BACKGROUND
mesenchymal stromal cells  offer great potential for therapeutic application since they combine a number of biological properties such as multilineage differentiation, stromal support, immunomodulation, and secretion of trophic factors  <cit> .
due to the low frequency within tissues, in general ex vivo expansion is required to achieve a clinically-relevant cell dose.
this has to comply with good manufacturing practice  guidelines.
fetal bovine serum  used in many protocols, however, is critically rated by the regulatory authorities due to the possible transmission of extraneous agents as well as the risk of triggering host immune responses comprising the therapeutic success  <cit> .
a chemically-defined medium sufficiently mimicking serum compounds to provide growth and attachment factors, buffering and detoxifying agents, is still under development  <cit> .
currently human supplements, including platelet derivatives and human serum from autologous, allogeneic or cord blood sources, are assessed in pre- and clinical studies to replace fbs  <cit> .
we have previously demonstrated that pooled human serum , human platelet lysate  and thrombin-activated platelet releasate in plasma  are promising alternatives to fbs and support the main characteristics of msc  <cit> .
interestingly, hs, tprp and phpl all promoted the proliferation of msc from adipose tissue  to a comparable extent  <cit> .
however, bone marrow-derived msc  proliferation was significantly enforced solely by phpl  <cit> .
the different proliferative responses raised three questions: i) which bioactive proteins differ in the lysate and the releasate, ii) do these proteins exert different effects on la- and bm-msc, and  can we extrapolate these findings to optimize chemically-defined msc media?
human serum and platelet granules contain various growth factors capable of promoting cell proliferation and tissue regeneration  <cit> .
identification and characterization of these factors was markedly achieved by bioinformatic approaches integrating proteomic data sets from plasma, serum, the entire platelet proteome and specific subproteomes with functional data  <cit> .
these analyses have provided a comprehensive list of platelet and plasma proteins and as such contributed significantly to our current biomolecular understanding of these components and their function in the human body.
they support a systems biology view on platelet protein function, network modules and enable evaluation of upcoming data sets  <cit> .
aiming to answer our questions we analyzed phpl and tprp with a differential proteomic approach and related the data to an in-depth catalog of human platelet proteins  <cit> .
upon integrating our own data with existing datasets on platelet factors differentially released upon activation  <cit> , resulting potential bioactive proteins were functionally assessed on la- and bm-msc in vitro proliferation.
in addition, to validate our system we assessed the effects of selected reference cytokines.
we chose cytokines described to enhance the proliferation of msc in the presence of fbs asking whether these exert similar or different effects when added to phpl or tprp supplement.
RESULTS
bioactive factors promoting msc expansion
human platelet derivates support the expansion of msc from different tissues without changing the differentiation capacity and immunoregulatory properties when compared to fbs supplementation   <cit> .
confirming previous results, phpl-supplemented medium significantly promoted bm-msc proliferation compared to tprp- and fbs   <cit> .
in contrast, phpl and tprp accelerated the proliferation of la-msc in a similar way when compared to fbs   <cit> .
the specifically enhanced proliferative response of bm-msc towards phpl was dose-dependent, with  <dig> % phpl comparable to 10% tprp or 10% fbs.
interestingly, in tprp dose dependency was not obvious within the range of 10-5%.
evaluating different batches, there were no significant differences in the proliferative response .
these data clearly indicate that phpl and tprp contain different bioactive factors which specifically affect bm-msc proliferation.
mass spectrometric identification of differential proteins in phpl and tprp
to detect and identify bioactive factors in phpl and tprp, a differential proteomic 2d-dige approach was performed .
after labelling phpl and tprp with cydyes and subsequent 2d separation,  <dig> differentially occurring proteins were quantified by delta 2d imaging software .
selected differential proteins of phpl and tprp were picked from the preparative gel  and subjected to maldi-tof analysis.
in total,  <dig> plus one differential protein species were identified .
six proteins appeared higher in tprp whereas  <dig> proteins were more abundant in phpl.
