many potential therapies for severe and/or chronic wounds fail as a result of poor vasculature .
hence , strategies to improve blood vessel supply into a wound bed are thought to promote wound healing .
transplantations of mesenchymal stem cells ( mscs ) have shown great potential as a therapeutic agent for the treatment of a range of disorders , including wound healing , and have become the subject of numerous clinical trials . however , whilst the safety of msc transplantation does not seem to be an issue , the effectiveness of such treatment has exhibited considerable variability . this variation in effect is problematic when translating preclinical research into msc - based clinical therapy .
we recently demonstrated that human msc - conditioned medium ( msc - cm ) was stimulatory to epidermal and fibroblast cell adherence and migration .
other reports suggest that msc are pro - angiogenic also through their paracrine activity on endothelial cells . whilst there are reports that msc are capable of endothelial differentiation , engraftment into new vasculature is low in vivo .
hence , these and other studies have contributed to recent thought that the predominant regenerative activity of mscs is due to their secretion of factors that stimulate endogenous cells at wound sites . in this investigation , we have examined the effects of msc - cm on endothelial cells , using the cell line eahy-926 as a model system .
we report that msc - cm promotes endothelial cell adhesion and migration , but that these effects show considerable donor - donor variability .
further , we have identified extracellular matrix ( ecm ) proteins that are secreted by mscs using maldi / tof - tof mass spectrometry .
we provide data to suggest that ecm composition plays a major role in the donor - donor variation we have seen .
these findings demonstrate proof of principle of the need to screen the msc secretome in order to optimise the application of mscs in the clinic .
cell culture and mass spectrometry of conditioned medium eahy-926 endothelial cells were maintained in dmem / f12 culture medium ( invitrogen , paisley , uk ) supplemented with 10% ( v / v ) fetal calf serum ( fcs ) ( invitrogen ) and 1% ( v / v ) penicillin and streptomycin ( invitrogen ) , incubated at 37c and in a humidified atmosphere containing 5% ( v / v ) co2 .
passaging was performed at ~90% confluence and cells were re - seeded at 1 x 10 cells / cm .
mscs were isolated from the iliac crest biopsies of bone marrow donors following ethical approval and with informed consent , as previously described .
the adherent cell population obtained was consistent with the characteristics of mscs laid out by the international society for cellular therapy ( isct ) . conditioned medium
was generated from msc cultures of equal cell number in serum free conditions , using dmem / f12 supplemented with insulin , transferrin and selenium ( invitrogen ) .
protein content of msc cm was determined by maldi - tof / tof mass spectrometry as previously described .
characterization of msc after three passages in culture , bone marrow derived cells were assessed by immunoprofiling for cd markers and by examining their differentiation potential to form osteoblasts , adipocytes and chondrocytes , by staining with alkaline phosphatase , oil red o , and toluidine blue respectively as per the criteria established for a msc phenotype by the isct .
coating of culture plates culture plates were coated with msc - cm or type - i collagen , decorin , or fibronectin ( all sigma - aldrich ) .
protein solutions were diluted in pbs to 0.2mg / ml and added to each well ( 50l for 96-well plates , 500l for 24-well plates ) .
these were refrigerated for 24 hours before being rinsed with pbs immediately prior to use .
cell adherence / spreading coated 24-well tissue culture plates were seeded with 2x10 eahy-926 endothelial cells in dmem / f12 supplemented with 1% penicillin / streptomycin ( sigma - aldrich ) and 1% its - x ( sigma - aldrich ) and incubated for 2 hours at 37c and 5% co2 before digital images were captured ( progres cf , jenoptik ) and analysed using image - j software .
scratch assays were established using previously published methods , in protein - coated tissue culture plates ( as described above ) .
cell migration was automatically captured and analysed at hourly intervals using an incucyte live - cell imaging system ( essen bioscience ) .
data were tested for significance using the mann - whitney u test . those differences that fell within a 95%
, 99% or 99.9% confidence interval were considered to be significant , indicated by asterisks within figures ( * p<0.05 * * p<0.01 * * * p<0.001 ) .
bone marrow cells obtained from iliac crest biopsies showed characteristics consistent with those expected of msc ( figure 1 ) .
msc - cm coating of culture plates resulted in the significant enhancement of the spreading of eahy-926 endothelial cells upon the culture surface .
this result was subject to a marked inter - donor variability , with conditioned media generated by both msc-1 and msc-3 resulting in a significantly greater degree of eahy-926 endothelial cell spreading than that generated by msc-2 ( figure 2a , 2b ) .
eahy-926 endothelial cell adherence on fibronectin coated plates was most advanced after two hours , compared to plates coated in either type i collagen or with decorin ( figure 3 ) .
on type i collagen - coated plates , the presence of msc - cm appeared to enhance the rate of eahy-926 endothelial cell migration into scratch - wounds compared to unconditioned media ( figure 4a , left ) .
