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I. INTRODUCTION
1. Intercellular signaling through extracellular nucleotides
1.1. History
The role of nucleotides as messengers of intercellular signaling was first demonstrated in 1929 by Drury and Szent-Györgyi 1. Since ATP is mainly described as an intracellular energy storing molecule, the concept that it could exhibit a dual function was met with considerable skepticism. However, since the early 70’s, Geoffrey Burnstock undertook an extensive research to determine the role of this messenger 2. He first proposed the existence of “purinergic nerves” that release ATP as a neurotransmitter 3–6. Afterward numerous findings demonstrated that ATP acts on cells from the extracellular space where it serves in the development and daily operation of many organs and tissues 7. With numerous
collaborators, Burnstock identified two subtypes of purinoreceptors: P1 and P2 receptors which respond to adenosine and ATP, respectively. Later, distinction between receptors that are activated by adenyl nucleotides (ATP and ADP) and by uracil nucleotides (UTP and UDP) was suggested. In the early 1990s, the genes coding for the purine and pyrimidine receptors were cloned 8. To date, the P2 receptors are distributed in two famiies : the
P2X1,2,3,4,5,6,7 (ion channels responsive to ATP) and the P2Y1,2,4,6,11,12,13,14 (G-protein coupled
receptors responding to ATP, ADP, UTP and UDP). Knockout-mice have been generated for almost all P2 receptors 9–14. The phenotypic analysis of these mouse strains highlight the extended number of functions these extracellular nucleotide receptors drive.
1.2. The nucleotides as extracellular signaling molecules
Figure 1: Activation of inflammasome complex by extracellular ATP
ATP released from cells undergoing necrosis binds to the P2 receptor on neighboring macrophages and other cells. Activation of P2 receptor initiates signaling through several pathways, which result in the activation of NLRP3 inflammasome. Caspase-1 activation stimulates the inflammatory response by the cleavage of pro-IL-1! and secretion of the mature IL-1-!.
Figure 2: Exocytosis of ATP
In excitatory cells, VNUT transporter is present on secretory vesicles and is responsible for vesicular storage of nucleotides. V-ATPase, acting as vesicular proton pump, provides the driving force for accumulation of neurotransmitters and ATP within synaptic vesicles. In response to calcium ions influx, the accumulated nucleotides are released by exocytosis with the neurotransmitters in the synapse.
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1.2.1. Nucleotides release
1.2.1.1. ATP release from dying cells
Following cell injury and plasmic membrane disruption, nucleotides are released by passive diffusion. In this case, they are often seen as a pro-inflammatory danger signals or DAMPS (danger-associated molecular patterns). For example, through the P2X7 receptor expressed
on macrophage, eATP initiates signaling resulting in the Nlrp3 protein activation 18. Consequently, this activation leads to the formation of the inflammasome complex and downstream the maturation of pro-IL-1β into IL-1β (for detail see Figure 1). Moreover, Elliot et al have recently proposed a model in which ATP and UTP released during apoptotic bodies formation act as “a find me” signal through the P2Y2 signaling leading to attraction of phagocytes for the engulfment and clearance of such apoptotic bodies 19.
1.2.1.2. Regulated ATP release
• Secretory pathway
In secretory cells (neurons, mast cells, pancreatic B cells,…), nucleotides are stored within granules with neurotransmitters and other mediators. The VNUT transporter (Vesicular Nucleotide Transporter) carries out the transfer of cytosolic ATP into these secretory granules 20 . In response to cytoplasmic calcium elevation, their content is released through the canonical pathway of vesicle exocytosis (for detail see Figure 2).
• Conductive release
Three discernible types of pathways have been identified as regulation ATP release mechanisms. They involve anions channels, hemichannel and transporters (Figure 3). Numerous studies illustrate that non lytic release of ATP occurred in practically every cell type when subjected to physical stress such as shear stress, mechanical loading, plasma membrane stretch, hypoxia and cell swelling.
o Anion channel
Figure 4: Extracellular metabolism of ATP
The nucleotide-hydrolyzing pathway comprises three ectoenzymes families, NPP, E-NTPDase and ecto-5'-nucleotidase:
NTPDase : ATP ! ADP + Pi ; ADP! AMP +Pi NPP: ATP ! AMP + 2 Pi
5 o Hemichannel
Two subtypes of hemichannel can be distinguished: the connexins and pannexins.
Two hexameric oligomers (connexon) of connexins each inserted in the plasma membrane of adjacent cells form a channel at the level of gap junction. This channel connects the cytosol of the two cells allowing the passage of small molecules including ATP. In some case, connexons can localize to non-junctional regions of the plasma membrane and allow therefore the release of ATP out of the cell.
In many publications, the term hemichannel is used to describe the pannexin. Nonetheless, pannexin oligomers cannot form intercellular channel and primilary form membrane channel (pannexon). To date, three pannexin isoforms have been identified (Panx1, 2, 3). Panx1 and Panx3 are widespreadly expressed in human and rodent tissues while Panx2 appears to be more restricted to several areas of the brain 22,23. Studies targeting pannexins with specific pharmacological inhibitors suggest the involvement of pannexin (mainly Panx1) in the release of ATP under a broad spectrum of physiological conditions. For example, expressed in mammalian epidermis, Panx1 has been demonstrated to be involved in keratinocyte differentiation 24. Also expressed in immune cells such as monocytes, Panx1 have been found to promote ATP release and thereby to modulate different immune functions 22,25.
o Transporter
The ABC transporters (ATP Binding Cassette) use energy of ATP hydrolysis to facilitate transport of hydrophobic molecules across the plasma membrane. Some of these proteins (SUR(sulfonylurea receptor), P-glycoprotein) use ATP as energy source but also allow its transport 26.
1.2.2. Extracellular metabolism
In the extracellular space, nucleotides tri, di-and mono phosphates are rapidly hydrolyzed by ecto-nucleotidases 27 (Figure 4). Three families of these hydrolases can be distinguished:
1.2.2.1. Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family
To date, eight E-NTPDases (NTPase 1-8) have been identified 28,29. Each of them is
Protein name Additional name Localization Preferential degradation
NTPDase 1 CD39 Extracellular
ATP#AMP; ADP!AMP
NTPDase 2 CD39L1 Extracellular ATP#ADP
NTPDase 3 CD39L3 Extracellular ATP#AMP
NTPDase 4 LALP70 Intracellular
NTPDase 5 CD39L4 Intracellular
NTPDase 6 CD39L2 Intracellular
NTPDase 7 LALP1 Intracellular
NTPDase 8 Extracellular ATP#AMP
Table 1: Family of E-NTPDases
6 regarding the hydrolysis of ADP into AMP, E-NTPDase2 (also called CD39L1) facilitates the accumulation of ADP or UDP from ATP or UTP (for detail see Table 1).
1.2.2.2. Ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) family
The seven identified E-NPPs (NNP1-NPP7) are capable of hydrolyzing pyrophosphate and
phosphodiester bonds in a variety of extracellular compounds including nucleotides, lysophospholipids and choline phosphate esters. NNP1,2 ,3 hydrolyze ATP into either AMP or
ADP 27,30.
