msc are multipotent cells , capable of differentiating , in vitro , into different lineages , including osteocytes , chondrocytes , adipocytes , muscle cells , cardiomyocytes , and neural precursor .
more recently , the capacity of human msc ( hmsc ) to suppress both innate and adaptive immunity has been described [ 2 , 3 ] as well as their poor immunogenicity . as a result
, the therapeutic potential of hmsc as immunoregulatory agents is currently being explored in several phase i / ii clinical trials [ 46 ] .
a number of recent studies have focused on the influence of hmsc on dc functions [ 2 , 7 , 8 ] .
can be generated from cd34 stem / progenitor cells and from cd14 monocytes [ 10 , 11 ] .
the in vitro interaction between hmsc and either cd34 or cd14 dc progenitors inhibits the generation of functional dc [ 2 , 7 , 8 ] , skewing their differentiation toward phenotypically abnormal dc , which express lower level of cd1a , cd40 , cd80 , cd86 , and cd83 .
different mechanisms are responsible for the effects of hmsc on dc differentiation including the involvement of both soluble factors   and cell - to - cell contact interactions [ 12 , 13 ]
. taken together , these studies demonstrated the profound immunosuppressive effects of hmsc on dc .
however , the topic of whether the immunological properties of hmsc persist after in vitro differentiation into hdoc has been not deeply investigated .
indeed , it has only been shown that hdoc suppress t cell proliferation elicited by allogeneic cells [ 14 , 15 ] .
in fact , the characterization of the immunological properties of hdoc may be of crucial importance for cell - based tissue - engineering therapeutic strategies .
although hmsc have been shown to contribute to repair bone defects in vivo either through infusion or local implantation [ 16 , 17 ] , more recent strategies aim to in vitro differentiate hmsc into hdoc before implantation , in order to optimize bone regeneration [ 18 , 19 ] .
therefore , the evaluation of the interactions between hdoc and the host allogeneic immune system , in particular of antigen presenting cells ( apc ) such as dc , may be important in view of clinical trials . since the preparation of hdoc for cell - based tissue - engineering strategies may be different [ 18 , 19 ]
, it is interesting to evaluate whether different culture systems may affect the capacity of doc of modulating the immune response . for these reasons
, here we characterized the immunological properties of hdoc manipulated in two different ways , which mimic the use of hdoc as cell suspension or as adherent cells , by showing their capacity to modulate the phenotype and the functions of allogeneic dc .
human mscs ( hmscs ) were isolated from bm aspirates of healthy donors after obtaining written informed consent .
the characteristics of the donors are shown in table s1 ( see supplementary material available online at http://dx.doi.org/10.1155/2015/526195 ) . to induce hdoc
, hmscs were seeded at 3.1  10 cells / cm and grown in osteogenic differentiation medium ( lonza ) containing l - glutamine , mcgs , dexamethasone , ascorbate , -glycerophosphate , and penicillin / streptomycin .
the osteogenic differentiation was analysed by cytological staining and by the evaluation of runx2 mrna levels ( figure s1 ) .
briefly , cells were fixed in 10% pfa in pbs for 15  min at room temperature ( rt ) , rinsed with pbs and distilled water , and then stained with 40  mm alizarin red solution ( sigma aldrich ) ph 4.2 , for 75  min at rt with gentle agitation .
after washing , alizarin red was extracted from fixed cells by incubating in 10% ( w / v ) cetylpyridinium chloride ( cpc ) solution ( sigma aldrich ) in 10  mm sodium phosphate for 15  min at rt with gentle agitation .
after 2 weeks hmsc and hdoc were lysed and total rna was extracted using rneasy micro kit ( qiagen ) and reverse transcribed using a promega improm ii kit and random examers in 20  l final volume .
quantitative real - time pcr was performed using an abi prism 7900 sequence detection system ( applied biosystems ) .
the relative level of runx2 mrna was calculated by subtracting ct values of the control gene ( gapdh ) from the ct values of the specific gene ( runx2 ) .