eahy-926 endothelial cells in msc - cm closed scratch - wounds to a significantly greater degree than those in unconditioned media over a 12-hour time course ( figure 4b , left ) . once again , the degree of this msc - cm mediated enhancement of eahy-926 endothelial cell migration was subject to inter - donor variability . conditioned medium generated by msc-1 ( and msc-3 ) elicited a greater degree of scratch - wound closure than msc-2 over 12 hours ( figure 4b , left ) .
cells isolated from bone marrow meet the minimum criteria for identification as msc.cells are immunoreactive for cd73 , cd90 and cd105 , whilst lacking immunoreactivity for cd14 , cd34 and cd45 , as shown by histograms obtained by flow cytometry , and readily differentiate to form osteoblasts , adipocytes and chondrocytes in vitro .
representative phase contrast images are shown of cells following tri - lineage differentiation and staining with alkaline phosphotase , oil red o , and toluidine blue ( top , left to right ) .
msc - cm coating of culture plates influences eahy-926 endothelial cell adherence and spreading.a : msc - cm coating of culture plates influences eahy-926 endothelial cell adherence and spreading .
after 4 hours in culture eahy-926 endothelial cells were observably more spread on culture plates coated with msc - cm than on plates coated with unconditioned ( control ) medium .
b : after 4 hours in culture the average cell area was significantly greater for eahy-926 endothelial cells on msc - cm coated culture plates and 389m2 than those cells on unconditioned ( control ) medium coated culture plates .
data shown are means  sem in relative units ( * * * = p<0.001 mann whitney u test ) .
ecm protein coating of culture plates influences eahy-926 endothelial cells adherence and spreading.after 4 hours in culture eahy-926 endothelial cells were observably more spread on culture plates coated with fibronectin than on plates coated with type i collagen or decorin .
b : after 4 hours in culture the average cell area was significantly greater for eahy-926 endothelial cells on fibronectin compared to those cells on either type i collagen or decorin .
data shown are means  sem in relative units ( * * = p<0.01 , * * * = p<0.001 , mann whitney u test ) .
eahy-926 endothelial cells closed scratch wounds faster in msc - cm than in unconditioned medium on type i collagen , fibronectin , and decorin coated culture plates . 
 a : eahy-926 cells on fibronectin coated culture plates closed scratch wounds significantly faster than those on either type i collagen or decorin .
b : after 12 hours eahy-926 endothelial cells in msc - cm had closed scratch wounds by a significantly greater degree than those in unconditioned control medium .
12 hours post - scratching , eahy-926 endothelial cells on plates coated fibronectin had closed scratch wounds by a significantly greater degree than those on either type i collagen or decorin . on fibronectin and decorin , medium conditioned by msc-2
on type one collagen , medium conditioned by msc-2 was associated with significantly reduced scratch closure compared to msc-1 .
all three were found to contain fibronectin , collagen type i , collagen type vi , and lumican , whilst cartilage oligomeric matrix protein ( comp ) and sparc were present in two out of three msc - cm and laminin , decorin , heparan sulphate proteoglycan ( hspg ) and igfbp-1 were each only observed in one msc - cm ( table 1 ) . of these ,
hence , not only was there clear inter - donor variability in the ecm components of the msc secretome , but the presence of these proteoglycans seemed to be associated with a reduction in the efficacy of msc - cm . when type i collagen , decorin and fibronectin were used as culture substrata , the degree of eahy-926 endothelial cell spreading appeared to be similar upon both type i collagen and decorin and greatly enhanced upon fibronectin ( figure 2a , 2b ) .
similarly , scratch - wound closure by eahy-926 endothelial cells in unconditioned media appeared to be similar upon type i collagen and decorin , but markedly greater upon fibronectin ( figure 4a , 4b ) . in the presence of each ecm substrate
the presence of msc - cm resulted in significantly enhanced scratch wound closure compared to unconditioned media ( figure 4b ) .
as seen previously upon type i collagen coated culture plates , the degree of closure upon both decorin and fibronectin coated plates was either significantly or near significantly ( p=0.057 by mann whitney u test ) less in the presence of medium conditioned by msc-2 , compared to medium conditioned by either msc-1 or msc-3 ( figure 4b ) .
mass spectrometry of msc - cm from 3 separate donors.maldi-tof/tof mass spectrometry of msc - cm detected variable protein content between media conditioned by msc from three different patient donors .
we have previously shown that msc - cm promotes the migration of skin cells in a wound healing model , and identified numerous potentially beneficial factors that may contribute to this effect .
in vivo wound healing is , however , a complex process influenced by a host of cellular events , including angiogenesis . if msc can stimulate endothelial cells as suggested here and elsewhere this supports their potential use in the treatment of cutaneous wounds . in these experiments ,
this is similar to previous studies in which msc - cm has been shown to stimulate angiogenesis , supporting the investigation of msc - cm as a pro - angiogenic agent .
although it is possible for proliferation of eahy-926 endothelial cells to have contributed somewhat to the closure of the scratch wounds , these scratch assay experiments were performed over the course of 12 hours .
the reported doubling time for these cells is over 25 hours . to significantly affect the rate of scratch wound closure , those cells at the leading edge of the scratch margins
would be required to undergo repeated doublings , and this is unlikely to have had a major influence on scratch wound closure over the time course of these studies .