1.2.2.3. 5’-Nucleotidase
Seven 5’-nucleotidases have been characterized. Only one (CD73) is linked to the outer plasma membrane (called CD73) 17. 5’-Nucleotidase catalyzes hydrolysis of AMP to
adenosine. Because NTPDase1 hydrolyzes ATP to AMP without any significant accumulation of ADP, fast appearance of adenosine is facilitated. On the contrary, since 5’-nucleotidase is inhibited by ADP, the formation of adenosine is prevented by NTPDase2 alone.
Extracellular adenosine can stimulate P1 receptors but can also be taken back up into the cells through an equilibrative (ENT) or concentrative nucleoside transporter (CNT)31.
1.3. Extracellular nucleotides receptor
Figure 6: P2X receptor
Individual P2X receptor subunits have two transmembrane domains. P2X receptors are ATP-gated cation channel receptors, gating Na+, K+, and Ca++.
:B?B43X:!! ]8*'R*&+!4=)8*+7<)!! 4$N"! 2D.)8')!*5!+&D&(*9(*6)/&(0/!5))-D0'R!,8!+=)!R,-8)7! KA^!60()!,85)/+,(,+7!KJE/)-&'+,*8!*5!.<)/6!,8! )_0'&(0+)H^/)-&')-!+=/*6D*.,.!0..*',0+)-!>,+=!,8_&/7!*5!+=)!>0((!*5!.60((!0/+)/,*().!KM! 4$N$! Y6<0,/)-!.780<+,'!50',(,+0+,*8!,8!=,<<*'06<0(!,8+)/8)&/*8). K\!^,6<0,/)-!+0.+)!^/)-&')-! <)/,.+0(.,.!*5!.60((!,8+).+,8)!KQ! 4$NK! 255)'+)-!8*',')<+,*8^!,6<0,/)-!+)6<)/0+&/)!.)8.,+,@,+7! 1V^!055)'+)-!@*,-,89!/)5()C!,8!&/,80/7! .7.+)6!1"! 4$N1! :)-&')-!=,<<*'06<0(!`34^!/)-&')-!'=/*8,'!<0,8! 1$^,6<0,/)-!5(*>!.)8.,+,@,+7!*5!D(**-!@)..)(! ,6<0,/)-!5(*>!.)8.,+,@,+7!*5!D(**-!@)..)(!^-)'/)0.)-!@0.*-,(0+0+,*8!1K! 4$NM! Y6<0,/)-!,66&8)!/).<*8.)! "\^!-)'/)0.)-!8)&/*<0+=,'!'=/*8,'!<0,8^!,6<0,/)-!.0(,@0! </*-&'+,*8^!0D8*/60(!D*8)!5*/60+,*8!08-!/).*/<+,*8!11a1J!!
Table 2: P2X knockout Phenotype
Figure 7: P2Y receptor structure
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1.3.1. P2 receptor family 1.3.1.1. P2X receptor family
P2X receptors are ligand-gated ion channels responsive to ATP. This family comprises 7 receptors (P2X1, 2, 3, 4, 5, 6, and 7). P2X receptors are 379 to 595 amino acids proteins, and
exhibit a tertiary structure characterized by two hydrophobic transmembrane domains joining an extracellular domain and N- and C-terminal intracellular domains (Figure 6) 47,48. Functional, P2X receptors are homo- or heterotrimeric proteins. To date, six homotrimers
(P2X1,2,3,4,5,7) and three heterotrimers (P2X2/3, P2X4/6, P2X1/5) have been characterized 47,49.
Activation of P2X receptors leads to Na+ influx, K+ efflux and to a varying extent Ca2+ influx leading to the cell membrane depolarization. Membrane depolarization activates voltage-gated calcium channel and downstream cascades involving both MAPK and calcium pathways 47,50. P2X activation is rapidly followed by its desensitization, thereby the cellular
response-time is generally very brief 51.
P2X receptors are widely distributed and through phenotype analysis of KO strains, functional responses have been reported in many tissues especially in the central and peripheral nervous system 52 (Table 2).
1.3.1.2. P2Y receptor family
• Structure of P2Y receptors
To date, eight human P2y subtypes genes (P2y1,2,4,6,11,12,13,14 ) have been cloned and display 21-48% identity of the amino acid sequence 34,53–55. Human P2Y receptors contain 328 (P2ry6) up to 377 (P2ry2) amino acids, corresponding to a predicted molecular mass of
41 to 53 kDa. The P2Y receptor structure has been modeled using the rhodopsin G protein coupled receptor (GPCR) as a template. This model suggests that the various P2Y receptor subtypes contain the typical features of GPCR, including 7 hydrophobic transmembrane regions (TM) connected by 3 extracellular loops (EL) and 3 intracellular loops (IL). The aminoterminal end is extracellular while the carboxyterminal one is cytoplasmic (Figure 7). Nucleotides bind on the external side in the cavity delimited by TMs. Four basic residues (H121 in TM3, H266 and K269 in TM6, R299 in TM7 for P2Y1 for example) positioned near
the external surface have been proposed to be involved in ligand binding. 56. The eight P2Y
Figure 8: GPCR Activation
Without any signal, GDP attaches to the " subunit. Ligand binding activates the G protein, and GTP physically replaces the GDP bound to the " subunit. As a result, the G protein subunits dissociate into two parts: the GTP-bound " subunit and !# dimer. When GTP is hydrolyzed back to GDP, the subunits reassociate into an inactive heterotrimer.
Figure 9: PLC! pathway
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Figure 10: cAMP pathway
Figure 11: Rho pathway
G12/13 $-subunits signal through Rho family GEFs. Activation of typical Rho proteins is controlled by GEFs, which catalyze the exchange of guanine diphosphate (GDP) for guanine triphosphate (GTP). GAPs inactivate typical Rho GTPases by enhancing their ability to hydrolyse bound GTP to GDP. Numerous proteins have been identified as targets of RhoA such as ROCK (Rho-associated protein kinase)
Figure 12: PI3K pathway
9 receptors share a Y-Q/K-X-X-R motif in TM7, whereas in P2Y12,13,14 receptors, this motif is
substituted with K-E-X-X-L34. This sequence specify as well as G-proteins coupling define two P2Y subgroups.
• Coupling of P2Y receptors to G proteins
Like other GPCR, P2Y receptor stimulation leads to activation of heterotrimeric G proteins composed of α, β and γ subunits. Gα subunit contains 2 domains, a GTPase domaine involved in the binding and hydrolysis of GTP and a helical domain that buries the GTP within the core of the protein. In the inactive state, the Gα subunit is associated to GDP. Upon activation, GDP is released and GTP binds Gα. Gα-GTP subunits dissociate from Gβγ and from the receptor. Both complexes are then free to activate downstream effectors. After hydrolysis of GTP, Gα-GDP is reassociated with βγ (Figure 8)57,58.
Classically, G proteins are divided into four families based on their association to the α subunit type: Gq, Gi/o,Gs, G12/13. Each Gα family activates a distinct profile of effectors 59
. Briefly:
- Gαq family proteins activate the phospholipase C-β (PLCβ) proteins producing inositol 1,4,5 triphosphate (IP3) and diacyglycerol (DAG) from phosphatidylinositol-4,5-bisphosphate ( for detail see Figure 9).
- Gs family proteins activate adenyl cyclase (AC), which generates cyclic adenosine 5’-monophosphate (cAMP) from ATP. cAMP is known to activate the protein kinase A (PKA), cyclic nucleotide gated channel and the guanine nucleotide exchange factor EPAC (EPAC1 and EPAC2) 60,61 (for detail see Figure 10).