primers probe for runx2 , hs00231692_m1 , and gapdh , hs00266705_g1 were purchased from applied biosystems . for immunological assays , hdoc
were used either in adhesion on the same plate used for differentiation ( adh - doc ) or after harvesting and replating ( det - doc ) . for harvesting ,
monolayers of hdoc were washed with washing buffer ( 0.5% bsa , 5  mm edta in pbs ) , incubated with trypsin ( 0.25% trypsin with 0.1% edta , euroclone ) , and collected with a cell scraper . where indicated the cellular component was lysed by incubating the monolayer with 0.1  m nacl , 0.01  m tris , 0.2% edta , and 0.1% tritonx-100 . in all the assays ,
the same number of adh - doc and det - doc was used . for each experiment ,
the cell number was evaluated the day before the assays , by counting 3 wells of adh - doc harvested separately .
the mean values of these 3 counts were used to plate det - doc . then , adh - doc and det - doc were cultured overnight in complete medium ( rpmi 1640 medium ( lonza ) supplemented with 10% heat - inactivated fbs ( gibco - invitrogen ) , 2  mm l - glutamine , 100  u / ml penicillin , and 100  g / ml streptomycin ( mp biomedicals ) ) at 37c in 5% co2 , before coculturing with dc and cd3 t cells .
the experimental strategy is showed in a schema ( figure s2 ) . the same strategy and the same conditions
indeed , the number of det - msc to plate was established by counting independently 3 wells of adh - msc .
buffy coats were obtained from healthy donors and were used to isolate mononuclear cells ( mnc ) , by gradient centrifugation ( lymphoprep ; 1.077  g / ml ; nycomed pharma ) .
after separation , cd14 monocytes and cd3 t cells were purified from total mnc by magnetic separation columns ( miltenyi biotec ) , according to manufacturer 's instructions .
monocyte - derived dcs ( mo - dcs ) were generated by a 5-day culture of cd14 cells in complete medium plus 50  ng / ml granulocyte - macrophage colony - stimulation factor ( gm - csf ) and 800  u / ml il-4 ( both by endogen ) , at 37c in 5% co2 . for maturation , day 5 mo - dcs were cultured with gm - csf and il-4 and incubated for 48 hours in presence of a cocktail of cytokine made of 10  ng / ml tnf , 10  ng / ml il-6 , 10  ng / ml il-1 , and 1  g / ml pge2 ( all by endogen ) . to detect lymphocytes proliferation
, cd3 t cells were labelled with carboxyfluorescein succinimidyl ester ( cfse , 2.5  m from sigma aldrich ) before plating . to test the capacity of adh - doc and det - doc to modulate the dc allostimulatory capacity , allogeneic mo - dc , either immature or mature ,
were irradiated ( 3000  cgy ) and plated ( 1  :  1 ) at 37c on either adh - doc or det - doc in presence of third party cfse - labeled cd3 t cells ( 1  :  10 ) . as a positive control ,
cd3 t cells were cultured with mo - dc and , as a negative control , with medium alone . for a second set of experiments ,
cd3 t cells were added to the upper chamber of a 0.4  m pore polycarbonate filter in 24-well transwell chambers ( corning costar ) to keep them separated from dc and from either adh - doc or det - doc , which were plated in the lower chamber of the transwell system .
after 5 days , the cells in the upper chamber were collected and analyzed using bd facscantoii equipment ( bd biosciences ) . to test the capacity of adh - doc and det - doc to modulate the dc capacity to induce in cd3 t cells the treg cell or the th17 cell phenotype , allogeneic mo - dc , either immature or mature ,
were plated ( 1  :  1 ) on either adh - doc or det - doc in presence of allogeneic cd3 t cells ( 1  :  20 ) . as a positive control , cd3 t cells were cultured with mo - dc , as a negative control , with medium alone . for a second set of experiments ,
cd3 t cells were added to the upper chamber of a 0.4  m pore polycarbonate filter in 24-well transwell chambers ( corning costar ) , while dc and either adh - doc or det - doc were added in the lower chamber .