one protein out of  <dig>  annexin a <dig>  was not satisfactory identified by ms  and initially not considered as identified, but subsequent western blot analysis confirmed differential expression .
a) b)
a)mascot software analysis by using swissprot data base.
ratio phpl/tprp: < <dig> higher abundant in tprp, > <dig> higher abundant in phpl.
pmf criteria: human source, one missed tryptic cleavage, methionine oxidation, score limit > <dig>  b)despite the fact that potential ms identification of annexin a <dig> is below required scoring , this non-significant ms hit was included into the list because the suggested high ratio and protein id was confirmed by western blotting.
verification of selected differential proteins
first, the 2d-dige results were verified by sds-page and western blot analysis in ∅ alb/igg-samples.
fibrinogen, apoa <dig>  clic and also of annexin a <dig> were differentially concentrated in ∅ alb/igg phpl and tprp samples .
second, all proteins were analyzed in the eight different phpl and tprp batches  used as 10% supplement.
clic <dig> was not detectable in the 10% supplemented medium and thus not further analysed.
fibrinogen and apoa <dig> were non-uniformly concentrated .
to define the concentration of fibrinogen and apoa <dig>  one batch was compared to a dilution series of known concentrations of fibrinogen and apoa <dig> in sds-page and western blotting.
by comparing band intensities a concentration of approximately 500 μg/ml fibrinogen in 10% phpl and approximately 80 μg/ml apoa <dig> in 10% tprp-supplemented medium was rated .
influence of selected identified proteins on msc proliferation
since none of the identified proteins was yet known as a mitogen for msc, those described to affect the proliferation of other cell types were further analyzed  <cit> .
to assess their bioactivity, bm- and la-msc were cultured in 5% phpl, tprp and fbs, respectively, plus an increasing concentration of the selected candidate proteins.
to reduce variability, phpl and tprp batch  <dig> was chosen for all these experiments.
of note, none of the single candidate proteins was able to affect proliferation in completely supplement-free conditions .
fibrinogen
125 μg/ml fibrinogen significantly stimulated la-msc proliferation in all three supplements compared to the unstimulated control .
bm-msc exhibited strong donor variation ranging from stimulation to inhibition especially in phpl.
fibrinogen can form a matrix to support cell adhesion, migration, but also proliferation  <cit> .
it has already been shown to dose-dependently control bm-msc proliferation, to reduce it at high concentrations depending on the fbs concentration  <cit> .
by differential dige/ms, we found a differential pattern of fibrinogen species, thus spliced, truncated and posttranslational modified proteins  <cit> .
single batch western blotting analysis failed to confirm dige/ms differential fibrinogen expression.
probably the primary anti-fibrinogen antibody may not be selective for all the individual isoform epitopes identified by dige/ms proteomics.
the higher concentration of fibrinogen gamma chain species in phpl well fits to the thrombin-induced conversion of fibrinogen to fibrin, consequently removed as clot in tprp.
by applying platelet-specific systems biology analysis several interaction partners of fibrinogen gamma such as icam <dig>  or integrin beta  <dig> , known to be involved in rapid platelet aggregation, have been identified .
apolipoprotein a1
only in fbs msc proliferation became significantly affected by apoa <dig>  which selectively promoted la-msc but inhibited bm-msc proliferation .
these observations illustrate the differences in msc tissue source and supplements, respectively.
apoa <dig>  a known major component of high density lipoprotein , has been reported to promote proliferation and to inhibit apoptosis of endothelial and vascular smooth muscle cells and to induce cardiac differentiation of embryonic stem cells  <cit> .
several functional interaction partners like apo a <dig> binding protein and lipopolysaccharide binding protein are depicted in figure 3f after performing platelet-specific systems biology analysis  <cit> .
atp
apart from the proteins identified by the proteomic approach, the atp-based celltiter-glo assay indicated higher atp concentrations in tprp supplement .
extracellular atp  has been described to affect various cellular features such as proliferation, apoptosis and arrest of growth, especially in neural cells  <cit> .