- Gi family proteins inhibit some isoforms of adenylyl cyclase (I, III, V, VI, and VIII), are regulator of K+ channels opening and inhibit voltage-dependent Ca2+ channels
- G12/13 proteins activate Rho-guanine nucleotide exchange factors (Rho-GEFs) that activate small monomeric GTPase of the Rho sub-family. Active RhoA regulates multiple downstream effectors including Rho kinases (ROCK1/2) (Figure11). Initially Gβγ was thought to prevent noise or spontaneous Gα activation in absence of receptor stimulation 62. However Gβγ is now known to activate several effectors such as
PLCβ2, 3, PI3K (phosphoinositide 3 kinase) and GIRKs K+ channels. The Figure 12
Receptor
family RECEPTOR G PROTEIN MAIN EFFECTOR P2Y1 Gq/11 PLCβ +
P2Y2 Gq/11 PLCβ +
Go PLCβ +
RAC +
P2Y1-‐like G12 RHOA+
P2Y4 Gq/11 PLCβ + Go PLCβ + P2Y6 Gq/11 PLCβ + G12/13 RHOA+ P2Y11 Gq/11 PLCβ + Gs AC + Go PLCβ + P2Y12 Gi AC -‐ G12/13 RHOA+
P2Y12-‐like P2Y13 Gi/o AC-‐
G12/13 RHOA+
P2Y14 Gi/o AC-‐
PLCβ +
10 RECEPTOR AGONISTS ANTAGONISTS EXPRESSION Knockout phenotype
P2Y1 ADP ADPβ 2MeSADP SURAMIN PPADS MRS2279 MRS2179 Brain Prostate Endothelial Cell Smooth Muscular Cell Platelet
Inhibition of platelet aggregation 63
Reduction of atherosclerotic lesions 64
P2Y2 UTP ATP UTPgS MRS 2698 MRS27G8 SURAMIN AR-C 126313 Brain Lung Spleen Heart Immune Cell Endothelial Cell Smooth Muscular Cell Epitheliale Cell
Decrease of pulmonary eosinophil infiltration in a murine model of
asthma 65
Decrease of neutrophil and monocyte
chemotactism 66
Inhibition of chloride secretion in
bronchial epithelial cells67
P2Y4 UTP ATP UTPgS SURAMIN PPADS Intestin Lung Heart Liver Endotheliale Cell
Inhibition of chloride secretion in
intestinal epithelial cells 11,67
Decrease of heart size (angiogenesis
defect) 68 P2Y6 UDP UDPβS MRS 2578 PPADS Intestin Kidney Placenta Spleen Endotheliale Cell Smooth Muscular Cell Leukocyte
Decrease of vessel contraction 9
P2Y11 ATP ATPγS BzATP ARC 67085MX SURAMIN Intestin Spleen Immune Cells P2Y12 ADP 2-‐MeSADP ADPβS AR-C69931MX Platelet
Smooth Muscular Cell Microglial Cell
Inhibition of platelet aggregation 69
Modulation of dentritic cell function 70
P2Y13 ADP 2-‐MeSADP ADPβS AR-C69931MX Brain Spleen Liver Defect in reverse cholesterol transport10 P2Y14 UDP-Glucose Bone Marrow Intestin Placenta Stomach
Alteration of stomach contraction 12
11 Individual P2Y receptor subtypes have been linked to one or more of the four subfamilies of G protein (Table 3). The P2Y1-like sub-family activates mainly the PLCβ signaling pathway
through coupling with Gαq protein. The P2Y11R is coupled both to Gαs and Gαq. The other
sub-family of P2Y, the P2Y12-like family is coupled to Gαi protein 51,71.
• Responsiveness to different nucleotides
According to their agonist profile, P2Y receptors are subdivided into receptors responding to adenine di or trinucleotides (P2Y1, P2Y11, P2Y12, P2Y13, P2Y4 (in mouse)) and to uridines
nucleotides (P2Y6, P2Y4 (in mouse and human)). It is to be noted that P2Y2 receptor belongs
to both subtypes and that P2Y14R is activated by UDP-glucose. In order to identify the
physiological role of P2Y receptor subtypes, numerous pharmacological studies were performed using P2YR agonists and antagonists. Nevertheless, lack of selective agonists and/or antagonists for any of the cloned P2YR led researchers to generate P2Y knockout mice. P2Y-deficient mice have been generated for all P2Y receptors except P2Y11 (not
present in murine genome) and to date, many physiological roles of these P2Y receptors have been described especially in the vascular, immune and intestinal systems 72,73. The most prominent results are listed in Table 4.
o P2Y13 receptor (P2Y13R)
In 2001, the human GPR86 orphan receptor (also called GPR94 or SP174) was identified as a P2Y receptor and therefore renamed P2Y13 receptor 74. Located in the 3q24 region, the 1002
bp open reading frame of P2ry13 gene encodes a protein of 333 amino acids. It displays significant sequence similarities with the human P2ry12 receptor (48% of amino acid identity). The cloning of murine and rat P2ry13 was performed based on homology with the human sequence. The murine sequence is located on 3D chromosome, near the P2ry12 and P2ry14 receptor sequences and displays 75% identity with its human ortholog 75.
The human P2ry13 is highly expressed in the spleen, brain, bone marrow, lymph nodes, liver and adult brain. The P2ry13 messenger is also detected in placenta, lung, thymus and small intestine but with a lower intensity. The murine and rat tissue distribution of P2ry13 messenger is close to human, with a high expression in the spleen, brain, liver and bone marrow, but it is also detected in the pancreas 76.
Using a P2Y13R- transfected 1321N1 cell line, pharmacological assays have highlighted that
12 expressing the P2ry13 receptor, ADP and its analogues inhibit the forskolin-stimulated accumulation of cAMP in a pertussis toxin sensitive way 75. Consequently, the P2Y13
receptor has been classified in the P2Y12-like family due to its sequence similarity with
P2Y12,its coupling to Gαi and its high affinity for ADP. More recently, RhoA/ROCK 1
signaling activation downstream of P2Y13R has been observed in human hepatocytes 77.
Although the Rho signaling activation is often associated to the G12/13 subunit, any evidence
of its coupling to P2Y13R has not been demonstrated yet.
Since P2Y13R is mainly expressed in immune cells, numerous authors suggested that it might
play a role in hematopoiesis and the immune system but to date no data have confirmed this hypothesis.
During his thesis in our lab, Dr. Hakem Ben Addi generated a strain of P2Y13 deficient mice 10. These mice display no apparent phenotype and reproduce normally. P2Y
13-deficient mice
exhibit a decrease in hepatic HDL cholesterol uptake, hepatic cholesterol content, and biliary cholesterol output, while their plasma HDL and other lipids levels are normal. These changes result from a significant decrease in the rate of reverse cholesterol transport 10,77,78. Consequently, it is reasonable to suggest its involvement in the development of atherosclerosis.
The second phenotype was described by the staff of Allison Gartland in UK, who showed that this receptor is involved in bone turnover. The details will be introduced in section 3 of this introduction.
o P2Y12 receptor (P2Y12R)
The P2Y12 R and P2Y13R receptors display notable similarities 79,80. They are both:
- activated by ADP and its stable analogues - coupled to Gαi protein
- coupled the Rho pathway
- highly expressed in immune cells
The most prominent role of the P2Y12R was demonstrated in platelets aggregation 56,81,82. The
Figure 13: Overview of the extracellular nucleotides/nucleosides signaling
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1.3.2. Purinergic signaling as a complex network
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2. Mesenchymal stem cell
Stem cells can be distinguished from other cells types by two characteristics: - They are undifferentiated cells capable of self–renewing
- They are able to differentiate into different cell types during the embryonic development and throughout the lifetime.
