mesenchymal stem cells ( mscs ) are one of the most interesting types of adult stem cells that are isolated , cultured , manipulated ex vivo , and have the ability of self - renewal and differentiation into various mesodermal cell lineages ( 1 - 3 ) . since the first description of mscs by friedenstein et al in 1976 as the clonal and plastic adherent cells , the interest in mscs rapidly grew with expanding knowledge about their exceptional characteristics and usefulness in the cell therapy applications ( 4 , 5 ) . several clinical case reports ( 6 ) have concluded the successful treatment of bone and cartilage defects , vascular ischemia and coronary artery disease , and of chronic skin wounds upon local administration of mscs to sites of injury . the injected cells were well tolerated and some spectacular healing results were obtained ( 7 ) . these results indicated that the immunosuppressive effects should be considered whenever msc transplantation takes place . human mscs may participate in cell therapy protocols by producing a broad range of mediators such as transforming growth factor- , hepatocyte growth factor ( hgf ) , and nitric oxide ( 8 , 9 ) . intravenous administration of murine mscs improves the outcome of neural ( 10 ) and lung ( 11 ) injury in experimental animal models primarily through paracrine effects and a shift from the production of pro - inflammatory to anti - inflammatory cytokines at the site of injury . in addition , several studies have shown the immunosuppressive effect of mscs through mechanisms identified for inhibition of proliferation and differentiation of immune cells ( 12 ) . the primary source of mscs in adult individuals is bone marrow ( bm ) , where they are immersed in the stroma at a low frequency . in humans , there is one msc per 34,000 nucleated cells ( 8) . although present in very low numbers , mscs are easily isolated from bm and are capable of substantial proliferation and expansion in culture . however , due to the possibility of donor morbidity , high degree of viral exposure , and invasiveness of procedures , the need to identify alternative sources to provide mscs with immunomodulatory properties has emerged ( 2 ) . mscs in adult individuals are also isolated and efficiently expanded from other body tissues such as adipose tissue ( at ) , which is derived from the embryonic mesenchyme , represent a rich source of mscs , and provides an abundant and accessible source of adult stem cells with minimal patient discomfort ( 13 - 16 ) . a recently reported alternative tissue source of mscs is the connective tissue wharton s jelly ( wj ) of human umbilical cord . these cells have the potential to be expanded , and can be obtained by a less invasive method , without posing harm to the mother or infant . moreover , it is a biological waste and can be used as an abundant source of mscs ( 3 , 14 , 17 , 18 ) . in this study , we compared the growth suppression effect of mscs derived from adult human bm , at , and wj on peripheral blood mononuclear cells ( pbmcs ) . human mscs were obtained from 5 ml bm aspirates from the iliac crest of normal donors within the age range of 1945 years . they were donors of bm to a related patient after obtaining approval of the ethics committee , shiraz university of medical sciences , shiraz , iran . written informed consent was also obtained allowing analysis of the clinical data and testing mentioned in this study . each sample of aspirate was diluted 1:1 with dmem - low glucose ( invitrogen , merelbeke , belgium ) and layered over about 5 ml of ficoll ( lymphoprep ; oslo , norway ) . the isolation method was according to a previously reported method ( 19 ) by some modifications , which will be stated completely . after centrifugation at 939 g for 20 min , the mononuclear cell layer was removed from the interface . cells were suspended in dmem and centrifuged at 338 g for 15 min then resuspended in basal dmem medium containing 10% fetal calf serum ( invitrogen , merelbeke , belgium ) , 1% penicillin ( invitrogen , merelbeke , belgium ) , 1% streptomycin ( invitrogen , merelbeke , belgium ) , and 2 mm glutamine ( invitrogen , merelbeke , belgium ) . the cells were seeded at a density of 80.000/cm in 25 cm t - flasks and maintained at 37 c in an atmosphere of 5% co2 . after 4 days , the non - adherent cells were removed and the media was changed every 3 days . in order to expand mscs , the