in msc, spontaneously released atp has been associated with decreased proliferation, reversible by p <dig> and p2y <dig> antagonists  <cit> .
whereas this inhibitory effect of high atp concentrations of  <dig> nm was reproducible in all three supplements for bm-msc  <cit> , la-msc in fbs appeared refractory to growth inhibition .
the distinct effects of eatp on different cell types have been attributed to engagement of different purinergic receptors  <cit> .
besides the current experimental limitations , we are confident that the combination of differential proteomics, database comparison and functional testing proves to be a suitable method to identify relevant factors affecting msc proliferation.
despite the few factors tested, we were able to identify some with differential effects on msc proliferation depending on the supplement and msc tissue source.
thus, the major hurdle in developing a chemically-defined medium is the combination and interaction of different factors which can best be modeled by multifactorial design experiments  <cit> .
influence of selected reference cytokines on msc proliferation
cytokines were not identified within the current 2d dige approach probably due to their often low concentration in plasma.
therefore, within a second set of experiments selected cytokines, known to foster msc proliferation in fbs  <cit> , were added to the different supplements and msc proliferation was measured.
here we a) tested the effect of adding different concentrations of cytokines, b) evaluated growth factor receptor expression and c) the concentration of cytokines in the culture medium/conditionened medium.
we decided to use 50 ng/ml hgf, 10 ng/ml igf- <dig> and 25 ng/ml bfgf  <cit> .
cytokines failed to induce a proliferative response in msc cultured with 5% platelet derivatives , whereas control cells in 5% fbs responded adequately.
bfgf inhibited bm-msc proliferation in the human supplements.
comparable findings have been observed by cheon et al.
testing the effects of egf, fgf and its  on at-msc proliferation in fbs or human serum.
in both settings, the combination of its and growth factors induced proliferation, whereas fgf on its own was highly effective in fbs only  <cit> .
addition of egf, bfgf and pdgfbb to 3% platelet poor plasma, however, reached a proliferation rate comparable to fbs  <cit> , advising a fine tuning in multifactorial design studies.
since cytokine effects depend on their concentration as well as the receptor expression, flow cytometry analysis was performed to assess expression of growth factor receptors.
fgfrii and hgfr were expressed at a significantly lower number of la-msc in fbs  .
interestingly, bfgf only induced proliferation in fbs, where both msc types had the lowest proportion of cells expressing fgfrii .
the higher proportion of fgfrii expression in the human supplements possibly rendered msc unresponsive to additional bfgf.
pdgfr-α expression appeared to be slightly higher in fbs, whereas pdgfr-β and tgf-βrii were expressed similar to fgfrii and hgfr .
with the exception of pdgfr-β in fbs, no differences between bm- and la-msc were observed.
in previous studies, we addressed the cytokine content in phpl, tprp and conditioned media of msc but failed to explain differential effects of phpl and tprp on msc proliferation  <cit> .
we now further analyzed the levels of selected candidates, including hgf, igf- <dig>  bfgf, pdgf-aa, ab and bb and tgf-βii in fresh medium with 10% supplement , in medium incubated for 24 h without cells  or in medium conditioned by msc .
tgf-βii was not detectable in any of the samples.
igf- <dig> concentrations remained largely unaffected in all conditions .
bfgf was detectable only in the phpl setting, significantly diminished by bm-msc .
hgf was only detectable in cm with apparent differences between bm- and la-msc in phpl, but not in tprp .
pdgf-aa levels were comparable in phpl at all four conditions, but in tprp values dropped in control medium to yield significant differences compared to phpl.
pdgf-ab levels, although initially comparable between phpl and tprp, became strongly reduced in tprp control medium.
similar data were obtained for pdgf-bb, with differences in phpl cm between bm- and la-msc .
individual experimental conditions such as donor variability, but obviously also the msc tissue origin, can largely affect the outcome as indicated in this study.
reported concentrations of cytokines in the supplements vary enormously and so do the concentrations used to stimulate msc  <cit> .
numerous data indicate a strong positive effect of bfgf on msc proliferation and osteogenic differentiation, either alone or in combination with tgf-β <dig>  <cit> .
platelet lysate-induced bm-msc proliferation can be inhibited by 75% by combining neutralizing antibodies against pdgf-ab/bb, tgf-β <dig> and bfgf.
stimulation with these three factors alone, however, was insufficient to promote proliferation  <cit> .
this is in line with the data presented herein.