These two characteristics are summarized by the term “stemness”.
3 subtypes of stem cells can be distinguished according to their long-term ability to maintain stemness and the number and type of differentiated cell that they give rise to 83:
• Unipotential stem cells: they give rise to only one mature/differentiated cell type (ex: keratinocyte stem cell differentiates only into keratinocyte)
• Multipotent stem cells: they are able to yield at least two or more differentiated fetal or adult cell types. Different adult stem cells have been described: neural stem cells (NSC), hematopoietic stem cell (HSC) and mesenchymal stem cell (MSC)
• Pluripotent stem cell: they can maintain stemness indefinitely and give rise to progenies that differentiate into all tissues including germinal tissues. These cells are the embryonic stem cells (ES).
2.1. Definition
MSCs described as multipotent cells able to differentiate into several cell lineages of mesodermal origin. Based on the current models, 4 criteria define the MSC identity (Figure 14)84–87:
- Their capacity of self –renewal
- Their capacity to differentiate in vitro into, at least, osteoblasts, chondrocytes and adipocytes
- Their capacity to adhere on plastic culture dishes in standard culture conditions
Figure 15: The leukaemia inhibitory factor (LIF) receptor (LIFR)–STAT3 pathway
15 It should be noted that the term "mesenchymal stromal cell” frequently replaces the term "mesenchymal stem cell". In this present work we consider that stromal cells refer to all adherent cell of the bone marrow.
2.1.1. Self-renewal of MSC:
The cell self –renewal refers to a biological process that preserves the undifferentiated stem state of one daughter cell after duplication. The self-renewal and differentiation activity of stem cells is controlled by their surrounding microenvironment, which is referred as stem cell niche. Composed of stem cells and other cells types, the niche regulates the secretion of cytokines and growth factors driving the balance between self-renewal and differentiation. The mains factors implicated in MSC stemness maintenance are the leukemia inhibitory factor (LIF), FGF2, Wnt3a and other growth factors such as EGF and HGF 52,88,89. The
canonical Wnt signaling is known to inhibit MSC proliferation and concomitantly to enhance their differentiation. Nevertheless, numerous studies demonstrated that the Wnt3a isoform has the opposite effect and therefore enhances the MSC stemness maintenance. 90. LIF is the
best-known factor to maintain the stem state of MSCs. In further detail, upon the binding to a two-part receptor complex (LIFR/gap130), LIF triggers activation of transcription factor STAT3 that forms a nucleoprotein complex with the Nanog-Sox2-Oct4 transcriptions factors. Nanog, Sox2 and Oct4 constitute the core regulatory network that controls downstream gene expression important for maintaining multipotency and inhibiting differentiation 91–93 (Figure 15).
To maintain tissue homeostasis, stem cells undergo asymmetrical divisions by which a single stem cell gives rise to different daughter cells, one maintaining the stem cell feature and the other entering the differentiation process. Two types of asymmetric division can be distinguished 94:
• The stem cell fate of the two daughter cells is determined during cell division
Source tissue Differentiation potential Osteoblast Adipocyte
Bone marrow Cardiomyocyte
Chondrocyte Hepatocyte Myocyte Neuron Mesenglial cell Muscle Adipocyte Osteoblast Endothelial cell Chondrocyte Neuron
Adipose tissue Chondrocyte
Osteoblast Adipocyte Myocyte Dermis Osteoblast Chondrocyte Adipocyte Myocyte Blood Adipocyte Osteoblast Fibroblast
Synovial membrane Adipocyte
Chondrocyte Osteoblast Myocyte Dentine Adipocyte Chondrocyte Osteoblast
16 Another important issue is that at each cell division the telomeres decrease in length and as a result cells become subject to age-dependent senescence. To overcome this loss, stem cells express telomerase, which elongates the telomere. Adult stem cells, including MSCs, express low but sufficient level of this enzyme allowing cell division during all the life of the organism. On the contrary ES cells produce high level of telomerase so that their telomeres never shorten and therefore are theoretically immortal 83,96.
2.1.2. Multipotent differential potential
MSC are able to differentiate in vitro into several cell lineages including osteoblast, adipocyte, chondrocyte and myocyte. MSC can also differentiate in vitro into cells of non-mesoderm origin, such as neuron, hepatocyte, pneumocyte, skin and gut epithelial cells. MSC reside in diverse tissues and possess the ability to generate specific cell type for these tissues (see Table 5) 95. Bone marrow is considered to be the best accessible and enriched
source of MSCs. Contrary to bone marrow stem cells, MSCs from other tissues (i.e. adipose tissues, periosteum, synovial membrane, peripheral blood, dentine) possess a limited capacity to differentiate into several lineage. In addition to adult tissues, MSC can be obtained from several birth-associated tissues including placenta, amnion, umbilical cord and cord blood 97. However, the concept of multipotency now needs to be called into question 98. According to several searchers, it becomes clear that although MSCs are able to differentate into several lineages in vitro, some in vivo assays correlate poorly with this model. As a matter of fact, it appears that these cells are not capable to differentiate into all mesodermal lineages in vivo and the potency of differentiation is dependent of the tissue.
2.1.3. Specific surface antigen expression
MARKER TYPE ANTIGENS HUMAN MOUSE NEGATIVE CD45 - - CD117 - CD11b - - CD31 - - POSITIVE STRO-1 + SCA-1 + CD105 + + CD106 + + PDGFRα + CD90 + + CD73 + CD29 + + CD13 +
Table 6: Surface antigens commonly identified during isolation of human and murine
17 during clonal expansion. Therefore, the STRO-1 marker is no longer considered as a general MSC marker. In combination with negative markers, MSCs are identified with a myriad of positive markers such as CD73 (5’-nucleotidase), CD105 (endothelial cells, bone marrow cell subset), CD106 (endothelial cells, bone marrow cell subset), PDGFRα, CD90 (thymocyte, fibroblast, endothelial cells, HSCs and MSCs), Sca-1 (hematopoietic and mesenchymal stem cell), Nestin ( MSCs and NSCs) …
Surface antigens commonly identified during isolation of MSCs are summarized in Table 6. This table also resumes the observed differences between murine and human markers.
2.1.4. Adherence to plastic in standard culture condition
Density gradients centrifugation using ficoll and percoll reagents are commonly used to isolate MSC from human bone marrow whereas direct plating is commonly used for cells from rats, mice and rabbit. The presence of MSC into bone marrow was first demonstrated by the research group of Friedienstein in the 1970’s by seeding the whole bone marrow samples in culture plastic dishes and removing non-adherent cells 102–104. The adherent cells, also called fibroblast like cells, were able to form cell clusters derived from single cell, defined as fibroblast colony forming units (CFU-F). After a few days of culture in specific culture conditions, these adherent cells can differentiate into mature cells of mesenchymal lineages such as osteoblasts, adipocytes…
2.2. Function
In the bone tissues, the main function of MSC is to provide a source of osteoblast progenitors. Furthermore MSCs also provide a “healthy” environment for hematopoietic stem cells, assuring their maintenance, proliferation, differentiation and function.