adhered monolayer was detached with trypsin - edta ( invitrogen , merelbeke , belgium ) for 5 min at 37 c , after 14 days for the first passage and every 45 days for successive passages . during in vitro passaging , the cells were seeded at a density of 5 - 1010 cells / cm and expanded for several passages until they no longer reached confluence . at - mscs were extracted from breast ats of healthy human adult donors with cosmetic mammoplasty surgery after obtaining the fully informed consent as described previously ( 20 , 21 ) . briefly , fragments of ats washed with phosphate buffered saline ( pbs ) , minced and digested with 0.2% collagenase type i ( gibco , usa ) at 37 c . the resulting cell suspension was centrifuged and the pellet including the adherent stromal cells was put on a ficoll gradient ( biosera , uk ) to separate the stromal vascular fraction ( svf ) . the separated cells were resuspended in dmem culture medium ( gibco , usa ) containing 10% fetal bovine serum ( gibco , usa ) and 1% penicillin / streptomycin ( biosera , uk ) . umbilical cords from healthy infants were transferred to our laboratory within 424 hr after delivery . a longitudinal section was made through the umbilical vein and the endothelial cells were scratched and discarded . each piece was put into a 100 mm petri dish and cultured in the presence of -mem containing 10% fcs , 0.1% l - glutamine , and 0.1% penicillin / streptomycin for 810 days . upon confluency , the cells were passaged . at each passage , the cells were counted and analyzed for viability by trypan blue staining analysis . flow cytometric analysis and functional ability of differentiation into osteocyte and adipocyte were achieved in response to specific culture conditions . the identification of adherent cells was performed by flow cytometric analysis . at the third passage , the cells were detached from the culture flasks with trypsin - edta and counted . about 110 cells were incubated on ice for 30 min with goat serum , resuspended in pbs and pelleted by centrifugation . subsequently , the cells were stained for 30 min at 4 c with fluorescein isothiocyanate ( fitc)-coupled or phycoerythrin ( pe)-conjugated cd34 , cd45 , cd90 , cd73 , cd80 , and hla - class ii . isotype - matched mouse monoclonal antibodies ( bd - pharmingen , usa ) were used to rule out non - specific staining of the cells . the labeled cells were thoroughly washed with pbs and analyzed on an facscalibur machine ( bd biosciences , usa ) using the cell quest as data acquisition software . the potential of at - mscs to differentiate into chondrogenic , osteogenic , and adipogenic lineages was examined as described before ( 20 , 21 ) . briefly , 110 at - mscs were used for differentiation when the cultures were 6080% confluent by using appropriate differentiation kits ( stempro differentiation kit , gibco , usa ) . after 2 weeks the cells were stained with alizarin red s , safranin , and oil red o for evaluating osteogenic , chondrogenic , and adipogenic differentiation , respectively ( merck , germany ) . the potentials of the bm - mscs and wj - mscs to differentiate into osteogenic and adipogenic lineages were examined . for osteogenic differentiation , osteogenic medium consisted of dmem supplemented with 10 m / l dexamethasone ( sigma - aldrich , st . louis , usa ) , 10 mm / l glycerol phosphate ( sigma - aldrich , st . louis , usa ) , 3.7 g / l sodium bicarbonate ( sigma - aldrich , st . louis , usa ) , and 0.05 g / l ascorbic acid ( sigma - aldrich , st . adipogenic medium consisted of dmem supplemented with 1 m / l hydrocortisone ( sigma - aldrich , st . louis , usa ) , 0.05 g / l ascorbic acid , 0.05 g / l indomethacin ( sigma - aldrich , st . peripheral blood sample was collected from a healthy donor and pbmcs were prepared by density gradient centrifugation using ficoll - hypaque media and were labeled with 5 nm cfse dye ( invitrogen molecular probe , usa ) . then cells were incubated at 37c for 10 min ; 2 ml fetal bovine serum ( fbs ) ( gibco , usa ) was added to the cells and centrifuged at 1800 rpm for 10 min . afterward , rpmi culture media with 2% fbs was added to the cell pellet and centrifuged again at 1800 rpm for 10 min . next cfse labeled pbmcs were counted and added into wells at a concentration of 110 cells per well . 