vascular smooth muscle cell ( smc ) differentiation is an essential component of vascular development .
a variety of human vascular diseases can be traced to a defect in smooth muscle development or proliferation [ 13 ] . in mice with defective smooth muscle development ,
they include mouse neural crest stem cell line monc-1 , mouse embryonic fibroblast 10t1/2   and mouse embryonic stem cells . under certain culture conditions ,
these cells express smc - specific markers that indicate smc differentiation , which include contractile apparatus - associated proteins such as calponin-1 and smooth muscle -actin ( sm -actin ) .
however , several problems arise when using these cell models for the study of smc differentiation .
first , because most of the cells used in smc differentiation studies are of mouse origin , there may be important interspecies differences in the differentiation environment , intracellular molecules involved in the differentiation process and underlying signaling mechanisms promoting smc differentiation .
second , some cell lines are not naive cells but immortalized cell lines derived from a primary culture . the regulating mechanisms involved in smc differentiation may be changed in the immortalized cell lines . as an alternative , simper et al .   have described smc progenitor cells in human peripheral blood mononuclear cells . however
, based on the experience of our lab and several other labs , progenitor cells isolated from human peripheral blood can hardly proliferate under in vitro culture condition and usually deteriorate after 3 weeks , and as a result , it is impossible to maintain the cell line for serial analysis and meaningful comparisons . also , the cell samples isolated from donors can only be used one time due to the lack of proliferative properties . the variation in blood samples from donors of different ethnicities , ages , genders and medical backgrounds
multi - lineage progenitor cell lines ( mlpcs ) are karyotypically normal multi - potent progenitor cells obtained from post - partum human umbilical cord blood .
it is possible to continue to expand these cells , and they can differentiate down specific lineages beyond 20 passages . their high purity and proliferative features make mlpc an ideal tool for the study of smc differentiation .
transforming growth factor 1 ( tgf-1 ) is thought to play a key role in smc differentiation and is known to coordinately upregulate a variety of smc differentiation markers in cultured smc from mature blood vessels   as well as pluri - potential stem cells . in this study , we investigated the differentiation of mlpc into smc lineage cells , which was induced by tgf-1 .
we found that the mrna levels of a variety of smc - specific markers were increased during this process , whereas endothelial cell - specific markers and epc marker cd34 were decreased in tgf-1-treated mlpc .
recombinant human tgf-1 was obtained from r&d systems ( minneapolis , mn , usa ) .
the mesenchymal stem cell medium bulletkit was obtained from cambrex - walker ( walkersville , md , usa ) .
iq sybr green super - mix kit was obtained from bio - rad ( hercules , ca , usa ) .
all of the primers were synthesized by sigma genosys ( the woodlands , tx , usa ) .
mlpcs were cultured in mesenchymal stem cell basic medium to maintain their undifferentiated status and were then subcultured by using trypsin - edta reagent in the regular way .
mlpcs at passage 4 to passage 6 were used in the experiment . to induce cell differentiation ,
mlpcs were seeded at 1.510 in each well on a 6 well plate containing mesenchymal stem cell basic medium plus 1% fbs with or without tgf-1 ( 1 ng / ml or 5 ng / ml ) .
mlpcs were cultured for 7 days , and cell rna was then harvested for pcr analysis .
the genomic dna contamination in rna preparation was removed by using the dna - free kit ( ambion , austin , tx ) , and a lack of detectable genomic dna was confirmed by pcr .
total rna ( 0.5 g ) was reverse - transcribed into cdna using the iscipt cdna synthesis kit ( bio - rad ) following the manufacturer s instruction .
primers for all tested genes were designed using the beacon designer 2.1 software ( bio - rad ) .
the quality of individual pairs of primers was confirmed by running conventional pcr before real - time pcr to make sure there were no detectable primer dimers or non - specific products yielded .
the real - time pcr reaction mixture included the following : 250 nm primers , 50 ng cdna , and iq sybr green supermix ( 0.2 mm of each dntp , 25 u / ml itaq dna polymerase , sybr green i , 10 nm fluorescein , 3 mm mgcl2 , 50 mm kcl , and 20 mm tris - hcl ) . using the icycler iq real - time pcr detection system ( bio - rad ) ,
pcr cycling conditions were set as follows : 95c for 90 seconds , 40 cycles at 95c for 20 seconds , and 60c for 1 minunte .