2.2.1. Bone tissues
Figure 16: Bone structure
The long bone comprises the trabecular bone and cortical bone.The medullar cavity is covered by an endosteal lining tissue composed of a single layer of “bone-lining cells” (osteoblasts and osteoclasts). Bone consists of a mineral phase mainly composed of
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Figure 17: Bone remodeling
The remodeling cycle is initiated by the activation of the bone lining osteoblasts. Monocyte fusion and maturation form the bone resorbing osteoclasts. The osteoclasts dissolve the inorganic matrix by creating an acidic microenvironment and degrade the organic matrix with specific enzymes. Matrix degradation leads to the release of factors (!) involved in osteoblast differentiation. New osteoblasts complete the cycle with the synthesis and deposition of new bone matrix.
Figure 18: Osteoclast maturation
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2.2.1.1. Bone and bone marrow: structure and composition
Bone is classified into two types: trabecular bone (also called spongy bone or cancellous bone) and cortical bone (also called compact bone). Trabecular bone gives supporting strength to the ends of the weight-bearing bone. Cortical bone is denser and is found in the shaft of long bones and forms the outer shell around spongy bone at the end of joints
.
Bone consists of a mineral phase mainly composed of hydroxyapatite crystals and an organic phase made up largely of collagen which forms the osteoid matrix. The most important non-collagenous organic constituents of bone matrix are four proteins: osteocalcin (OC), bone sialoprotein (BSP), osteopontin (OP) and osteonectin (ON). They are produced by bone cells and appear to be multi-functional (adhesion, regulation …). The bone cavities are covered by an endosteal lining tissue composed of a single layer of “bone-lining cells” (osteoblasts, osteoclasts and osteoprogenitors). Osteocytes (quiescent mature osteoblast) located deep in the bone matrix maintain contact between osteoid and endosteal lining (Figure 16).
The bone marrow is found within the central cavities of axial and long bones. It consists of hematopoietic tissue islands and adipose cells surrounded by vascular sinuses. Bone marrow contains four mains cell types: endothelial cells, stromal cells, hematopoietic cells and MSCs.
2.2.1.2. Bone remodeling
Bone is in continuous remodeling which involves the resorption of mineralized bone by osteoclasts followed by the formation of new bone matrix by osteoblasts (Figure 17) 105–107.
Bone remodeling (also called bone turnover) allows adjusting the extracellular calcium homeostasis and is mainly regulated by the parathyroid hormone (PTH). In response to a low serum calcium level, parathyroid glands secrete PTH, which stimulates indirectly the bone resorption by osteoclasts. PTH/PTH-related protein (PTHrP) receptors are located on osteoblasts, which then signal to osteoclast precursors (monocyte-derived cells) to stimulate their fusion, differentiation and activation108. Upon PTH binding, osteoblasts increase the production of M-CSF (macrophage colony–stimulating factor) 109 and RANK (receptor
degrade the bone matrix through the release of acidic agents and proteolytic enzymes. Bone resorption leads to the release of several growth factors stored in the matrix, which are
Figure 19: BMP and Wnt signling
WNT signaling: In absence of signal, !-catenin is hyperphosphorylated via CKI$ and GSK3!. Phosphorylated !-catenin is a target for ubiquitination and degradation by the proteasome. Binding of Wnt ligand to a Frizzled/LRP-5/6 receptor complex leads to activation of the Dvl (Dishevelled) protein which allows the disorganization of the APC-GSK-3!$ Axin complex and therefore the stabilization of hypophosphorylated !-catenin, which interacts with TCF/LEF proteins in the nucleus to activate transcription.
20 responsible for the recruitment, and the differentiation of MSC into osteoblast (for details see next section). Mature osteoblasts produce the new bone matrix, initially not calcified and then they promote its mineralization, thus completing the bone remodeling process
2.2.1.3. Differentiation of MSCs
2.2.1.3.1. Osteogenesis
Osteoblast differentiation is a highly regulated process which is characterized by the following sequence of events: commitment of MSC into osteoblast, maturation of pre-osteoblast into pre-osteoblasts and terminal differentiation of pre-osteoblast into osteocyte 108,111.
• Commitment of MSC into pre-osteoblast
During bone remodeling, osteoclastic bone resorption induces release of factors stored in the calcified bone matrix. These factors induce MSCs recruitment and their differentiation. Commitment of MSCs to osteoprogenitors is controlled by the transcription factor Runx2 (Runt-related transcription factor 2) (also called Cbaf1)108,112,113. This master switch is required to initiate the osteogenic differentiation but also to suppress the chondrogenic potential of uncommitted progenitors 114. The expression and activation of Runx2 is regulated through numerous signaling pathways. The more important are BMP (Bone Morphogenic Protein) and WNT signaling (Figure 19)115–117.
o BMP signaling
Figure 20: Runx2 phosphorylation pathways
(A) In the Smad-independent pathway phosphorylation of TAK1 by the BMP ligand-receptor complex leads to signal transduction through the p38 MAP kinase pathway, resulting in phorphorylation of Runx2.
Cross-talk between RhoA-ROCK (C) and the ERK-MAPK (B) pathway stimulates phosphorylation of ERK 1/2 (p44/42 MAPK) through MEK (MAPK kinase), which regulates the phosphorylation of the osteogenic transcription factor RUNX2
21 To date, twenty BMPs have been discovered but it is generally accepted that the BMP2, BMP4 and BMP6 are the main members involved in the osteoblast differentiation 118.
o WNT signaling
The WNT (WiNgless and inTegration site) ligand is a secreted glycoprotein that binds to LPR accessory protein and Frizzled receptor. In the absence of WNT-ligand, the cytoplasmic protein β−catenin is constitutively phosphorylated by CK1 (Casein Kinase 1) and/or GSK-3β associated to the APC (Adenomatosis Polyposis Coli) and Axin. This phosphorylation favours its ubiquitination and subsequent proteosomal degradation through the β-TCP/SKP pathway. In the presence of WNT ligand, the co-receptor LRP5/6 (lipoprotein receptor-related protein) displays the ligand to the Frizzled receptor. This leads to the activation of the Dishevelled (Dvl) protein which allows the disorganization of the APC-GSK-3β−axin complex and accumulation of the β-catenin that allows its translocation into the nucleus where it interacts with the TCF/LEF transcription factors to control gene expression 119.
Beside these two factors, many others have been described as inducing RUNX2 expression: they are FGFs, PTH, and HHG.
• Maturation of pre-osteoblasts into osteoblasts
Differentiation of pre-osteoblast into osteoblast is necessary to suppress the adipogenic potential of osteoprogenitor cells. The transcription factor Osx (Osterix) is required for this final commitment.
Runx2 protein function is regulated on multiple levels including post-translational modifications 120. Phosphorylation is one of the most important post-translational
modifications required for Runx2 protein activity. Indeed, several studies have demonstrated that Runx2 phosphorylation leads to its DNA binding on the Runx2 consensus sequence (OSE2, Osteoblast Sequence Element). Runx2 is phosphorylated and activated by MAPK/ERK pathway.
Figure 21: Osteoblast differentiation
22 Moreover, it has been shown that the non-canonical pathway of BMP signaling can induce RUNX2 activation. Indeed, upon BMP2 activation, BMPR triggers the TGF-β–activated kinase 1 (TAK1). TAK1, as a mediator of the p38 MAPK pathway, regulates osteoblast differentiation through Runx2 phosphorylation 124,125,105 (Figure 20).