210 mscs were plated in culture plates 24 hr prior to co - culture with pbmcs . in this way pbmcs were stimulated with pha ( gibco , uk ) and the effect of mscs on lymphocyte proliferation was studied in both direct and indirect ( using 0.4 m transwell plates , corning , usa ) culture system after 3 , 5 , and 7 days post culture by flow cytometry and compared to pha - activated pbmcs . all experiments were carried out with 3 at , 2 wj , and 2 bm samples . human mscs were obtained from 5 ml bm aspirates from the iliac crest of normal donors within the age range of 1945 years . they were donors of bm to a related patient after obtaining approval of the ethics committee , shiraz university of medical sciences , shiraz , iran . written informed consent was also obtained allowing analysis of the clinical data and testing mentioned in this study . each sample of aspirate was diluted 1:1 with dmem - low glucose ( invitrogen , merelbeke , belgium ) and layered over about 5 ml of ficoll ( lymphoprep ; oslo , norway ) . the isolation method was according to a previously reported method ( 19 ) by some modifications , which will be stated completely . after centrifugation at 939 g for 20 min , the mononuclear cell layer was removed from the interface . cells were suspended in dmem and centrifuged at 338 g for 15 min then resuspended in basal dmem medium containing 10% fetal calf serum ( invitrogen , merelbeke , belgium ) , 1% penicillin ( invitrogen , merelbeke , belgium ) , 1% streptomycin ( invitrogen , merelbeke , belgium ) , and 2 mm glutamine ( invitrogen , merelbeke , belgium ) . the cells were seeded at a density of 80.000/cm in 25 cm t - flasks and maintained at 37 c in an atmosphere of 5% co2 . after 4 days , the non - adherent cells were removed and the media was changed every 3 days . in order to expand mscs , the adhered monolayer was detached with trypsin - edta ( invitrogen , merelbeke , belgium ) for 5 min at 37 c , after 14 days for the first passage and every 45 days for successive passages . during in vitro passaging , the cells were seeded at a density of 5 - 1010 cells / cm and expanded for several passages until they no longer reached confluence . at - mscs were extracted from breast ats of healthy human adult donors with cosmetic mammoplasty surgery after obtaining the fully informed consent as described previously ( 20 , 21 ) . briefly , fragments of ats washed with phosphate buffered saline ( pbs ) , minced and digested with 0.2% collagenase type i ( gibco , usa ) at 37 c . the resulting cell suspension was centrifuged and the pellet including the adherent stromal cells was put on a ficoll gradient ( biosera , uk ) to separate the stromal vascular fraction ( svf ) . the separated cells were resuspended in dmem culture medium ( gibco , usa ) containing 10% fetal bovine serum ( gibco , usa ) and 1% penicillin / streptomycin ( biosera , uk ) . umbilical cords from healthy infants were transferred to our laboratory within 424 hr after delivery . a longitudinal section was made through the umbilical vein and the endothelial cells were scratched and discarded . each piece was put into a 100 mm petri dish and cultured in the presence of -mem containing 10% fcs , 0.1% l - glutamine , and 0.1% penicillin / streptomycin for 810 days . upon confluency at each passage , the cells were counted and analyzed for viability by trypan blue staining analysis . flow cytometric analysis and functional ability of differentiation into osteocyte and adipocyte were achieved in response to specific culture conditions . the identification of adherent cells was performed by flow cytometric analysis . at the third passage , about 110 cells were incubated on ice for 30 min with goat serum , resuspended in pbs and pelleted by centrifugation . subsequently , the cells were stained for 30 min at 4 c with fluorescein isothiocyanate ( fitc)-coupled or phycoerythrin ( pe)-conjugated cd34 , cd45 , cd90 , cd73 , cd80 , and hla - class ii . isotype - matched mouse monoclonal antibodies ( bd - pharmingen , usa ) were used to rule out non - specific staining of the cells . the labeled cells were thoroughly washed with pbs and analyzed on an facscalibur machine ( bd biosciences , usa ) using the cell quest as data acquisition software . the potential of at - mscs to differentiate into chondrogenic , osteogenic , and adipogenic lineages was examined as described before ( 20 , 21 ) . briefly , 110 at - mscs were used for differentiation when the cultures were 6080% confluent by using appropriate