CONCLUSIONS
human supplements, such as platelet derivatives, are emerging as alternatives to fbs in clinical-scale msc manufacturing.
observed differences in the proliferative response of bm- and la-msc towards platelet lysate versus platelet releasate prompted us to initiate a proteomic screen of differentially expressed proteins.
although the identified differential proteins have not been previously shown to affect msc expansion, bioactivity testing confirmed differential effects of identified proteins and selected cytokines on bm- and la-msc in the context of supplement .
we are confident that combining already existing data on msc mitogens and platelet and plasma proteome databases with the current data on identified proteins and their differential bioactivity will provide a platform to identify novel factors to optimize and standardize bm- and la-msc culture conditions for safer clinical application.

 mesenchymal stromal cells offer great potential for therapeutic application since they combine a number of biological properties such as multilineage differentiation, stromal support, immunomodulation, and secretion of trophic factors. due to the low frequency within tissues, in general ex vivo expansion is required to achieve a clinically-relevant cell dose. this has to comply with good manufacturing practice guidelines. fetal bovine serum used in many protocols, however, is critically rated by the regulatory authorities due to the possible transmission of extraneous agents as well as the risk of triggering host immune responses comprising the therapeutic success. a chemically-defined medium sufficiently mimicking serum compounds to provide growth and attachment factors, buffering and detoxifying agents, is still under development. currently human supplements, including platelet derivatives and human serum from autologous, allogeneic or cord blood sources, are assessed in pre- and clinical studies to replace fbs. we have previously demonstrated that pooled human serum, human platelet lysate and thrombin-activated platelet releasate in plasma are promising alternatives to fbs and support the main characteristics of msc. interestingly, hs, tprp and phpl all promoted the proliferation of msc from adipose tissue to a comparable extent. however, bone marrow-derived msc proliferation was significantly enforced solely by phpl. the different proliferative responses raised three questions: i) which bioactive proteins differ in the lysate and the releasate, ii) do these proteins exert different effects on la- and bm-msc, and can we extrapolate these findings to optimize chemically-defined msc media? human serum and platelet granules contain various growth factors capable of promoting cell proliferation and tissue regeneration. identification and characterization of these factors was markedly achieved by bioinformatic approaches integrating proteomic data sets from plasma, serum, the entire platelet proteome and specific subproteomes with functional data. these analyses have provided a comprehensive list of platelet and plasma proteins and as such contributed significantly to our current biomolecular understanding of these components and their function in the human body. they support a systems biology view on platelet protein function, network modules and enable evaluation of upcoming data sets. aiming to answer our questions we analyzed phpl and tprp with a differential proteomic approach and related the data to an in-depth catalog of human platelet proteins. upon integrating our own data with existing datasets on platelet factors differentially released upon activation, resulting potential bioactive proteins were functionally assessed on la- and bm-msc in vitro proliferation. in addition, to validate our system we assessed the effects of selected reference cytokines. we chose cytokines described to enhance the proliferation of msc in the presence of fbs asking whether these exert similar or different effects when added to phpl or tprp supplement.  bioactive factors promoting msc expansion human platelet derivates support the expansion of msc from different tissues without changing the differentiation capacity and immunoregulatory properties when compared to fbs supplementation. confirming previous results, phpl-supplemented medium significantly promoted bm-msc proliferation compared to tprp- and fbs. in contrast, phpl and tprp accelerated the proliferation of la-msc in a similar way when compared to fbs. the specifically enhanced proliferative response of bm-msc towards phpl was dose-dependent, with % phpl comparable to 10% tprp or 10% fbs. interestingly, in tprp dose dependency was not obvious within the range of 10-5%. evaluating different batches, there