DNA binding proteins that interact and cooperate with Runx2 to activate gene expression are abundant. The most important transcription factor partners/gene expression regulators of Runx2 are AP1 (c-Fos and c-Jun heterodimer), BMP-responsive SMADs (SMAD1 and SMAD5), androgen and glucocorticoid receptors, several CAATT enhancer binding proteins (C/EBPs), Dlx5 (distal-less homeobox 5), Hes1(hairy and enhancer of split-1), Msx2 (Msh homeobox1), and Oct-1 ( octamer-binding protein1) 127,128.
The Runx2-null mutant mice are characterized by the complete lack of bone formation129. Such deficiency is also observed in Osx KO mice while the Runx2 expression is normal 130. This demonstrates that Osx acts downstream of Runx2 to induce osteogenic differentiation and is essential for bone formation. Finally, Osx regulatory region of the OSX promoter contains an OSE2 element, that binds Runx2 131.
Osx is a transcriptional activator that drives the expression of osteogenic target genes such as osteopontin (OP) and alkaline phosphatase (ALP), collagene I (COLI), osteocalcin (OC), bone sialoprotein (BSP) 131–133. Expression of these proteins is characteristic of the mature osteoblast phenotype.
• Differentiation of osteoblast into osteocytes
23 Osteoblast differentiation of MSCs in vitro is induced by the presence of dexamethasone (a
synthetic analog of glucocorticoids), ascorbic acid (commonly called vitamin C) and β-glycerophosphate 137. Dexamethasone is a synthetic ligand binding the glucocorticoid nuclear receptor. This receptor acts as a transcription factor that directly binds to DNA and regulates the expression of Runx2. Ascorbic acid also promotes transcription of Runx2, nonetheless its mechanism of action is not documented in the literature. The phosphate from
β-glycerophosphate serves as a source for the substrate for the hydroxyapatite synthesis.
2.2.1.3.2. Adipogenesis
In mammals, two kinds of adipose tissue can be distinguished: the white and brown adipose tissue. The white adipose tissue (WAT) serves as a depot of stored energy (in the form of triacylglycerides (TAG)) whereas brown adipose tissue (BAT) provides source of heat in order to maintain body temperature in small mammals and in newborn humans 138,139,140. WAT is located beneath the skin (subcutaneous fat), around internal organs (visceral fat), in bone marrow (yellow bone marrow) and in the breast tissue. Fatty acids reach the adipocyte via the blood circulation and can enter into the cell via passive diffusion or through specific transport such as FATP1 (Fatty acid transport protein 1) and CD36 141. After entry into the adipocyte, fatty acids are esterified by acyl-CoA-synthase and used for the TAG synthesis on the endoplasmic reticulum (ER). Moreover, adipocytes take up glucose via Glut-4
transporters, which are translocated to the plasma membrane upon insulin stimulation. Glucose is converting into glycerol via the glycolysis and glyceroneogenesis reactions and serves for the synthesis of TAGs. For storage, TAG are incorporated into lipid droplets, which are form at the ER142.
In the newborn mammals, there is no yellow fat in the bone marrow. With aging, the MSCs potential of differentiation changes according to the production of signaling molecules within a modified marrow micro-environment resulting in the increase of adipocyte number 99. First, marrow adipocytes were thought to be metabolically inert just filling the marrow void
vacated by bone loss. However, a new theory has emerged suggesting a role of these cells as an energy source or as a regulator of adjacent tissues by the production of paracrine and autocrine factors such as leptin, adiponectine, TNFα, IL6, MCP1…143–145
24 components of adipogenic stimuli used to promote adipogenesis in both MSC and pre-adipocyte. Elevation of cytosolic cAMP is known to promote Cebp/β transcription through the CBP/aCREB complex (Figure 22). Moreover, insulin, acting on IGF-1 (insulin-like growth factor) receptor, has been shown to enhance adipocyte differentiation through the PI3K/PKB pathway.
As well as osteogenesis, adipogenesis is characterized by sequential changes in the expression of specific genes that determine the adipogenic phenotype 146–148.
• Commitment of MSC into pre-adipocyte
The commitment of MSC into the adipocyte lineage is characterized by the expression of C/EBPβ (CCAAT/enhancer-binding protein) 149,150. In the canonical pathway, cAMP accumulation favours the protein kinase A-mediated phosphorylation and activation of the transcription factor CREB (cAMP Response Element Binding protein) that stimulates the transcription of C/EBPβ gene. Once activated by phosphorylation, this transcription factor induces the expression of C/EBPα and PPARγ through the CEBP/β regulatory element in the proximal promoter of these genes. PPARγ are members of the nuclear receptor superfamily and exist as 2 isoforms (PPARγ1 and PPARγ2) 151. Both are transcribed from the same gene
through alternative splicing and both control adipogenesis. Nevertheless PPARγ2 isoform is known to be expressed exclusively in adipocyte lineage and has a greater specificity to induce adipogenic differentiation. It has been also shown that PPARγ2 is more tardily expressed than PPARγ1 152,153.
• Maturation of pre-adipocyte into adipocyte
Figure 22: Adipocyte differentiation
Adipogensis is characterized by sequential changes in the expression of specific genes that determine the adipogenic phenotype. cAMP elevating agent promote pre-adipocyte differentiation through the expression on CEBP/!. CEBP/! induces the expression of PPAR#. In maturation phase, PPAR#2 triggers expression of genes required for complete adipocyte differentiation.
Figure 23: Balance between osteogenesis and adipogenesis
Schematic representation of MSCs differentiation into osteoblasts or adipocytes. Cell
25 demonstrated that in cells lacking PPARγ, the adipogenic potential was not rescued by CEBP/α suggesting that CEBPα promotes adipogenesis in a PPARγ-dependant manner
146,147,149 (Figure 22).
2.2.1.3.1. Balance between adipogenesis and osteogenesis
Under bone loss conditions such as osteoporosis, the decrease in osteoblasts number is correlated with an increased adipocyte formation 154. This phenomenon suggests that adipocytes are generated at the expense of osteoblasts from a common precursor. Numerous studies suggest that lineage fate determination of MSC is regulated through several secreted factors in the bone marrow microenvironment. These factors mediate the cross-talk between adipogenesis and osteogenesis. Among these factors we can mention BMP2/4 155, Wnt10b
156,157, FGFs, VEGF 158 which are known as factors triggering osteogenic differentiation but
which also inhibit adipocyte differentiation. Adipokines such as leptin and adiponectine
159,160, secreted by adipose tissue, act on MSCs by enhancing osteoblasts differentiation and
inhibiting adipocyte differentiation. Other non-secreted factors are involved in this balance. One of these is the PPARγ transcription factor which has been demonstrated to inhibit osteogenesis through down-regulation and trapping of Runx2 161,162. Conversely, Runx2 represses adipogenesis, and Runx2–/–cells show enhanced lipid droplet accumulation. The RhoA signaling pathway has also been demonstrated as an importance balance regulator. Indeed RhoA can induce MSCs differentiation towards the osteogenic lineage while it inhibits adipogenesis 121,123 (Figure 23).
2.2.1.3.2. Transdifferentiation
Transdifferentiation is a process whereby a pre-committed cell switches into a cell type of a different lineage. Several studies have shown that MSCs have the ability to differentiate into pre-adipocytes, dedifferentiate, and subsequently differentiate into osteoblasts in vitro 163,164.