differentiation kits ( stempro differentiation kit , gibco , usa ) . after 2 weeks the cells were stained with alizarin red s , safranin , and oil red o for evaluating osteogenic , chondrogenic , and adipogenic differentiation , respectively ( merck , germany ) . the potentials of the bm - mscs and wj - mscs to differentiate into osteogenic and adipogenic lineages were examined . for osteogenic differentiation , osteogenic medium consisted of dmem supplemented with 10 m / l dexamethasone ( sigma - aldrich , st . louis , usa ) , 10 mm / l glycerol phosphate ( sigma - aldrich , st . louis , usa ) , 3.7 g / l sodium bicarbonate ( sigma - aldrich , st . louis , usa ) , and 0.05 g / l ascorbic acid ( sigma - aldrich , st . adipogenic medium consisted of dmem supplemented with 1 m / l hydrocortisone ( sigma - aldrich , st . g / l ascorbic acid , 0.05 g / l indomethacin ( sigma - aldrich , st . peripheral blood sample was collected from a healthy donor and pbmcs were prepared by density gradient centrifugation using ficoll - hypaque media and were labeled with 5 nm cfse dye ( invitrogen molecular probe , usa ) . then cells were incubated at 37c for 10 min ; 2 ml fetal bovine serum ( fbs ) ( gibco , usa ) was added to the cells and centrifuged at 1800 rpm for 10 min . afterward , rpmi culture media with 2% fbs was added to the cell pellet and centrifuged again at 1800 rpm for 10 min . next cfse labeled pbmcs were counted and added into wells at a concentration of 110 cells per well . 210 mscs were plated in culture plates 24 hr prior to co - culture with pbmcs . in this way pbmcs were stimulated with pha ( gibco , uk ) and the effect of mscs on lymphocyte proliferation was studied in both direct and indirect ( using 0.4 m transwell plates , corning , usa ) culture system after 3 , 5 , and 7 days post culture by flow cytometry and compared to pha - activated pbmcs . all experiments were carried out with 3 at , 2 wj , and 2 bm samples . using the same previous approach , single cell - derived colonies of msc - like cells which were rapidly grown and exhibiting homogeneous morphology were selected for culture expansion . bm cells rapidly generated a confluent layer of cells possessing an elongated and fibroblastic shape . their surface phenotype was determined by flowcytometry ; also , we have characterized a population of cells resident within human bm cultures that were negative for the major histocompatibility complex ( mhc ) class ii , and cd80 ( b7 - 1 ) costimulators for t lymphocyte activation . however , they were positive for the msc - candidate markers cd90 and cd73 ( figure 1a ) . a. the cells were negative for cd80 , hla - dr , cd45 , and cd34 ( the black histogram shows the profile of the isotype control ) , but were positive for cd90 and cd73 ( the shaded area shows the profile of the negative control ) . b. human bm - mscs before differentiation c. the adipose droplet in differentiated cells after incubating with adipogenic media ( 10x ) d. osteogenic differentiation was positive for alizarin red staining ( 10x ) lipid droplets in differentiated adipocytes were located by oil red o - staining . likewise , alizarin red s staining confirmed the presence of calcium deposits in osteocytes , whereas undifferentiated mscs were negative in staining procedure ( figures 1b and c ) . these results indicate that the isolated cells have the basic properties of mscs . at - mscs emerged with a spindle - shaped appearance in the culture as depicted in figure 2a . results of osteocyte differentiation demonstrated the accumulation of calcium in the culture ( figure 2b ) . differentiation to chondrocytes caused morphological changes in at - mscs into rounder and cuboidal shapes as shown in figure 2c . differentiation to adipocytes caused the formation of well - formed lipid vesicles in at - mscs as presented in figure 2d . the flow cytometry analysis for stem cell - specific markers revealed that at - mscs express cd44 and cd73 ( higher than 98% of all cells ) . they were negative ( higher than 90% of all ascs ) for the expressions of mhc ii , cd80 , cd45 , and cd34 ( figure 2e ) . at - mscs with a spindle - shaped appearance in culture before treating for differentiation ( a ) . these cells emerged in culture with the ability to differentiate into osteocytes ( b ) , chondrocytes ( c ) , and adipocytes ( d ) . results are representative of two independent experiments . filled histograms