mass spectrometry of msc - cm revealed numerous factors ( including fibronectin and collagen ) in medium conditioned by each of the three msc examined and some , including laminin and decorin , in one or two but not all three of the msc - cm samples .
of these ecm components , fibronectin , collagen and laminin are known to promote or support angiogenesis .
unusually , in these experiments collagen did not seem to induce any observable cell response when compared to decorin .
fibronectin and collagen contain protein motifs are known to mediate angiogenesis by integrin receptor signaling in vivo and in vitro .
decorin inhibits endothelial cell migration and tubule formation in vitro 
  and inhibits the pro - angiogenic effects of vegf .
lumican interferes with 21 receptor activity and inhibits angiogenesis both in vitro and in vivo .
whilst lumican was present in each of the msc - cm used in these investigations , decorin was only found within the msc - cm that consistently showed significantly less enhancement of eahy-926 endothelial cell migration . as the method ( maldi - tof / tof mass spectrometry ) used to detect these protein components within msc - cm was not quantitative
, it was not possible to determine whether differences in concentrations of each ecm component were related to efficacy of the conditioned media .
msc - cm coating of culture plates enhanced eahy-926 endothelial cell adherence , as did coating with fibronectin .
cell adherence to these substrata was examined in an indirect fashion , by assessing the average area of cells in the immediate - early period after seeding . as cells settle upon a permissive substrate they spread out from the rounded morphology observed in suspension to adopt a flattened morphology that is usually seen in vitro in adherent cell populations .
after prolonged periods of time , cells in culture , including endothelial cells , synthesize matrix molecules that may also promote cell adhesion , potentially masking the initial effects of the original substrate being investigated . of the matrix proteins detected in msc - cm by maldi - tof / tof mass spectrometry ,
fibronectin was observed to have a profound effect upon cell adherence , similar to those findings concerning cell migration .
however , the addition of msc - cm to endothelial cell scratch assays performed upon fibronectin coated culture plates resulted in a further enhancement of endothelial cell migration , suggesting that fibronectin may not be solely responsible for the entire effect .
further experiments investigating the effects of msc secreted growth factors and cytokines individually , and in combination , may reveal their relative contribution to the enhancement of endothelial cell migration .
the relatively low number of individual donors examined during this study is a clear limitation , and although the results presented support the conclusions that ( i ) msc donor variation and secretome composition may account for differential effects of mscs on endothelial cells , and ( ii ) that this variation should be taken into consideration in msc - based regenerative medicine , subsequent investigation of greater numbers of samples is required to further authenticate these findings .
angiogenesis depends on endothelial cell migration and the actions of endothelial cell chemotactic factors , e.g. vegf and il-8 , and ecm proteins such as collagens , fibronectin and laminin .
many of these pro - angiogenic factors have been found to be present within the msc secretome both within this investigation and in previous studies .
these experiments have showed that msc - cm enhances both the rate of endothelial cell migration and the adherence of these cells to their culture surface , and that this effect was , in part , mediated by the presence of fibronectin .
the effects of msc - cm upon endothelial cell migration were not entirely induced by fibronectin , as suggested by the further enhancement of eahy-926 endothelial cell migration by msc - cm in the presence of exogenous fibronectin .
factors such as interleukin ( il)-8 , vegf , and laminin have all been shown to be stimulatory to endothelial cells and these factors have been shown to be present within msc - cm .
it seems likely that these known mediators of angiogenesis might contribute to the effects of msc - cm upon eahy926 endothelial cells observed in these investigations .
overall , the data presented here support the hypothesis that msc may stimulate the formation of new vasculature , and that this may be an important aspect of msc - mediated enhancement of wound healing .
 mesenchymal stem cells ( mscs ) stimulate angiogenesis within a wound environment and this effect is mediated through paracrine interactions with the endothelial cells present . here 
 we report that human msc - conditioned medium ( n=3 donors ) significantly increased eahy-926 endothelial cell adhesion and cell migration , but that this stimulatory effect was markedly donor - dependent . 
 maldi - tof / tof mass spectrometry demonstrated that whilst collagen type i and fibronectin were secreted by all of the msc cultures , the small leucine rich proteoglycan , decorin was secreted only by the msc culture that was least effective upon eahy-926 cells . 
 these individual extracellular matrix components were then tested as culture substrata . 
 eahy-926 cell adherence was greatest on fibronectin - coated surfaces with least adherence on decorin - coated surfaces . 
 scratch wound assays were used to examine cell migration . 
 eahy-926 cell scratch wound closure was quickest on substrates of fibronectin and slowest on decorin . 
 however , eahy-926 cell migration was stimulated by the addition of msc - conditioned medium irrespective of the types of culture substrates . 
 these data suggest that whilst the msc secretome may generally be considered angiogenic , the composition of the secretome is variable and this variation probably contributes to donor - donor differences in activity . 
 hence , screening and optimizing msc secretomes will improve the clinical effectiveness of pro - angiogenic msc - based therapies . 