**5. Cell-to-cell communication through exosomes**

Cell-to-cell communication is required to guarantee proper coordination among different cell types within tissues. Cells may communicate by soluble factors (Majka, Janowska-Wieczorek et al. 2001), adhesion molecule mediated cell-to-cell interactions including cytonemes con‐ necting neighbouring cells enabling ligand–receptor-mediated transfer of surface-associated molecules, or by tunnelling nanotubules which establish conduits between cells. These would allow the transfer of both surface molecules and cytoplasmic components (Rustom, Saffrich et al. 2004; Sherer and Mothes 2008). Recently published studies have suggested that cells may also communicate by spherical membrane fragments called micro-vesicles (MVs) or exosomes (Ratajczak, Wysoczynski et al. 2006). MVs or exosomes were since long considered to be inert cellular debris, and the frequently observed vesicles by electron microscopy in the interstitial space of tissues or in blood were considered the consequence of cell damage or a result of dynamic plasma membrane turnover (Siekevitz 1972). It was suggested that the circular plasma membrane fragments released from human cells might possibly result from a specific process, and it was shown that they carried functional membrane enzymes in the same ratio as the membrane of the cells they originated from (De Broe, Wieme et al. 1977). However, only a few recent studies have assigned a defined function to the vesicles/exosomes released to the microenvironment by various cell types. Two distinct processes of shedding vesicles from the cells have been described. MVs may originate from the endosomal membrane compartment that, subsequent to fusion with the plasma membrane, is extruded from the cell surface of activated cells as exosomes (Heijnen, Schiel et al. 1999; Rozmyslowicz, Majka et al. 2003). Otherwise, MVs may take origin by direct budding from the cell plasma membrane as shedding vesicles (Cocucci, Racchetti et al. 2009).

Released MVs may remain in the extracellular space in the proximity of the place of origin, or they may enter into the circulation. One has hypothesized that, under normal healthy condi‐ tions and disease states, micro-vesicles contain microRNAs, contributing to biological homeo‐ stasis in general (Hunter, Ismail et al. 2008; Gordeladze, Djouad et al. 2009; Gordeladze 2011). It may also be hypothesized that MVs or exosomes, when present in disproportional amounts within a tissue during development of certain diseases, in fact may affect all adjacent cell types, thus altering their phenotypes and main functional properties (Camussi, Deregibus et al. 2010).

that X-ray analyses and histology (visual parameters) were not significantly altered when cells were manipulated by cytokines (IL-1, IL-6, IL-17, and TNFα). However, molecular parameters were dramatically changed. X-ray analyses and histology were performed on excised legs 21 days subsequent to cell deposition. **B**. Differentiated chondrocytes (21 days) exposed to cytokines (initial 14 days) or pre-microRNAs (initial 14 days) and osteoclasts injected into the tibial muscle of SCID mice. Molecular markers of chondrocytes and osteoclasts (PBMCs differentiated for 10 days) were ana‐ lysed by Q-PCR or colorimetric methods (GAG = glucosaminoglycan/DNA). Nd = Not determined. All values are given relative to control conditions (chondrocytes alone or chondrocytes + osteoclasts). **C**. Differentiated osteoblasts (21 days) exposed to cytokines (initial 14 days) or antago-microRNAs (initial 14 days) and osteoclasts were injected into the tibial muscle of SCID mice. Molecular markers of osteoblasts and osteoclasts (PBMCs differentiated for 10 days) were analysed by Q-PCR or colorimetric methods (Ca2+-analyses in HCl-extracts). All values are given related to control

Cell-to-cell communication is required to guarantee proper coordination among different cell types within tissues. Cells may communicate by soluble factors (Majka, Janowska-Wieczorek et al. 2001), adhesion molecule mediated cell-to-cell interactions including cytonemes con‐ necting neighbouring cells enabling ligand–receptor-mediated transfer of surface-associated molecules, or by tunnelling nanotubules which establish conduits between cells. These would allow the transfer of both surface molecules and cytoplasmic components (Rustom, Saffrich et al. 2004; Sherer and Mothes 2008). Recently published studies have suggested that cells may also communicate by spherical membrane fragments called micro-vesicles (MVs) or exosomes (Ratajczak, Wysoczynski et al. 2006). MVs or exosomes were since long considered to be inert cellular debris, and the frequently observed vesicles by electron microscopy in the interstitial space of tissues or in blood were considered the consequence of cell damage or a result of dynamic plasma membrane turnover (Siekevitz 1972). It was suggested that the circular plasma membrane fragments released from human cells might possibly result from a specific process, and it was shown that they carried functional membrane enzymes in the same ratio as the membrane of the cells they originated from (De Broe, Wieme et al. 1977). However, only a few recent studies have assigned a defined function to the vesicles/exosomes released to the microenvironment by various cell types. Two distinct processes of shedding vesicles from the cells have been described. MVs may originate from the endosomal membrane compartment that, subsequent to fusion with the plasma membrane, is extruded from the cell surface of activated cells as exosomes (Heijnen, Schiel et al. 1999; Rozmyslowicz, Majka et al. 2003). Otherwise, MVs may take origin by direct budding from the cell plasma membrane as