represent the specific markers ( cds ) and the unfilled were isotype control ( 40x ) . wj - mscs were positive for the expression of cd90 , cd73 , cd44 , cd105 , and cd106 , but were negative for the expression of cd34 and cd144 ( figure 3a ) . adipose vacuoles and calcium deposition in culture showed the ability of wj - mscs for differentiation ( figure 3b and c ) . c and d. differentiation ability of wj - mscs to adipocytes and osteocytes , respectively in order to show the inhibitory effects of the extracted cells , three different types of mscs were co - cultured with normal allogeneic human pha - activated pbmcs . the proliferation of pbmcs was assessed by flow cytometry of cfse stained cells and compared to each other and to the growth of pbmcs in the absence of mscs , 3 , 5 , and 7 days post co - culture . based on the results , data of days 3 and 7 were compared between different groups . as depicted in figure 4 , the proliferation of pbmcs reduced to 6.2 , 7 , and 15.4- fold ( 74% , 76% , and 83% growth suppression , respectively ) in cultures with at - mscs , wj - mscs , and bm - mscs , respectively , compared to the pha - activated cells at day 3 post culture . the meansem frequencies of dividing cells were 14.85.6 , 12.63.4 , 5.70.4 , and 88.3 for at - mscs , wj - mscs , bm - mscs , and non - co - cultured cells , respectively . when the growth suppression was indirectly assessed by using transwell culture system , it is revealed that at - mscs , wj - mscs , and bm - mscs caused growth reduction in pbmcs to 3 , 8 , and 8-fold ( 59% , 77% , and 77% growth suppression ) , respectively , lower than pha - activated cells . the meansem frequencies of dividing cells were 296.4 , 11 6.4 , and 117.6 for at - mscs , wj - mscs , and bm - mscs , respectively ( figure 4 ) . growth suppression of pbmcs after coculture with at , bm , and wj - mscs . ( a ) typical representation of flow cytometry plots showing growth suppression of pbmcs in coculture with mscs after 3 days . ( b ) growth suppression of pbmcs in coculture with mscs based on the mean frequency of dividing pbmcs no significant difference was found between the data of days 3 and 5 , but results of day 7 were different . at day 7 the growth suppression effect of all types of mscs was lower than that of day 3 because it showed 60% , 49% , and 84% reduction 7 days post culture with at- , wj- , and bm - mscs , respectively , compared to day 3 . similar effects were seen in indirect cultures for at- and wj- , but not bm - msc because 27% and 4% reduction in growth suppression effect of at- and wj - msc was observed compared to day 3 . using the same previous approach , single cell - derived colonies of msc - like cells which were rapidly grown and exhibiting homogeneous morphology were selected for culture expansion . bm cells rapidly generated a confluent layer of cells possessing an elongated and fibroblastic shape . their surface phenotype was determined by flowcytometry ; also , we have characterized a population of cells resident within human bm cultures that were negative for the major histocompatibility complex ( mhc ) class ii , and cd80 ( b7 - 1 ) costimulators for t lymphocyte activation . however , they were positive for the msc - candidate markers cd90 and cd73 ( figure 1a ) . a. the cells were negative for cd80 , hla - dr , cd45 , and cd34 ( the black histogram shows the profile of the isotype control ) , but were positive for cd90 and cd73 ( the shaded area shows the profile of the negative control ) . b. human bm - mscs before differentiation c. the adipose droplet in differentiated cells after incubating with adipogenic media ( 10x ) d. osteogenic differentiation was positive for alizarin red staining ( 10x ) lipid droplets in differentiated adipocytes were located by oil red o - staining . likewise , alizarin red s staining confirmed the presence of calcium deposits in osteocytes , whereas undifferentiated mscs were negative in staining procedure ( figures 1b and c ) . at - mscs emerged with a spindle - shaped appearance in the culture as depicted in figure 2a . results of osteocyte differentiation demonstrated the accumulation of calcium in the culture ( figure 2b ) . differentiation to chondrocytes caused morphological changes in at - mscs into rounder and cuboidal shapes as shown in figure 2c . differentiation to adipocytes caused the formation of well - formed lipid vesicles in at - mscs as presented in figure 2d . the flow cytometry analysis for stem cell - specific markers revealed that at - mscs express cd44 and cd73 ( higher than 98% of all cells ) . they were negative ( higher than 90% of all ascs ) for the expressions of mhc ii , cd80 , cd45 , and cd34 ( figure 2e ) . at - mscs with a spindle - shaped appearance in culture before treating for differentiation ( a ) . these cells emerged in culture with the ability to differentiate into osteocytes ( b ) , chondrocytes ( c ) , and adipocytes ( d ) . results are representative of two independent experiments . filled histograms represent the specific markers ( cds ) and the unfilled were isotype control ( 40x ) . wj - derived cells were seen as spindle - shaped cells in the culture . figure 3 shows the phenotypic and differentiation characteristics of these cells . wj - mscs were positive for the expression of cd90 , cd73 , cd44 , cd105 , and cd106 , but were negative for the expression of cd34 and cd144 ( figure 3a ) . adipose vacuoles and calcium deposition in culture showed the ability of wj - mscs for differentiation ( figure 3b and c ) . in order to show the inhibitory effects of the extracted cells , three different types of mscs were co - cultured with normal allogeneic human pha - activated pbmcs . the proliferation of pbmcs was assessed by flow cytometry of cfse stained cells and compared to each other and to the growth of pbmcs in the absence of mscs , 3 , 5 , and 7 days post co - culture . based on the results , data of days 3 and 7 were compared between different groups . as depicted in figure 4 , the proliferation of pbmcs reduced to 6.2 , 7 , and 15.4- fold ( 74% , 76% , and 83% growth suppression , respectively ) in cultures with at - mscs , wj - mscs , and bm - mscs , respectively , compared to the pha - activated cells at day 3 post culture . the meansem frequencies of dividing cells were 14.85.6 , 12.63.4 , 5.70.4 , and 88.3 for at - mscs , wj - mscs , bm - mscs , and non - co - cultured cells , respectively . when the growth suppression was indirectly assessed by using transwell culture system , it is revealed that at - mscs , wj - mscs , and bm - mscs caused growth reduction in pbmcs to 3 , 8 , and 8-fold ( 59% , 77% , and 77% growth suppression ) , respectively , lower than pha - activated cells . the meansem frequencies of dividing cells were 296.4 , 11 6.4 , and 117.6 for at - mscs , wj - mscs , and bm - mscs , respectively ( figure 4 ) . growth suppression of pbmcs after coculture with at , bm , and wj - mscs . ( a ) typical representation of flow cytometry plots showing growth suppression of pbmcs in coculture with mscs after 3 days . ( b ) growth suppression of pbmcs in coculture with mscs based on the mean frequency of dividing pbmcs no significant difference was found between the data of days 3 and 5 , but results of day 7 were different . at day 7 the growth suppression effect of all types of mscs was lower than that of day 3 because it showed 60% , 49% , and 84% reduction 7 days post culture with at- , wj- , and bm - mscs , respectively , compared to day 3 . similar effects were seen in indirect cultures for at- and wj- , but not bm - msc because 27% and 4% reduction in growth suppression effect of at- and wj - msc was observed compared to day 3 . the specific characteristics of mscs , including their extensive proliferative potential and the ability to differentiate into various cell types , like bone , fat , and cartilage , make them an attractive tool in regenerative medicine . apparently , these cells might be simply isolated from various tissues and expanded in culture in large numbers , which gives the opportunity to create tissue - engineered constructs for re - introducing them into patients ( 22 ) . further interest in the clinical application of mscs has been raised by the observation that these cells are strongly immunosuppressive in vitro and in vivo . mscs exist in almost all tissues , including bm , fat , muscle , tooth root , hair follicles , placenta , dermis , umbilical cord , wj , lung , liver , and spleen ( 1 , ) . to date the mscs derived from bm have been applied in cell - based therapies , including the treatment of osteogenesis imperfecta , intracoronary transplantation in patients with acute myocardial infarction ( 23 - 25 ) , and the treatment of graft - versus - host disease after allogeneic bm transplantation ( 26 ) . although bm has