melting curve analysis was performed on the icycler over the range 5595c by monitoring iq sybr green fluorescence with increasing temperature ( 0.5c increment changes at 10 seconds intervals ) .
sample cycle threshold ( ct ) values were determined from plots of relative fluorescence units ( rfu ) versus pcr cycle number during exponential amplification so that sample measurement comparison was possible .
standard curves for all primer amplifications were generated by plotting average ct values against the logarithm starting quantity of target template molecules ( series dilution of cdna template : 50 , 10 , 2 , 0.4 , and 0.08 ng ) , followed by a sum of least squares regression analysis .
the gene expression in each sample was normalized to gapdh expression as [ 2^(ctgapdhctgene ) ] .
data from the control and tgf-1-treated groups were analyzed using a paired student s t test ( one tail , minitab software , sigma breakthrough technologies , inc .
recombinant human tgf-1 was obtained from r&d systems ( minneapolis , mn , usa ) .
the mesenchymal stem cell medium bulletkit was obtained from cambrex - walker ( walkersville , md , usa ) .
iq sybr green super - mix kit was obtained from bio - rad ( hercules , ca , usa ) .
all of the primers were synthesized by sigma genosys ( the woodlands , tx , usa ) .
mlpcs were cultured in mesenchymal stem cell basic medium to maintain their undifferentiated status and were then subcultured by using trypsin - edta reagent in the regular way .
mlpcs at passage 4 to passage 6 were used in the experiment . to induce cell differentiation ,
mlpcs were seeded at 1.510 in each well on a 6 well plate containing mesenchymal stem cell basic medium plus 1% fbs with or without tgf-1 ( 1 ng / ml or 5 ng / ml ) .
mlpcs were cultured for 7 days , and cell rna was then harvested for pcr analysis .
the genomic dna contamination in rna preparation was removed by using the dna - free kit ( ambion , austin , tx ) , and a lack of detectable genomic dna was confirmed by pcr .
total rna ( 0.5 g ) was reverse - transcribed into cdna using the iscipt cdna synthesis kit ( bio - rad ) following the manufacturer s instruction .
primers for all tested genes were designed using the beacon designer 2.1 software ( bio - rad ) .
the quality of individual pairs of primers was confirmed by running conventional pcr before real - time pcr to make sure there were no detectable primer dimers or non - specific products yielded .
the real - time pcr reaction mixture included the following : 250 nm primers , 50 ng cdna , and iq sybr green supermix ( 0.2 mm of each dntp , 25 u / ml itaq dna polymerase , sybr green i , 10 nm fluorescein , 3 mm mgcl2 , 50 mm kcl , and 20 mm tris - hcl ) . using the icycler iq real - time pcr detection system ( bio - rad ) ,
pcr cycling conditions were set as follows : 95c for 90 seconds , 40 cycles at 95c for 20 seconds , and 60c for 1 minunte .
melting curve analysis was performed on the icycler over the range 5595c by monitoring iq sybr green fluorescence with increasing temperature ( 0.5c increment changes at 10 seconds intervals ) .
sample cycle threshold ( ct ) values were determined from plots of relative fluorescence units ( rfu ) versus pcr cycle number during exponential amplification so that sample measurement comparison was possible .
standard curves for all primer amplifications were generated by plotting average ct values against the logarithm starting quantity of target template molecules ( series dilution of cdna template : 50 , 10 , 2 , 0.4 , and 0.08 ng ) , followed by a sum of least squares regression analysis .
the gene expression in each sample was normalized to gapdh expression as [ 2^(ctgapdhctgene ) ] .
data from the control and tgf-1-treated groups were analyzed using a paired student s t test ( one tail , minitab software , sigma breakthrough technologies , inc . , san marcos , tx ) .
after 7 days of exposure to tgf-1 , several smc - specific markers were dramatically increased .
the addition of tgf-1 ( 1 ng / ml ) to mesenchymal stem cell medium significantly increased the mrna levels of sm22 , calponin-1 , sm -actin , caldesmon , tropomyosin and myosin light chain kinase ( mlck ) to 1215.5% , 1974.6% , 567% , 429.7% , 567% and 162.8% , respectively , when compared to controls ( medium only ) ( p<0.05 , figure 1a  f ) .
tgf-1 ( 5 ng / ml ) also increased the mrna levels of these markers significantly ( p<0.05 ) , but the effect was less than that of the tgf-1 treatment at 1 ng / ml ( figure 1a  f ) .