cell cultures were incubated at 37c for 5 days ; then the t cells were harvested from the upper chamber and stained for immunophenotype via tricolor immunofluorescence , which was performed using fluorescein isothiocyanate- ( fitc- ) conjugated anti - human cd4 ( clone rpa - t4 ) , phycoerythrin- ( pe- ) conjugated anti - human foxp3 ( clone 206d ) , and allophycocyanin- ( apc- ) conjugated anti - human cd25 ( clone bc96 , biolegend ) . for cell - surface staining , 1  10 cells/100  l were incubated in the dark for 20  min at rt with mabs in phosphate - buffered saline- ( pbs- ) 1% bovine serum albumin .
subsequently , for foxp3 intracellular staining , cells were incubated at rt in the dark for 20  min with fix / perm buffer followed by 15  min with perm solution and additional 30  min with the mab .
after 2 washes , samples were analyzed using bd facscantoii equipment ( bd biosciences ) .
immature and mature mo - dc were incubated for 5 days either alone or with adh - doc or det - doc ( 1  :  1 ) .
dual - color immunofluorescence was performed using the following panel of mabs : pe- or fitc - conjugated anti - human hla - dr ( bd pharmingen ; clone l242 ) , cd86 ( biolegend ; clone it2.2 ) , and cd80 ( biolegend ; clone 2d10 ) .
immature and mature mo - dc were incubated for 5 days either alone or with adh - doc or det - doc ( 1  :  1 ) .
supernatants were collected and tested for the release of tgf-1 ( drg diagnostics , marburg , germany ) il-6 and il-10 ( thermo scientific , erembodegem , belgium ) , according to the manufacturer 's instructions .
 = p < 0.05 ,  = p < 0.01 , and  = p < 0.001 , bonferroni corrected .
we first compared the suppressive capacity of hdoc on dc - mediated t cell proliferation .
in particular , we used hdoc either after detachment from culture plates ( det - doc ) or as adherent cells ( adh - doc ) to mimic , in vitro , the conditions in which they are used therapeutically ( i.e. , cultured and then detached to be injected as cellular suspension or grown adherent on a scaffold and implanted without detachment ) .
as shown in figures 1(a ) and 1(b ) , both det - doc and adh - doc inhibited the allostimulatory capacity of dc ( kruskal wallis p < 0.0001 ) .
however , the inhibition induced by adh - doc is much stronger than det - doc
thus , we asked why det - doc partially lost their ability to inhibit dc allostimulation . to investigate the mechanism(s )
involved in such process , we first asked whether det - doc were less viable than adh - doc , since they were harvested after treatment with trypsin and collected with a cell scraper .
we could not find a reduction of viability in harvested det - doc ( data not shown ) .
then , we asked whether the extracellular matrix secreted by hdoc ( routinely discarded to obtain det - doc ) could itself inhibit the allostimulatory capacity of dc .
therefore , we lysed the cellular component of hdoc and recovered the extracellular matrix to perform the same assay . as shown in figure 1(c ) , the extracellular matrix had no inhibitory capacity by itself .
then , we asked whether the procedure used to detach cells from the plates influenced by itself the tolerogenic capacity of detached doc .
thus , we compared the capacity of undifferentiated hmsc used after detachment from culture plates ( mimicking det - msc ) with that of cells used as adherent cells ( mimicking adh - msc ) .
as shown in figure 1(d ) , both det - msc and adh - msc were able to inhibit the dc allostimulation at the same level , suggesting that the detachment procedure , by itself , had no influence on the tolerogenic capacity of the cells .
finally we investigated whether the different capacity to inhibit dc allostimulation of det - doc and adh - doc was mediated by soluble factors rather than by a cell - to - cell contact - dependent mechanism(s ) .
therefore , we performed the same suppression assay by separating cd3 t cells with a 0.4  m pore transwell chamber , which allows the migration of only soluble factors .