Released MVs may remain in the extracellular space in the proximity of the place of origin, or they may enter into the circulation. One has hypothesized that, under normal healthy condi‐ tions and disease states, micro-vesicles contain microRNAs, contributing to biological homeo‐ stasis in general (Hunter, Ismail et al. 2008; Gordeladze, Djouad et al. 2009; Gordeladze 2011). It may also be hypothesized that MVs or exosomes, when present in disproportional amounts within a tissue during development of certain diseases, in fact may affect all adjacent cell types, thus altering their phenotypes and main functional properties (Camussi, Deregibus et al. 2010).

conditions (osteoblasts alone or osteoblasts + osteoclasts).

488 Regenerative Medicine and Tissue Engineering

shedding vesicles (Cocucci, Racchetti et al. 2009).

**5. Cell-to-cell communication through exosomes**

**Figure 6. A**. The effect of exosomes-containing 100,000xg fractions of supernatants derived from activated human Th17 cells on human chondrocytes and osteoblasts in the presence and absence of osteoclasts. Chondrocytes (500,000 cells) derived from hMSCs were either exposed to 1 ml volume containing exosomes from 250,000 Th-17 cells or pre-microRNA 222 (which is highly expressed in Th17 cells, but not in chondrocytes) and analysed for marker gene and microRNA expressions using standard Q-PCR methods (Ambion) or immunohistochemistry (aggrecan and collagen2α1 positive surfaces). Osteoblasts (500,000 cells) derived from hMSCs were either exposed to 1 ml volume containing exosomes from 250,000 Th-17 cells or pre-microRNA 22 (which is highly expressed in Th-17 cells, but not in osteoblasts) and analysed for marker gene and microRNA expressions using standard Q-PCR methods (Ambion) or col‐ orimetric methods (mineralized surface visualized by Alizarin red S). **B**. The effect of exosomes-containing 100,000xg fractions of supernatants derived from activated human Th17 cells on human chondrocytes and osteoblasts in the presence and absence of osteoclasts. Differentiated osteoblasts (21 days) exposed to exosomes from Th17 cells (initial 14 days) or antago-microRNAs (initial 14 days) and osteoclasts were injected into the tibial muscle of SCID mice, and molecular markers of osteoblasts and osteoclasts (PBMCs differentiated for 10 days) were analysed by Q-PCR or colori‐ metric methods (Ca2+-analyses in HCl-extracts). All values are given related to control conditions (osteoblasts alone or osteoblasts + osteoclasts).

In one publication, it was shown that exosomes from healthy volunteers matched mononuclear cells and contained 420 known mature microRNAs (Hunter, Ismail et al. 2008). Hierarchical clustering of the data sets pointed to significant differences in microRNA expression between peripheral blood mononuclear cells (PBMCs) and plasma micro-vesicles. It was observed that 71 microRNAs co-expressed between micro-vesicles and PBMCs. Prediction of the gene targets and associated biological pathways regulated by the detected microRNAs demonstrated that the majority of these microRNAs expressed in the micro-vesicles from the blood were pre‐ dicted to regulate the differentiation of blood cells and their metabolic pathways (Hunter, Ismail et al. 2008). Interestingly, a small group of these microRNA species were also predicted to serve as important modulators of immune function. The microRNAs in question were hypothesized to be taken up by adjacent cells, and thus it may be asserted that exosomes from Th17 cells, known to be present in vast numbers in articular fluid of patients with rheumatoid arthritis (Adamopoulos and Bowman 2008; Nakashima and Takayanagi 2009; Raggatt and Partridge 2010; Nakashima, Hayashi et al. 2012) with rheumatoid arthritis, may disturb the phenotypic characteristics of osteochondral cells in inflamed joints.