in addition , we investigated the expression level of cd105 ( a tgf- accessory receptor ) in mlpcs .
we found that cd105 is expressed at a high level in mlpcs under control conditions ( relative mrna level is 0.156 , data not shown ) .
after 7 days , tgf-1 at 1 ng / ml and 5 ng / ml led to a significant downregulation of cd105 mrna levels to 61.7% and 70.8% of that of controls , respectively ( p<0.05 , figure 2 ) .
two ec - specific markers , ve - cadherin and vegfr-2 , were detected in mlpcs under control conditions .
after 7 days of exposure to tgf-1 at 1 ng / ml , there was a significant reduction in ve - cadherin and vegfr-2 mrna levels to 15.4% and 70.8% of control levels , respectively .
tgf-1 at 5 ng / ml also significantly decreased ve - cadherin and vegfr-2 ; mrna levels were 21.8% and 66.1% of controls , respectively ( p<0.05 , figure 3a and b ) .
we investigated the expression of cd34 , a marker of endothelial progenitor cells , in mlpcs .
cd34 was expressed at a low level in mlpcs under control conditions ( relative mrna level is 2.34e-05 , data not shown ) .
after 7 days , the addition of tgf-1 at 1 ng / ml and 5 ng / ml to the mesenchymal stem cell culture media significantly decreased the mrna level of cd34 to 28.7% and 25.1% of control levels , respectively ( p<0.05 , figure 4 ) .
the mrna level of cd146 was low in mlpcs under control conditions ( relative mrna level is 3.8e-05 , data not shown ) . after 7 days , tgf-1 at 1
ng / ml and 5 ng / ml significantly increased the mrna level of cd146 to 2430% and 2605% of that of the controls , respectively ( p<0.05 , figure 5 ) .
after 7 days of exposure to tgf-1 , several smc - specific markers were dramatically increased .
the addition of tgf-1 ( 1 ng / ml ) to mesenchymal stem cell medium significantly increased the mrna levels of sm22 , calponin-1 , sm -actin , caldesmon , tropomyosin and myosin light chain kinase ( mlck ) to 1215.5% , 1974.6% , 567% , 429.7% , 567% and 162.8% , respectively , when compared to controls ( medium only ) ( p<0.05 , figure 1a  f ) .
tgf-1 ( 5 ng / ml ) also increased the mrna levels of these markers significantly ( p<0.05 ) , but the effect was less than that of the tgf-1 treatment at 1 ng / ml ( figure 1a  f ) .
in addition , we investigated the expression level of cd105 ( a tgf- accessory receptor ) in mlpcs .
we found that cd105 is expressed at a high level in mlpcs under control conditions ( relative mrna level is 0.156 , data not shown ) .
after 7 days , tgf-1 at 1 ng / ml and 5 ng / ml led to a significant downregulation of cd105 mrna levels to 61.7% and 70.8% of that of controls , respectively ( p<0.05 , figure 2 ) .
two ec - specific markers , ve - cadherin and vegfr-2 , were detected in mlpcs under control conditions .
after 7 days of exposure to tgf-1 at 1 ng / ml , there was a significant reduction in ve - cadherin and vegfr-2 mrna levels to 15.4% and 70.8% of control levels , respectively .
tgf-1 at 5 ng / ml also significantly decreased ve - cadherin and vegfr-2 ; mrna levels were 21.8% and 66.1% of controls , respectively ( p<0.05 , figure 3a and b ) .
we investigated the expression of cd34 , a marker of endothelial progenitor cells , in mlpcs .
cd34 was expressed at a low level in mlpcs under control conditions ( relative mrna level is 2.34e-05 , data not shown ) .
after 7 days , the addition of tgf-1 at 1 ng / ml and 5 ng / ml to the mesenchymal stem cell culture media significantly decreased the mrna level of cd34 to 28.7% and 25.1% of control levels , respectively ( p<0.05 , figure 4 ) .
the mrna level of cd146 was low in mlpcs under control conditions ( relative mrna level is 3.8e-05 , data not shown ) . after 7 days , tgf-1 at 1
ng / ml and 5 ng / ml significantly increased the mrna level of cd146 to 2430% and 2605% of that of the controls , respectively ( p<0.05 , figure 5 ) .