as shown in figure 1(e ) , neither det - doc nor adh - doc could inhibit dc allostimulation when dc are cultured without any contact with cd3 t cells .
therefore , in this setting , hdoc modified the allostimulatory capacity of dc , mainly by modulating cell - to - cell contact - dependent mechanism(s ) .
thus , we wanted to investigate which surface marker(s ) on dc may be affected by the inhibitory activity of hdoc [ 24 , 25 ] . to this end
, we cocultured dc with either det - doc or adh - doc , for 5 days and then analyzed dc phenotype ( kruskal - wallis p < 0.0001 for cd80 , p = 0.0012 for cd86 ) . to discriminate dc from hdoc we gated on hla - dr cells .
as shown in figure 2(a ) , the coculture of dc with det - doc or adh - doc resulted in the downregulation of cd80 .
notably , adh - doc and det - doc could downregulate this marker to a comparable extent .
conversely , det - doc were not able to downregulate cd86 , while this molecule was strongly downregulated by adh - doc , as shown in figure 2(b ) . taken together ,
our results demonstrate that , if hdoc are left in their own extracellular matrix ( adh - doc ) , they acquire a high capacity to inhibit the dc expression of molecules involved in t cell costimulation . on the other hand , if the interactions of hdoc with their extracellular matrix are destroyed ( i.e. , hdoc are harvested from their own extracellular matrix and seeded back in a new culture support , as for det - doc ) , they partly lose this capacity , resulting in lower inhibition of costimulatory molecules on dc and , consequently , of t cell proliferation .
dc have a crucial role not only in the activation of t cell response but also in the induction of tolerance by generating cd4cd25foxp3 t cell population .
therefore , we asked whether det - doc and adh - doc could modulate this function of dc . to test this hypothesis
, we cocultured dc with allogeneic t cells , in the presence or absence of either det - doc or adh - doc ( kruskal - wallis p < 0.0001 ) .
coculture of t cells with dc increased the percentage of cd4cd25foxp3 t cells , as compared to cd3 t cells alone ( figures 3(a ) and 3(b ) ) .
the addition of det - doc or adh - doc decreased the population of cd4cd25foxp3 t cells induced by dc ( figures 3(a ) and 3(b ) ) , with the adh - doc showing the greatest inhibitory effect ( p < 0.001 , bonferroni corrected ) .
then , we asked whether the different capacity to inhibit the dc - mediated cd4cd25foxp3 t cell generation of det - doc and adh - doc was mediated by soluble factors rather than by a cell - to - cell contact - dependent mechanism
. therefore , we performed the same assay by separating cd3 t cells with a 0.4  m pore transwell chamber .
as shown in figure 3(c ) , det - doc and adh - doc did not inhibit the capacity of dc to induce a cd4cd25foxp3 t cell population and there was no significant difference among different hdoc .
therefore , adh - doc had an enhanced inhibitory effect on dc to induce cd4cd25foxp3 t cell population , in comparison to det - doc , by modulating mechanism(s ) involved in cell - to - cell , contact - dependent suppression .
as shown in figure 2 , adh - doc were able to downregulate the expression of both cd80 and cd86 , while det - doc decreased only cd80 at a lower extent .
. taken together , these data demonstrate that hdoc inhibit the capacity of dc to induce cd4cd25foxp3 t cells .
however , such inhibitory capacity is markedly increased when hdoc are left adherent to the same plate used for differentiation ( adh - doc ) , whereas it is reduced when hdoc are detached from culture plates ( det - doc ) .
this effect may be mediated by different modulation of cd80 and cd86 costimulatory molecules on dc by the two different preparations of hdoc . since recent reports showed that there is a reciprocal relationship between cd4cd25foxp3 t cells and th17 cells in their development
[ 28 , 29 ] , we asked whether adh - doc and det - doc showed differential inhibitory capacity during th17 generation as well as during cd4cd25foxp3 t cell induction .