were no significant differences in the proliferative response. these data clearly indicate that phpl and tprp contain different bioactive factors which specifically affect bm-msc proliferation. mass spectrometric identification of differential proteins in phpl and tprp to detect and identify bioactive factors in phpl and tprp, a differential proteomic 2d-dige approach was performed. after labelling phpl and tprp with cydyes and subsequent 2d separation, differentially occurring proteins were quantified by delta 2d imaging software. selected differential proteins of phpl and tprp were picked from the preparative gel and subjected to maldi-tof analysis. in total, plus one differential protein species were identified. six proteins appeared higher in tprp whereas proteins were more abundant in phpl. one protein out of annexin a was not satisfactory identified by ms and initially not considered as identified, but subsequent western blot analysis confirmed differential expression. a) b) a)mascot software analysis by using swissprot data base. ratio phpl/tprp: < higher abundant in tprp, > higher abundant in phpl. pmf criteria: human source, one missed tryptic cleavage, methionine oxidation, score limit > b)despite the fact that potential ms identification of annexin a is below required scoring, this non-significant ms hit was included into the list because the suggested high ratio and protein id was confirmed by western blotting. verification of selected differential proteins first, the 2d-dige results were verified by sds-page and western blot analysis in ∅ alb/igg-samples. fibrinogen, apoa clic and also of annexin a were differentially concentrated in ∅ alb/igg phpl and tprp samples. second, all proteins were analyzed in the eight different phpl and tprp batches used as 10% supplement. clic was not detectable in the 10% supplemented medium and thus not further analysed. fibrinogen and apoa were non-uniformly concentrated. to define the concentration of fibrinogen and apoa one batch was compared to a dilution series of known concentrations of fibrinogen and apoa in sds-page and western blotting. by comparing band intensities a concentration of approximately 500 μg/ml fibrinogen in 10% phpl and approximately 80 μg/ml apoa in 10% tprp-supplemented medium was rated. influence of selected identified proteins on msc proliferation since none of the identified proteins was yet known as a mitogen for msc, those described to affect the proliferation of other cell types were further analyzed. to assess their bioactivity, bm- and la-msc were cultured in 5% phpl, tprp and fbs, respectively, plus an increasing concentration of the selected candidate proteins. to reduce variability, phpl and tprp batch was chosen for all these experiments. of note, none of the single candidate proteins was able to affect proliferation in completely supplement-free conditions. fibrinogen 125 μg/ml fibrinogen significantly stimulated la-msc proliferation in all three supplements compared to the unstimulated control. bm-msc exhibited strong donor variation ranging from stimulation to inhibition especially in phpl. fibrinogen can form a matrix to support cell adhesion, migration, but also proliferation. it has already been shown to dose-dependently control bm-msc proliferation, to reduce it at high concentrations depending on the fbs concentration. by differential dige/ms, we found a differential pattern of fibrinogen species, thus spliced, truncated and posttranslational modified proteins. single batch western blotting analysis failed to confirm dige/ms differential fibrinogen expression. probably the primary anti-fibrinogen antibody may not be selective for all the individual isoform epitopes identified by dige/ms proteomics. the higher concentration of fibrinogen gamma chain species in phpl well fits to the thrombin-induced conversion of fibrinogen to fibrin, consequently removed as clot in tprp. by applying platelet-specific systems biology analysis several interaction partners of fibrinogen gamma such as icam or integrin beta, known to be involved in rapid platelet aggregation, have been identified. apolipoprotein a1 only in fbs msc proliferation became significantly affected by apoa which selectively promoted la-msc but inhibited bm-msc proliferation. these observations illustrate the differences in msc tissue source and supplements, respectively. apoa a known major component of high density lipoprotein, has been reported to promote proliferation and to inhibit apoptosis of endothelial and vascular smooth muscle cells and to induce cardiac differentiation of embryonic stem cells. several functional interaction partners like apo a binding protein and lipopolysaccharide binding protein