mlpcs originate from human umbilical cord blood , are karyotypically normal , demonstrate proliferative features , and are highly pure . in the present study , we observed the differentiation of mlpcs to smc lineage in the presence of tgf-1 as evidenced by an upregulation of the mrna levels of both early and late phase smc - specific markers and adhesion molecule cd146 .
two ec markers , as well as epc marker cd34 and tgf-1 cell surface accessory protein cd105 , were decreased in tgf-1 treated mlpcs . as such , these data suggest that mlpcs represent a promising tool for the study of smc differentiation .
quiescent vascular smcs exhibit a phenotype characterized by the expression of several contractile apparatus - associated proteins ; these are used as markers to identify smc lineage differentiation .
we checked both early phase markers ( sm -actin , sm22 ) and late phase markers ( mlck , calponin-1 , caldesmon , and tropomyosin ) for smc differentiation in mlpcs .
we found that treatment with tgf-1 led to a dramatic increase in the mrna levels of all the tested markers in mlpcs after 7 days of culture ; this strongly indicates the differentiation of mlpcs into smc lineage in the presence of tgf-1 .
chen s et al .   reported that tgf-1 ( 5 ng / ml ) increases the expression of sm -actin , sm22 , calponin-1 and myosin in the neural crest stem cell line monc-1 in vitro .
.   also found that tgf-1 ( 2 ng / ml ) induces sm -actin , sm22 and smooth muscle myosin heavy chain ( smmhc ) in 10t1/2 mesenchymal cells in vitro . in our study , the mrna levels of all the tested smc markers in mlpcs were upregulated to a comparable level with tgf-1 treatment when compared to those found in neural crest stem cell line ( monc-1 ) or 10t1/2 mesenchymal cells .
the signaling pathway underlying tgf-1-induced smc - specific marker expression in mlpcs was not investigated in this study .
investigated differential tgf--smad signaling for early versus late smc marker expression ; sm -actin promoter activity was found to be dependent on both smad 2 and smad 3 whereas smooth muscle myosin heavy chain ( smmhc ) activity is smad2 dependent in mouse embryonic stem cell .
chen s et al .   also found that tgf- increased sm -actin and sm22 in the neural crest stem cell line through activation of smad2 and smad3 .
in addition , rhoa was reported to be essential in smad signaling during tgf--induced sm a - actin , sm22 and calponin expression in the neural crest stem cell line monc-1 . meanwhile ,
the pi3 kinase / akt pathway was found to be involved in tgf-1-induced sm -actin , sm22 and smmhc expression in 10t1/2 mesenchymal cells .
cd105 is a transmembrane accessory receptor for tgf-. by forming a heteromeric complex with distinct tgf- receptors , it modulates the access of tgf- to the signaling complex and the postponed cellular responses to tgf- .
we found that cd105 is expressed at a high level in mlpcs in the control condition ( relative mrna level is 0.156 ) , which may facilitate tgf-1/tgf- receptor signaling transduction . over a long duration of tgf-1 treatment ( 7 days ) ,
expression of cd105 was decreased , which may be due to a negative feedback mechanism .
the specific signaling mechanism involved in tgf-1-induced differentiation of mlpcs into smc will need to be further elucidated .
progenitor cells are primitive cells that possess the capacity to differentiate into multiple lineages under different conditions .
evidence has shown that progenitor cells from human peripheral blood can differentiate into ec when stimulated by shear stress   and into smcs in conditions of cyclic strain .
hence , we investigated the expression of two ec specific markers to confirm that the potential for ec lineage differentiation by the mlpcs was suppressed under the designated conditions of this study .
we found that the mrna levels of ve - cadherin and vegfr-2 were significantly decreased by tgf-1 on day 7 of culture .
vegfr-2 is the pivotal receptor mediating the mitogenic action of vegf ; it plays an essential role in angiogenesis , neovascularization and epc differentiation .
ve - cadherin is another ec - specific marker which has been shown to play important role in vasculogenesis and angiogenesis .
the downregulated ve - cadherin and vegfr-2 observed in the mlpcs provides further confirmation that tgf-1 induced smc lineage differentiation in the mlpcs .
similarly , chen s et al .   found that tgf-1 reduced epithelial markers while inducing smc - specific markers in the neural crest stem cell line monc-1 .
reported that tgf- inhibited proliferation and sheet formation of embryonic stem cell ( esc)-derived ecs .