26
2.3. Support of hematopoiesis
Beside their role as osteoblast precursors, MSCs support self-renewal, homing and differentiation of bone marrow hematopoietic stem cells (HSCs). All these processes depend on a healthy hematopoietic environment defined as the stem cell niche in 1978 by Schofield et al. 84,166–170.
Stem cell niches are not randomly located in the bone marrow but preferentially reside in trabecular bone and are relatively rare in the diaphysis regions of the long bones. Within the bone, two anatomical regions have been identified as preferential site for niche location: the endosteal surface and the sinusoidal walls 168.
HSCs produce all cells required to replenish the blood and immune systems. This process is highly regulated to maintain a steady number of leukocytes, platelets and red cells in the blood and/or to respond to homeostatic requirements. Consequently, in the niche, HSCs undergo one of several fates: quiescence, apoptosis, cycling either to self-renew or to generate a more committed hematopoietic progenitor cell and migration. These fates are largely controlled by specific local factors secreted by stem cell niche players. Cell components of these niches are: MSCs, osteoblasts, monocytes/macrophages, endothelial cells, and nervous cells.
The concept that MSCs regulate hematopoiesis inside the niche is well established. Numerous authors have published on this subject and the main knowledge will be introduced below. In contrast, the idea that cells of the hematopoietic lineage regulate MSCs fate has been little investigated. Nevertheless, it becomes clear that macrophages participate in MSCs and bone physiology. This new concept will be introduced at the end of this chapter.
2.3.1. Niche components 101,166,171
• MSCs
Two subpopulations of MSC have been identified to be closely associated with putative HSC: the Nestin+ MSCs and CAR cells (CXCL12- abundant reticular cells) 172,84 . Nestin is an intermediate filament protein that has been originally identified as a marker of neural progenitors but has been also detected in a wide range of progenitor cells. CXCL12 (also known as stromal cell-derived factor, SDF1) is a chemokine that acts on CXC-chemokine receptor 4 and is essential for homing and maintenance of HSCs.
27 Nestin+ MSCs are strictly perivascular and are typically found in more central areas of the bone marrow. These MSCs express high levels of HSC maintenance factors including CXCL12, SCF (Stem Cell Factor) and Ang1 (Angiopoietin 1).
Deletion of Nestin-expressing MSCs leads to a 50% reduction in the bone marrow HSC numbers and a proportional increase in HSC number in the spleen. This suggests that Nestin+ MSCs are involved in HSCs retention at a perivascular location within the bone marrow
89,166,173.
CAR cells express HSC maintenance proteins such CXCL12 and SCF. As Nestin+ MSCs, deletion of CAR cells leads to a decrease of in HSCs numbers in the bone marrow 174,175.
• Osteoblast
Bone forming cells are crucial players for homeostasis of HSCs in the area closed to the endosteum. Osteoblasts express several cell-signaling molecules such as BMP4, Jagged-1, Ang1, TPO (thrombopoietin), WNTs ligand, SCF and CXCL12 166,176,177.
• Macrophages/Monocyte
Macrophages are one of the major actors for bone marrow homeostasis 166. They are positive regulators of Nestin+ MSCs and are required to maintain the expression of HSCs retention
factors. These resident bone tissue macrophages found in the endosteum are termed as Osteomacs ( Osteal tissue macrophage).
• Endothelial cells
Bone marrow endothelial cells were found to express factors that promote hematopoiesis such as granulocyte- colony-stimulating (GM-CSF), granulocyte-macrophage colony- stimulating factor (M-CSF), SCF and IL-6 173,177.
In addition to these cytokines, endothelial cells express adhesion molecules such as E-selectin, P-E-selectin, vascular adhesion molecule 1 (VCAM-1), intercellular adhesion molecule (ICAM 1) but also expressed CXCL12, and Notch ligand (Jagged-1).
• Nervous system
• Soluble factor Bone marrow source Effects on HSCs or immune cells
Angiopoietin 1 Osteoblastic cells, nestin-‐expressing MSCs Maintenance of long-‐term repopulating
activity and quiescence
CXCL12 CAR cells, nestin-‐expressing MSCs,
endothelial cells, osteoblastic cells Homing and retention; maintenance of the HSC pool size
SCF Endothelial cells, osteoblastic cells, nestin-‐
expressing MSCs Maintenance of long-‐term repopulating activity
Notch ligands Endothelial cells, osteoblastic cells Increased expression on osteoblastic cells
after parathyroid hormone stimulation is associated with increased HSC numbers in the bone marrow; however, loss of this signaling pathway does not impair HSC function in the steady state
Thrombopoietin Osteoblastic cells Maintenance of long-‐term repopulating
activity and quiescence
WNT ligands Osteoblastic cells Conflicting findings: enhanced self-‐renewal
when used pharmacologically; however, loss of this signaling pathway does not impair HSC function in the steady state121;
inhibition by osteoblastic cell-‐specific expression of DKK1 increased HSC cycling and reduced HSC serial transplant capability
Table 7: Soluble factors produced by bone marrow niches that contribute to HSC and
immune cell maintenance 173
Figure 24: Stem cell niche in steady state 166
28
Figure 25: Stem cell niche upon stimulation 166
29 system (SNS) tone. For example, increase in SNS tone induces the down-regulation of CXCL12 expression by MSCc 172,178,179.
Here are the mechanisms of action and function of the major players responsible for the niche stability :
• CXCL12, also called SDF-1, is a chemokine that binds to its receptor CXCR4. Once stimulated, this GPCR activates several pathways including PI3K and MAPKinase. CXCL12 promotes homing and retention of HSCs
• Angiopoietin is one of the vascular growth factors. It binds the receptor tyrosine kinase (RTK) Tie2. Following this stimulation, PI3K pathway is activated and leads to cell survival and quiescence of HSCs
• SCF, also called Kit factor, is a growth factor binding the c-Kit receptor (RTK). It supports maintenance of long-term repopulation activity.
• Thrombopoietin is a glycoprotein hormone binding the thrombopoietin receptor (also called c-Mpl). This JAK/STAT associated receptor induces expression of genes involved in HSCs quiescence.
Function of cited factors is summarized in Table 7.
2.3.2. Stem cell niche as a unit
Under steady state, HSCs are in close contact with osteoblasts and Nestin+ MSCs which both supply HSCs maintenance and quiescence factors including CXCL12, SCF, Ang-1, and TPO. As a result, HSCs are retained in the endosteal niche. In the perivascular niche, CAR cells and endothelial cells secrete factors that promote self-renewal of active HSCs. Meanwhile OsteoMacs (resident bone tissue macrophages) support the maintenance and proliferation of MSCs and osteoblasts. The SNS inhibits MSC proliferation and induces circadian oscillations of CXCL12 expression (Figure 24).
30
2.4. Role of OsteoMacs in MSCs physiology
If the influence of MSCs and (pre)osteoblasts on the self-renewall and retention of HSCs into bone marrow niches is well documented, the role of HSCs on MSCs biology and
osteoblastogenesis is poorly understood. Jung et al. have shown that the co-culture of non adherent HSCs with bone marrow stromal cells increases the number of osteoblastic- and fibroblastic-CFU180.