are depicted in figure 3f after performing platelet-specific systems biology analysis. atp apart from the proteins identified by the proteomic approach, the atp-based celltiter-glo assay indicated higher atp concentrations in tprp supplement. extracellular atp has been described to affect various cellular features such as proliferation, apoptosis and arrest of growth, especially in neural cells. in msc, spontaneously released atp has been associated with decreased proliferation, reversible by p and p2y antagonists. whereas this inhibitory effect of high atp concentrations of nm was reproducible in all three supplements for bm-msc, la-msc in fbs appeared refractory to growth inhibition. the distinct effects of eatp on different cell types have been attributed to engagement of different purinergic receptors. besides the current experimental limitations, we are confident that the combination of differential proteomics, database comparison and functional testing proves to be a suitable method to identify relevant factors affecting msc proliferation. despite the few factors tested, we were able to identify some with differential effects on msc proliferation depending on the supplement and msc tissue source. thus, the major hurdle in developing a chemically-defined medium is the combination and interaction of different factors which can best be modeled by multifactorial design experiments. influence of selected reference cytokines on msc proliferation cytokines were not identified within the current 2d dige approach probably due to their often low concentration in plasma. therefore, within a second set of experiments selected cytokines, known to foster msc proliferation in fbs, were added to the different supplements and msc proliferation was measured. here we a) tested the effect of adding different concentrations of cytokines, b) evaluated growth factor receptor expression and c) the concentration of cytokines in the culture medium/conditionened medium. we decided to use 50 ng/ml hgf, 10 ng/ml igf- and 25 ng/ml bfgf. cytokines failed to induce a proliferative response in msc cultured with 5% platelet derivatives, whereas control cells in 5% fbs responded adequately. bfgf inhibited bm-msc proliferation in the human supplements. comparable findings have been observed by cheon et al. testing the effects of egf, fgf and its on at-msc proliferation in fbs or human serum. in both settings, the combination of its and growth factors induced proliferation, whereas fgf on its own was highly effective in fbs only. addition of egf, bfgf and pdgfbb to 3% platelet poor plasma, however, reached a proliferation rate comparable to fbs, advising a fine tuning in multifactorial design studies. since cytokine effects depend on their concentration as well as the receptor expression, flow cytometry analysis was performed to assess expression of growth factor receptors. fgfrii and hgfr were expressed at a significantly lower number of la-msc in fbs. interestingly, bfgf only induced proliferation in fbs, where both msc types had the lowest proportion of cells expressing fgfrii. the higher proportion of fgfrii expression in the human supplements possibly rendered msc unresponsive to additional bfgf. pdgfr-α expression appeared to be slightly higher in fbs, whereas pdgfr-β and tgf-βrii were expressed similar to fgfrii and hgfr. with the exception of pdgfr-β in fbs, no differences between bm- and la-msc were observed. in previous studies, we addressed the cytokine content in phpl, tprp and conditioned media of msc but failed to explain differential effects of phpl and tprp on msc proliferation. we now further analyzed the levels of selected candidates, including hgf, igf- bfgf, pdgf-aa, ab and bb and tgf-βii in fresh medium with 10% supplement, in medium incubated for 24 h without cells or in medium conditioned by msc. tgf-βii was not detectable in any of the samples. igf- concentrations remained largely unaffected in all conditions. bfgf was detectable only in the phpl setting, significantly diminished by bm-msc. hgf was only detectable in cm with apparent differences between bm- and la-msc in phpl, but not in tprp. pdgf-aa levels were comparable in phpl at all four conditions, but in tprp values dropped in control medium to yield significant differences compared to phpl. pdgf-ab levels, although initially comparable between phpl and tprp, became strongly reduced in tprp control medium. similar data were obtained for pdgf-bb, with differences in phpl cm between bm- and la-msc. individual experimental conditions such as donor variability, but obviously also the msc tissue origin, can largely affect the outcome as indicated in this study. reported concentrations of cytokines in the supplements vary enormously and so do the concentrations used to stimulate msc. numerous data indicate a strong positive effect of bfgf on msc proliferation and osteogenic differentiation, either alone or in combination with tgf-β. platelet lysate-induced bm-msc proliferation can be inhibited by 75% by combining neutralizing antibodies against pdgf-ab/bb, tgf-β and bfgf. stimulation with these three factors alone, however, was insufficient to promote proliferation. this is in line with the data presented herein.  