stimulation of esc - derived ecs with tgf- resulted in phosphorylation of both smad2 and smadl/5 .
the specific signaling mechanism underlying the tgf-1-induced reduction in ec markers in mlpc need to be further studied .
in addition to ec markers , we tested the epc marker cd34 in mlpcs . cd34 has been used as a hematopoietic stem cell marker ; endothelial progenitor cells are characterized by the expression of cd34 , cd133 and vegfr-2 . in this study
, cd34 was expressed at a low level in mlpc under the control conditions , and expression was further reduced by tgf-1 treatment ; this suggests that the tendency for ec lineage differentiation by the mlcps was suppressed under the designated conditions .
this in accordance with a previous finding that cd34 expression was downregulated during tgf-1 induced myofibroblast differentiation .
cd146 , melanoma cell adhesion molecule , is also expressed in smc and plays an important role in vasculogenesis and embryo development .
cd146 , when activated , induces association of p59 with cd146 , resulting in the phosphorylation of p125 and its binding with paxillin ; this finding suggests that cd146 is involved in outside - in signaling and may contribute to focal adhesion assembly , reorganization of the cytoskeleton , intercellular interaction , maintenance of cell shape , and control of cell migration and proliferation . in this study , cd146 was significantly increased by tgf-1 treatment in mlpcs , which may facilitate signaling transduction by tgf-1 and contribute to the differentiation of mlpcs into mature smc .
in conclusion , a variety of smc - specific markers , including early and late phase markers , were dramatically increased in mlcp treated with tgf-1 . meanwhile , two ec - specific markers as well as the epc marker cd34 were significantly decreased .
these data strongly indicate that mlcps differentiate into smc lineage cells in the presence of tgf-1 .
mlpcs are karyotypically normal , non - transformed , non - immortalized cells that are obtained from post - partum human umbilical cord blood . because they have been expanded from a single cell and have the capacity to differentiate into multiple lineages , they are highly pure and proliferative .
mlpcs offer significant advantages over other currently used cell models , such as 10t1/2 cells , the neural crest stem cell line monc-1 and smc progenitor cells from human peripheral blood for the study of smc differentiation .
this study demonstrates a novel cell model for smc lineage differentiation analysis , which may increase our understanding of smc differentiation and help contribute to the field of vascular repair and tissue engineering .
 summarybackgroundvascular smooth muscle cell ( smc ) differentiation is an essential component of vascular repair and tissue engineering . 
 however , currently used cell models for the study of smc differentiation have several limitations . 
 multi - lineage progenitor cells ( mlpcs ) originate from human umbilical cord blood and are cloned from a single cell . 
 the object of this study was to investigate whether mlpcs could differentiate into smcs in vitro with induction by transforming growth factor 1 ( tgf-1).material / methodsmlpcs were treated without or with tgf-1 ( 1 and 5 ng / ml ) in mesenchymal stem cell media plus 1% fbs for 7 days . 
 total rna was isolated from the mlpcs , and semi - quantitative real - time pcr was performed to test the following mrna levels : early and late phase smc - specific markers , two endothelial cell ( ec)-specific markers , endothelial progenitor cell ( epc ) marker cd34 , tgf-1 accessory protein cd105 , and adhesion molecule cd146.resultstgf-1 ( 1 ng / ml ) significantly increased the mrna levels of smc - specific markers sm22 , calponin-1 , sm -actin , caldesmon , tropomyosin and mlck as well as adhesion molecule cd146 . 
 the mrna levels of ec - specific markers ve - cadherin and vegfr-2 , epc marker cd34 and tgf-1 accessory protein cd105 were decreased significantly , after mlpc were treated with tgf-1 ( 1 ng / ml ) . 
 tgf-1 at 5 ng / ml showed similar effect on the expression of these genes.conclusionsthis study demonstrates that in the presence of tgf-1 , mlpcs undergo smc lineage differentiation indicating that mlpcs are a promising cell model for smc lineage differentiation studies , which may contribute to advances in vascular repair and tissue engineering . 
Background
Material and Methods
Chemicals and reagents
Cell culture
Real-time PCR
Statistical analysis
Results
TGF-1 increased the expression of SMC markers in MLPCs
TGF-1 decreased the expression of endothelial cell-specific markers in MLPCs
L2 TGF-
TGF-1 increased the expression of adhesion molecule CD146 in MLPCs
Discussion
Conclusions