adipocyte genes expression ... 48

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2.6. Fat content in bone marrow and total body: comparison between WT and P2Y

13

R

-‐/-‐

mice

49 3 Role of the P2Y

12

R in the differentiation of BMSCs into osteoblasts and adipocytes .. 52

3.1 Effect of P2Y

12

R deletion on osteoblast differentiation ... 52

3.1.1. Effect on P2Y12 deletion on osteoblast formation ... 52

3.1.2. Effect of P2Y12R deletion on osteogenic gene expression ... 52

3.2 Effect of P2Y

12

R deletion on adipocyte differentiation ... 52

3.2.1. Effect on P2Y12 deletion on adipocyte formation ... 52

3.2.2. Effect of P2Y12R deletion on adipocyte marker expression ... 52

3.3 Comparison of fat content in bone marrow between WT and P2Y

12-‐/-‐

mice ... 54

3.4 Cooperative function of P2Y

12

R and P2Y

13

R in osteoblast differentiation ... 54

4. Identification of cellular and molecular components involved in the pro-‐osteogenic

effect of the P2Y

13

R ... 56

4.1. Expression of P2Y13 messenger in human derived-‐ MSC ... 56

4.2. Purification of CD45

+

and CD45

-‐

cell populations ... 56

4.3. Expression of P2Y

13

messenger in CD45

+

and CD45

-‐

populations ... 58

4.4. Expression of CD39L1 and Pannexin 1 in CD45

+

_{and
CD45}

-‐

_{populations
...
58}

4.5. ATP release in CD45

+

_{and
CD45}

-‐

_{populations
...
58}

4.6. Role of the P2Y

13

R in the control of BMP2, FGF2 and Wnt10b ... 60

4.7. Expression of BMP2 in CD45 population ... 60

4.8. P2Y

13

signaling pathway ... 60

4.9. Effect of a neutralizing antibody against BMP2 ... 61

IV. DISCUSSION ... 62

1. Effect of ADPβS on osteogenic differentiation ... 62

2. Effect of P2Y

13

deletion on osteogenic differentiation ... 63

3. Effect of osteogenic stimulation on ATP release ... 63

4. Effect of P2Y

13

deletion on adipogenic differentiation ... 65

5. Comparison of in vitro observations and P2Y

13

bone phenotype ... 66

6. Role of P2Y

12

R in BMSCs differentiation ... 67

7. The P2Y

13

Table of Contents I. INTRODUCTION .............................................................................................................. 3 1. Intercellular signaling through extracellular nucleotides ...............................................