been represented as the main available and well - accepted source of mscs , alternative sources such as at and wj of human umbilical cord may potentially be used in tissue injuries ( 16 , 27 ) . bm- , at- , and wj - mscs have initially shown similarities in their immunomodulatory effects , but additional studies showed various disparities ( 28 , 29 ) . pr et al reported several differences between wj- , bm- , and at - mscs including a higher proliferation potential and higher production of chemokines , pro - inflammatory proteins , and growth factors in wj - msc . they showed that at - mscs are better sources for pro - angiogenic factors such as vegf and extracellular matrix components and metalloproteinases ( 30 ) . at - mscs showed a stronger inhibitory effect on cd4 and cd8 t cells . in contrast to bm- and at - mscs , umbilical cord matrix - mscs did not influence the activation or lymphoblast characteristics of b cells ( 31 ) . here , we compared the growth suppression effects of bm- , at- , and wj - mscs on pbmcs . initial studies on mscs showed that extracted cells from all three sources have the specific mesenchymal characteristics such as expression of surface markers and differentiation abilities . additionally , based on the results of this study , all kinds of mscs showed suppression effects on the proliferation of pbmcs . when the growth suppression was compared between different types of mscs , bm - mscs and then wj - mscs showed higher growth suppression effects on pbmcs compared to at - mscs . indirect culture experiments using the transwell system showed similar results because using at - ascs had the least effect on growth suppression of pbmcs compared to the culture supernatants of wj- and bm - mscs . when we compared the direct and indirect cultures of each msc to itself , it was revealed that the presence of at and bm - mscs , thus cell to cell contact , was more important than the secretary effects of these cells . on day 7 , the influence of at - mscs compared to other sources was similar to day 3 , but it seems that the effects of all types of mscs were generally diminished during the time . bochev et al indicated that at - mscs inhibited antibody production in pbmcs to a much greater extent than bm - mscs ( 32 ) . furthermore , they demonstrated that at - mscs are more capable of suppressing the differentiation of blood monocytes into dendritic cells ( dcs ) and the expression of costimulatory molecules in mature monocyte - derived dcs ( 33 ) . results of our study on the underlying effect of at- and bm - mscs on the proliferation of pbmcs were inconsistent with these reports because we showed that bm - mscs had a higher suppression effect than at - mscs which may be related to the source of at because they used liposuction , but our at - mscs originated from breast tissue . thus , mscs originating from different sources of adipose may not have similar immunomodulatory effects . the assessment of immune - suppression caused by bm - mscs and wj - mscs showed that although both mscs distort the cytokine profile from a pro - inflammatory to an anti - inflammatory phenotype , bm - mscs , but not wj - mscs increase the levels of immune suppressive cytokine il-10 and modulate the level of pro - inflammatory cytokine il-2 . interestingly , indoleamine 2,3-dioxygenase ( ido ) activity was higher in bm - mscs than wj - mscs when both cells were treated with interferon gamma ( ifn ) . also , bm - mscs secreted higher levels of prostaglandin e2 ( pge2 ) in comparison with wj - mscs ( 29 ) . these differences in secretion pattern of mscs and disparity in expressing distinct molecules ( 34 ) may reflect why mscs from different sources show specific growth suppression effects . our results of direct cultures are consistent with these data because we also observed the higher suppression effect of bm - mscs compared to wj - mscs . in contrast , the supernatant of wj - mscs was more effective than bm - mscs for reducing the proliferation of pbmcs . based on the present investigation , it is concluded that direct or cell - cell contact and indirect immunomodulatory effects of mscs are different . additionally , mscs from various sources and even different parts of the body have special characteristics , which undoubtedly impact their properties and effects . results of this study may contribute to the application of adult stem cells in regenerative medicine .