since th17 cells can be identified as cd4cd161cd196 t cells [ 30 , 31 ] , we analyzed the induction of cd4cd161cd196 t cells , after coculture of cd3 t cells with dc alone or incubated either with det - doc or with adh - doc ( kruskal - wallis p < 0.0001 ) .
coculture of t cells with dc increased the percentage of cd4cd161cd196 t cells , as compared to cd3 t cells alone ( figures 4(a ) and 4(b ) ) .
interestingly , the addition of det - doc enhanced the generation of cd4cd161cd196 t cells induced by dc , while the addition of adh - doc significantly decreased that population ( figures 4(a ) and 4(b ) ) .
then , we asked whether the different capacity to modulate the dc - mediated cd4cd161cd196 t cell generation of det - doc and adh - doc was mediated by cytokine secretion rather than by a cell - to - cell contact - dependent mechanism(s )
. therefore , we performed the same assay but after separating cd3 t cells from both dc and doc with a 0.4  m pore transwell chamber .
as shown in figure 4(c ) , when added in the upper chamber of the transwell , cd3 t cells differentiated into cd4cd161cd196 t cells similarly to cell - to - cell contact culture conditions .
therefore , we conclude that hdoc influence the generation of cd4cd161cd196 t cells by dc , by modulating soluble factor(s ) . to investigate which soluble factor(s )
involved in the differentiation of cd4cd161cd196 t cells was regulated by the presence of hdoc , we analyzed the supernatants of the coculture for the presence of tgf- and il-6 , since it has been previously shown that dc can induce th17 differentiation through the release of il-6 , which acts in concert with tgf- .
our results showed that il-6 secretion was enhanced in presence of det - doc , while it was not influenced by the presence of adh - doc ( figure 5(a ) , kruskal - wallis p < 0.0001 ) .
however , tgf- was decreased in the supernatants of both the cocultures , compared with the supernatant of the dc cultured alone ( figure 5(b ) , kruskal - wallis p < 0.0001 ) . to evaluate whether det - doc and/or adh - doc could modulate the extracellular milieu towards a tolerogenic environment , il-10 was also tested and no differences were found ( figure s3 ) .
taken together , these data demonstrate that hdoc can modulate the dc induction of cd4cd161cd196 t cells via il-6/tgf- regulation
. the different capacity of det - doc and of adh - doc to modulate the induction of the cd4cd161cd196 t cell phenotype may be due to the different balance between il-6 and tgf- secreted or induced by these cells , a difference that according to our results is generated by the way hdoc are manipulated .
the most efficient tissue source would be the autologous bone , but the donor - site morbidity , the inadequate supply , and the problems about size and shape make this source not always feasible .
allografts from cadaveric donors are increasingly used , but the risk of disease transmission and/or immune reaction limit their use [ 33 , 34 ] .
indeed , allogeneic peptides may be presented by apc , such as dc , on mhc - i and mhc - ii molecules and can activate cytotoxic cd8 t cells and cd4 helper t cells .
this activation can ( i ) mediate allograft rejection , ( ii ) dramatically inhibit bone generation , and ( iii ) lead to gradual , long - term immune response . among the other sources tested to prevent these adverse effects ,
msc have been shown to be immunoprivileged , that is , nontargeted by mhc - mismatched immune cells , to exert a suppressive effect on the host immune system   and to repair bone defects in vivo either through infusion or through local implantation [ 16 , 40 ] .
the main limitation for bone repair is the low capacity of engraftment of the in vitro - cultured msc .
since the basic functional unit for the repair of bone defects is the differentiated osteogenic cell ( doc ) derived from msc , it has been suggested that osteogenic differentiation of msc before implantation might be useful to optimize bone regeneration . in this way ,
the interval between implantation and subsequent osteogenesis in situ might be shortened , with a consequent reduction of loss of the implanted cells .