Beside their role as first line of defense against pathogens invasion, macrophages play important roles in various homeostasis processes as it is the case for bone marrow physiology (see previous section) In 2008, Pettit and collaborators identified a population of resident tissue macrophages called OsteoMacs at the level of the endosteum and the periosteum 181. These cells, that are not precursors of osteoclasts, produce a wide range of regulatory molecules involved in osteoblast differentiation and function like BMP2, 182. Production of TGF-β, Vitamin D3 and osteopontin has been also observed 183–185.
In co-culturing experiment, it has been shown that macrophages promote osteoblast mineralization activity suggesting a co-dependence between macrophages and osteoblasts 181. The importance of these OsteoMacs has been also established in vivo : in a transgenic mouse model in which macrophage depletion is induced, bone remodelling is severely altered 181.
2.5. MSCs and cell therapy/tissue engineering
MSC have been widely used in stem cell transplantation. Current research focuses on the use of MSCs for the purpose of regeneration of damaged tissues of the musculoskeletal system, such as cartilage, bone, ligaments, muscles and tendons 186,187. Although bone tissue is capable of self-regeneration, the natural bone healing process is in some cases insufficient.
In bone regenerative medicine, two strategies of stem cell application have been extensively used 188:
- Cell therapy : bone marrow stem cells are either isolated from the patient (autologous transplantation) or from other donors (allogenous transplantation). The cells are expanded in vitro and administered directly to the patient to substitute lost cells - Tissue engineering: stem cells are seeded into 3 dimensional scaffolds and
differentiated into the demanded cell type. The composed artificial tissue construct is subsequently implanted into the patients’ tissue lesion.
31 (0.001 to 0.01% of the nucleated marrow cells) and their limited accessibility reduce their
attractiveness in this field of research. Adipose tissue stem cells (ASC) represent another source for bone tissue engineering. Lipoaspirate contains a relatively high frequency of stem cells (1 to 5% of isolated nucleated cells) 189. Compared to bone marrow, lipoaspirate
isolation requires minimally invasive techniques. ASC cells differentiate into osteoblast-like cells in vitro in the presence of ascorbate, β-glycerophosphate, dexamethasone and vitamin D3. Because ASCs are able to form mineralized matrix in vitro as well as in 2D and 3D culture (with or without scaffold) , they were shown to have a significant healing potential in various studies when seeded and implanted into tissue defect 190.
MSCs have been also widely used to improve cardiac and vascular function through the induction of angiogenesis 191–193. Although MSCs can differentiate into functional endothelial cells and cardiomyocyte in vivo, they produce high level of neoangiogenic cytokines, especially VEGF (Vascular endothelial growth factor). Thereby, VEGF acts as a paracrine factor which contributes to the MSCs angiogenic properties.
3. EXTRACELLULAR NUCLEOTIDES SIGNALING IN BONE PHYSIOLOGY
3.1. Role of extracellular nucleotides signaling in human MSC
Over the past decade, it has been shown that extracellular nucleotides exert actions on MSC
194–198, although data collected so far are conflicting and indicate that pharmacological assays
are not sufficient to identify the receptors involved in these actions.
3.1.1. Expression of P2 receptor in MSC
In 2010, Ferrari et al. demonstrated that human marrow-derived MSCs expressed all P2YRs subtypes cloned so far 194. Later, the research staff of Dr. Edda Tobiasch compared the P2Rs expression between human adipose tissue-derived stem cells (ATSCs) and MSCs derived from dental follicle (DFCs)195. mRNA expression of all P2Y is detected in either cell type. However, P2Y1, 2, 4 and 14R are expressed in DFCs to a lower degree when compared to that
observed in ATSCs. Interestingly, P2Y1,2,4 and 14 expression is down-regulated following
osteogenic differentiation. In parallel, they observed a down-regulation of P2Y4 and 14
expression in ATSCs after adipogenic stimulation while P2Y11 is upregulated.
Noronha-Matos et al. also demonstrated the presence of P2Y2, 4 and 6 receptor proteins in
32 observed that P2Y2 and 4 receptor proteins expression increases with osteogenic treatment
whereas that of P2Y6 remains constant 198.
These data thus confirm that according to the tissue origin, MSCs do not display the same expression profile particularly regarding to the P2YRs expression.
3.1.1. ATP release and extracellular metabolism in MSC
To date, very few studies focused on the ATP release process in MSCs. However it is reported that hMSCs released spontaneously large amount of ATP 196,199. Moreover, when the cells are incubated with a non-specific hemichannel blocker, the ATP concentration decreased markedly. In contrast exocytosis and anion channel blockers have no effect on ATP release 196.
NTPDase 1, 2 and 3 and CD73 are expressed and functional in hMSCs 27,198. Interestingly,
osteogenic stimulation induces up-reguation of NTPDase 2 (CD39L1) 198. Unlike osteoblasts, undifferentiated MSCs expressed the 5'-nucleotidase (CD73)200. Because of this differentiated expression , it has been proposed that these enzymes assume the role of specific markers that allow distinction between differentiated osteoblasts and early undifferentiated MSCs 200.
3.1.2. Effect of extracellular nucleotides in MSC physiology
ATP modulates both proliferation and differentiation of MSCs.
In 2007, two contradictory papers were published, one demonstrating that ATP inhibits hMSCs proliferation 199 and the other showing the opposite effect 105. However, the two authors agree on the fact that ATP effect is associated with the stimulation of intracellular calcium signaling pathways. In 2010, Ferrari and colleagues provided new evidence of the inhibitory activity of ATP on hMSCs proliferation 194.
P2Y
RECEPTOR
DEXA AND µCT
ANALYSIS REF
P2Y1
Bone Mineral Density↓
201
Bone Mineral Content ↓ Trabecular volume ↓ P2Y2
Bone Mineral Content ↑ 201
Trabecular volume ↑
P2Y4 NOT TESTED
P2Y6
Length ↑ 202
Bone Mineral Content ↑ P2Y12
Bone Mineral Content ↑ 203
Trabecular volume ↑
P2Y13 Reduced Bone Turnover 204
P2Y14 NOT TESTED
33 Moreover, new studies also highlighted the role of eNTPs in the regulation of cytokine secretion by hMSCs 194,205,206. Indeed, these recent works showed that ATP treatment induces secretion of IL-2, INF-γ, CXCL12 and SCF.
3.2. Bone phenotype of P2Y13 knockout mice
As described in the two previous sections, extracellular nucleotides exert different effects on MSCs and osteoblast function. Nevertheless, the collected data are slightly conflicting. To distinguish which nucleotides and receptors could be involved, P2Y- deficient mice are very useful 201. Dexa (Dual energy x-ray absorbtiometry) and µCT (micro computed tomography) analysis were performed and many P2Y deficient mice exhibit a bone tissue alteration which is unique for each strain (for detail see Table 8)
Analysis of the microstructure of the bone reveals that P2ry13 gene deletion does not significantly alter length and total volume of the long bone. Compared to the WT, P2Y13
-/-mice exhibit a decreased trabecular bone volume (37,5 %) as well as a decrese number of trabecules (37,8%). Bone histomorphometry assay revealed that there are less osteoblasts and osteoclasts on the bone surface of the cortical bone (about 50% less).
In calvaria-derived osteoblasts, deletion of P2ry13 induces down regulation of regulatory genes (RANKL) involved in osteoclast differentiation. Decrease of RhoA/ROCKI signaling has been also observed. These results and the fact that P2Y13-/- mice have a reduced rates of