human supplements, such as platelet derivatives, are emerging as alternatives to fbs in clinical-scale msc manufacturing. observed differences in the proliferative response of bm- and la-msc towards platelet lysate versus platelet releasate prompted us to initiate a proteomic screen of differentially expressed proteins. although the identified differential proteins have not been previously shown to affect msc expansion, bioactivity testing confirmed differential effects of identified proteins and selected cytokines on bm- and la-msc in the context of supplement. we are confident that combining already existing data on msc mitogens and platelet and plasma proteome databases with the current data on identified proteins and their differential bioactivity will provide a platform to identify novel factors to optimize and standardize bm- and la-msc culture conditions for safer clinical application. 
to identify functionally bioactive factors affecting ex vivo msc expansion, a differential proteomic approach was performed and identified candidate proteins were evaluated within a bioassay. the differential proteomic approach identified novel platelet derived factors differentially acting on human msc proliferation. multilineage differentiation, immunomodulation and secretion of trophic factors render mesenchymal stromal cells highly attractive for clinical application. bone marrowdifferential proteomicsmesenchymal stromal cells2d-digemass spectrometryplatelets the candidate proteins fibrinogen and apolipoprotein a differentially affected la- and bm-msc proliferation. the data describe a new coherent approach to combine proteomic technologies with functional testing to develop novel, humanized, gmp-compliant conditions for msc expansion. human platelet derivatives such as pooled human platelet lysate and thrombin-activated platelet releasate in plasma have been introduced as alternatives to fetal bovine serum to achieve gmp-compliance. candidate proteins occured at higher concentrations in phpl compared to tprp and at higher concentrations in tprp. in a second set of experiments, reference cytokines known to foster proliferation in fbs were tested for their effects in the human supplements. complementary testing of reference cytokines revealed a lack of stimulation in the human supplements compared to fbs.
 multilineage differentiation, immunomodulation and secretion of trophic factors render mesenchymal stromal cells highly attractive for clinical application. human platelet derivatives such as pooled human platelet lysate and thrombin-activated platelet releasate in plasma have been introduced as alternatives to fetal bovine serum to achieve gmp-compliance. however, whereas both phpl and tprp support similar proliferation kinetics of lipoaspirate-derived msc, only phpl significantly accelerates bone marrow-derived msc expansion. to identify functionally bioactive factors affecting ex vivo msc expansion, a differential proteomic approach was performed and identified candidate proteins were evaluated within a bioassay.  two dimensional difference gel electrophoresis, maldi-tof analyses and complementary western blotting revealed differential protein species. candidate proteins occured at higher concentrations in phpl compared to tprp and at higher concentrations in tprp. the candidate proteins fibrinogen and apolipoprotein a differentially affected la- and bm-msc proliferation. in a second set of experiments, reference cytokines known to foster proliferation in fbs were tested for their effects in the human supplements. interestingly although these cytokines promoted proliferation in fbs, they failed to do so when added to the humanized system.  the differential proteomic approach identified novel platelet derived factors differentially acting on human msc proliferation. complementary testing of reference cytokines revealed a lack of stimulation in the human supplements compared to fbs. the data describe a new coherent approach to combine proteomic technologies with functional testing to develop novel, humanized, gmp-compliant conditions for msc expansion. bone marrowdifferential proteomicsmesenchymal stromal cells2d-digemass spectrometryplatelets 