however , differentiated osteoprogenitors may be not immunoprivileged and may not have the same suppressive effect on the host immune system as their undifferentiated msc progenitors . here
we show for the first time that the immunological properties of hmsc to modulate dc phenotype and functions can persist after in vitro differentiation into hdoc .
however , our data demonstrate that the capacity of hdoc of modulating recipient dc function depends on different processing protocols and experimental conditions ( i.e. , the presence or absence of extracellular matrix ) .
in fact , det - doc are less potent than adh - doc to inhibit the allostimulation of dc in a contact - dependent manner , and this is due to the partial downregulation of the costimulatory molecules expressed on dc .
indeed , det - doc do not modify the expression of cd86 , while they weakly downregulate cd80 . on the other hand , adh - doc
strongly downregulate cd80 and cd86 on dc , resulting in a more profound inhibition of t cell proliferation .
the different capacity of det - doc and adh - doc to downregulate cd80 and cd86 might also explain why adh - doc are more efficient than det - doc in inhibiting also the induction of cd4cd25foxp3 t cells by dc , in a contact - dependent manner .
similarly , adh - doc have an opposite effect on dc in the induction of cd4cd161cd196 t cells in comparison with det - doc .
in fact , adh - doc inhibit , while det - doc enhance , the capacity of dc to generate cd4cd161cd196 t cells in a cytokine - dependent manner , depending on their capacity to differently modulate the balance between tgf- and il-6 .
the same results were obtained also with mature dc ( data not shown ) , suggesting that the capacity of hdoc to modulate dc function is independent from the maturation state of dc .
overall , here we show that adh - doc strongly inhibit some of the main functions of dc , such as the induction of t cell proliferation , the generation of cd4cd25foxp3 , and the generation of cd4cd161cd196 t cells . on the other hand
we also show that det - doc weakly inhibit the dc capacity of inducing t cell proliferation and of generating cd4cd25foxp3 t cells , while they enhance the dc capacity of generating cd4cd161cd196 t cells .
thus , the coculture of either det - doc or adh - doc with dc results in altered apc functions .
in the present work , we showed that hdoc are able to modulate the function of dc and , therefore , the host immune response . moreover , by comparing hdoc as cellular suspension ( i.e. , det - doc ) to cells implanted on a scaffold ( i.e. , adh - doc ) , we showed that different manipulation techniques could result in different immunological properties of hdoc .
as a consequence , the culture system of hdoc can produce highly different immunological outcome .
. however , our report about different in vitro capacity of det - doc and adh - doc in modulating dc functions offers the rationale to specifically address , at the clinical level , the safety and immunomodulatory capacity of doc . indeed , better definition of the most suitable culture system for hdoc preparation may have relevant clinical implications for their implantation in the context of bone repair cell - based tissue - engineering clinical trials .
 
 in vitro differentiation of mesenchymal stromal cells ( msc ) into osteocytes ( human differentiated osteogenic cells , hdoc ) before implantation has been proposed to optimize bone regeneration . however , a deep characterization of the immunological properties of doc , including their effect on dendritic cell ( dc ) function , is not available . 
 doc can be used either as cellular suspension ( detached , det - doc ) or as adherent cells implanted on scaffolds ( adherent , adh - doc ) . by mimicking in vitro these two different routes of administration , we show that both det - doc and adh - doc can modulate dc functions . 
 specifically , the weak downregulation of cd80 and cd86 caused by det - doc on dc surface results in a weak modulation of dc functions , which indeed retain a high capacity to induce t - cell proliferation and to generate cd4+cd25+foxp3 + t cells . 
 moreover , det - doc enhance the dc capacity to differentiate cd4+cd161+cd196 + th17-cells by upregulating il-6 secretion . 
 conversely , adh - doc strongly suppress dc functions by a profound downregulation of cd80 and cd86 on dc as well as by the inhibition of tgf- production . in conclusion , we demonstrate that different types of doc cell preparation may have a different impact on the modulation of the host immune system . 
 this finding may have relevant implications for the design of cell - based tissue - engineering strategies . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions