BACKGROUND
adipose tissue has gained intense interest in the last decades.
early in the 19 s, adipose tissue was considered an agglomeration of fat-storing adipocytes surrounded by connective tissue.
only in  <dig>  shapiro and wertheimer suggested that adipose tissue was metabolically active  <cit> .
since then, several studies have improved our knowledge of adipose tissue metabolic, endocrine and immunologic functions.
fully differentiated adipocytes were first isolated using collagenase and flotation in  <dig> by rodbell  <cit> .
early in the 1990s, the first evidence of adipose tissue secretory function was reported  <cit> .
adipocytes secrete a wide variety of proteins, including adipokines, a family of proteins exclusively secreted by adipocytes  <cit> .
the first adipokine described in  <dig> was leptin, which confirmed that fat tissue has an important endocrine function  <cit> .
in  <dig>  zuk and colleagues described the procedure to process lipoaspirates  <cit> .
the same group published subsequently biochemical and molecular analysis of the adipose tissue-derived stem cells, confirming their differentiation capacity into different mesodermal cell types and the expression of specific genes  <cit> .
the possibility of stem cell isolation from the easily accessible and abundant adipose tissue was relevant in the stem cell and regenerative medicine field.
before researchers completely understood adipose tissue biology, fat was already used in cosmetic, plastic, and reconstructive surgeries.
in  <dig>  neuber performed the first clinical fat transplantation, using small pieces of autologous material for filling depressed scars  <cit> .
problems related to necrosis, cyst formation and reabsorption were usual, due to low revascularization, especially when large fat pieces were transplanted.
autologous fat transplantation has several advantages: it results in a satisfactory tissue repair, avoids large incision scars, causes no immune host responses, and avoids use of prosthesis.
its major disadvantage is the apparently unpredictable survival of the transplanted fat, since variable resorption rates have been reported .
in order to obtain the desired final volume of implant, multiple interventions may be required  <cit> .
mature adipocytes are fragile cells, and a large part may suffer extensive lesions with a loss of membrane integrity and release of fat.
sydney coleman developed in  <dig> an improved method for fat transplantation that minimizes the stress during tissue collection, by recommending transplantation of small fat tissue fragments in multiple steps, in order to facilitate revascularization and, therefore, tissue survival.
this tissue processing, called coleman’s method, resulted in reduced graft resorption  <cit> .
the discovery of stem cells in adipose tissue by zuk and colleagues in  <dig> complemented the understanding of the durability of fat transfer  <cit> .
matsumoto and coworkers developed in the year  <dig> a new technique to reduce tissue resorption, using the “cell-assisted lipotransfer”  <cit> .
the technique consisted in separating a part of the aspirated fat in a stromal vascular fraction enriched in msc.
the lipotransfer containing these cells allowed a higher survival and vascularization of the graft in pre-clinical and clinical trials  <cit> .
further studies have proposed alternative procedures to manipulate lipoaspirates, and overall recommendations for handling of samples and for characterization of transferred cells  <cit> .
the msc content in primary cell suspensions collected from lipoaspirates is however low.
immunophenotyping of cells in the stromal fraction indicates presence of blood-vessel components and associated elements, as well as pre-adipocytes and msc.
the latter two are characterized as cd45neg, cd34pos and/or cd105pos, and their content in the stromal fraction was reported to be below 5%  <cit> .
rigotti and coworkers reported in the year  <dig> that the content of msc in the adipose tissue stromal fraction was in order of 1%, but importantly, the level of clonogenic colony-forming units that possibly better reflects the quantity of cells able to participate in long-term tissue regeneration was tenfold lower  <cit> .
in contrast, when selected by plastic adhesion  and expanded in vitro, msc are selected and can represent 70-90% of the harvested cells.
the use of these cells offers new cues in regenerative procedures using much higher levels of msc in the “cell-assisted lipotransfers”.
the msc used in this procedure can be autologous, requiring a first harvest of fat tissue to establish cultures, and a second one to provide the tissue for the graft.
when the donor adipose tissue is limited, alternative sources of msc may be used, associated with autologous fat graft, in order to grant a long-term restoration of fat tissue under controlled conditions, maintaining the volume and the implant’s form.
among others, this may be the case of facial fat restoration in lipodystrophies or in chronic hiv infection  <cit> .
we hypothesized that msc from different origins other than adipose tissue can be used in assisting fat transplantation, especially if they show a high adipogenic differentiation potential.
msc obtained from the wharton’s jelly of the umbilical cord, a convenient source of cells with high proliferation capacity, may be proposed for allogeneic transplantation.
therefore, we characterized msc obtained from different sources  regarding their ability to differentiate into adipocytes.
characterization of msc and their ability to differentiate into adipocytes may be relevant in many other fields of regenerative medicine.
novel proposals for tendon and muscle regeneration deal with the use of msc for repair of such lesions of shoulder, which are particularly sensitive to the fatty degeneration of muscles  <cit> .
this is generally an irreversible process, in which adipocytes invade and replace the muscle tissue.
the origin of these adipocytes is a differentiation from resident msc, which in the absence of mechanical stimulation by muscle contraction suffer a decrease of wnt signaling, leading to transcription of adipogenic factors such as pparγ, c/ebpα and leptin  <cit> .
knowledge of which msc already express, or easily activate expression of adipogenic factors, and readily differentiate into adipocytes, may be relevant for the choice of msc to be used in such a therapy.
in the present study, we compared adipogenic differentiation in vitro of msc derived from bone marrow, adipose tissue and wharton’s jelly.
our main goal is to determine which source of msc is more appropriate for allogeneic transplantation, so they can be use together with adipose tissue for long-lasting effects after fat transplantation.
we quantified the gene expression of key pluripotent and differentiation markers, and the secretion of different proteins, and we compared these results with those recently obtained by our group regarding protein secretion of undifferentiated bm-msc, at-msc and wj-msc.
RESULTS
msc obtained from human bone marrow, adipose tissue and wharton’s jelly identity was confirmed: they adhered to the plastic surfaces of cell culture flasks with a fibroblast–like morphology, expressed the cell surface markers cd <dig>  cd <dig> and cd <dig> and negative for hematopoietic markers   <cit> .
msc also differentiated into all three mesodermal lineages: adipocytes, osteoblasts and chondroblasts .
all msc cultured in dmem supplemented with fbs and with the compounds recommended for adipocyte induction differentiated into adipocyte-like cells.
differentiation controls maintained in the standard medium were run in parallel for all conditions, and they were all negative, not presenting any visible lipid accumulation .
gene expression during differentiation into adipocyte-like cells
gene expression of pluripotent  and adipogenic  markers were studied, evaluating cell differentiation effects on gene expression.
appropriate reference genes were rpl13a for wj-msc and at-msc, and hprt <dig> for bm-msc  <cit> .
tert expression was not detected in any sample.
published data are contradictory regarding tert expression: some authors described tert mrna as a pluripotent marker in bm-msc  <cit>  but many others detected neither tert mrna  <cit>  nor tert activity  <cit>  in msc primary cultures.
data on mrna expression during adipogenic differentiation are shown in table  <dig>  a statistical analysis was performed comparing the expression of one gene for all cells and time points evaluated.
for all the genes, at-msc at day  <dig> expressed statistically significant higher amounts of the corresponding mrna.
regarding pluripotent markers, only pou5f <dig> was downregulated in bm-msc cultures.table  <dig>
relative gene expression  for human adipose tissue- , bone marrow-  and wharton’s jelly-mesenchymal stromal cells  when grown in low glucose media supplemented with 10% fetal bovine serum
bm-msc
at-msc
wj-msc
d10
d21
d10
d21
d10
d21
pparg
adipoq
cebpa
sox2
pou5f1
adipoq: adiponectin; at-msc: adipose tissue-derived msc; bm-msc: bone marrow-derived msc; cepba: ccaat/enhancer-binding protein alpha; d10: day 10; d21: day 21; msc: mesenchymal stromal cells; pou5f1: pou domain, class  <dig>  transcription factor 1; pparg: peroxisome proliferator-activated receptor gamma; sox2: sry -box 2; wj-msc: wharton’s jelly-derived msc; *not detected in initial sample ; **not detected in that particular sample .
negative values mean downregulation on mrna expression when compared to non-induced cultured cells, and positive values, upregulation.
statistical significance was assessed by one-way non-parametric anova followed by bonferroni post-hoc test to compare all pairs of data for a determined gene .
in a same row, a same letter means that both conditions are not significantly different; different letters  indicate statistically different values.
regarding adipogenic markers, adipoq was detected only at day  <dig> in wj-msc, but it showed the highest increase in at- and bm-msc .
highest upregulation of adipogenic markers in bm-msc and at-msc correlated with highest lipid accumulation in differentiation assays and with a lower expression levels of wj-msc.
protein secretion by adipocyte-like cells
results from cell culture supernatant quantification of secreted chemokines, pro- and anti-inflammatory cytokines, angiogenic and growth factors, extracellular matrix components and matrix metalloproteinases are shown in table  <dig>  in order to make results comparable, supernatant concentration was expressed in pg secreted per million cells and per day, since msc from different sources proliferated in significantly different rates when differentiated into the adipogenic phenotype.
final cell concentration was  <dig> .
<dig> cells/ml for bm-msc,  <dig> .
<dig> cells/ml for at-msc and  <dig> .
<dig> cells/ml for wj-msc.table  <dig>
protein secretion  of adipose tissue- , bone marrow-  and wharton’s jelly-mesenchymal stromal cells  when grown in low glucose media supplemented with 10% fetal bovine serum
bm-msc
at-msc
wj-msc
chemokines
ip-10
mcp-1
pro-inflammatory cytokines
il-6
il-7
il-8
il-12
anti-inflammatory cytokines
il-1ra
angiogenic factors
angiogenin
thrombospondin-2
plgf
afgf
vegf-d
endostatin
angiopoietin-1
vegf
growth factors
tgf-β1
tgf-β2
hgf
g-csf
pdgf-aa
extracellular matrix proteins
collagen i
collagen ii
collagen iii
collagen iv
elastin
fibronectin
heparan sulfate
laminin
aggrecan
matrix metalloproteinases
mmp1
mmp3
mmp7
mmp8
mmp13
afgf: acidic fibroblast growth factor; at-msc: adipose tissue-derived msc; bm-msc: bone marrow-derived msc; gcs-f: granulocyte-colony stimulating factor; hgf: hepatocyte growth facctor; il: interleukin; ip-10: interferon-gamma induced protein 10; mcp-1: monocyte chemotactic protein 1; mmp: metalloproteinase; nd: not determined; pdgf-aa: platelet derived growth factor aa; pg: picogram; plgf: placental growth factor; tgf: transforming growth factor; vegf: vascular endothelial growth factor; wj-msc: wharton’s jelly-derived msc.
protein concentration is expressed in pg secreted per million cells and per day.
anova and bonferroni tests  were applied for each quantified factor in order to determine significant differences between all three msc.
for a specific protein; different letters  mean statistically different expression levels.
only ip- <dig> and mcp- <dig> were detected in supernatants of cells induced to differentiate into adipocytes.
ip- <dig> secretion was higher for wj-msc, and mcp- <dig> was higher for bm-msc.
at-msc secreted the lowest concentration of both chemokines.
pro-inflammatory cytokines gm-csf, il-1β, tnf-α, ifn-γ, il- <dig>  il-2r, il- <dig> and il- <dig> were not detected in any of the supernatants of differentiated cells.
only il- <dig>  il- <dig>  il- <dig> and il- <dig> were detected in all supernatants.
again, at-msc secreted the lowest concentration of all  <dig> detected cytokines.
bm-msc secreted higher amounts of il- <dig> and il- <dig>  with concentration rates of  <dig>  ±  <dig>  and  <dig>  ±  <dig>  pg/ <dig> cells/day, respectively.
on the other side, wj-msc secreted  <dig>  ±  <dig>  pg il-6/ <dig> cells/day and  <dig> .3 ±  <dig>  pg il-8/ <dig> cells/day, higher secretion rates when compared to bm- and at-msc.
in the group of anti-inflammatory cytokines, only il-1ra was quantified in cell culture supernatants: the highest secretion rate was  <dig>  ±  <dig>  pg/ <dig> cells/day in wj-msc, but il-1ra was undetectable in at-msc supernatants.
il- <dig>  il- <dig>  il- <dig>  il- <dig> and ifn-α were not detected in any supernatant.
all the studied angiogenic factors were found in culture supernatants.
bm-msc secreted the highest concentrations of angiogenin, plgf, afgf, vegf-d, angiopoietin and vegf.
wj-msc showed the highest secretion rates for thrombospondin- <dig> and endostatin.
plgf was not detected in wj-msc supernatants and vegf-d was only detected in bm-msc cell cultures.
egf, bfgf, igf- <dig>  tgf-β <dig>  pdgf-ab and pdgf-bb were the only growth factors that were not detected in adipocyte-like cell supernatants.
g-csf was only detected in wj-msc, which also secreted the highest amounts of hgf , as compared to secretion rates of at-msc and bm-msc.
bm-msc showed the highest secretion rates of pdgf-aa  and both detected tgf forms, β <dig>  and β <dig> .
decorin and perlecan were not detected in any cell culture supernatant.
only bm-msc secreted collagen type ii and only at-msc secreted fibronectin in detectable concentrations.
wj-msc showed a poor extracellular matrix production, since several proteins were not detected in their supernatants .
wj-sc secreted the highest amounts of collagen type iii, at-msc secreted the highest amounts of collagen type i, and bm-msc collagen type iv.
bm-msc secreted highest heparan sulfate, laminin, aggrecan and elastin.
all the matrix metalloproteinases were detected in adipocyte-like cell supernatants, except for mmp <dig> in wj-msc and mmp- <dig> in at-msc.
mmp- <dig> was secreted in higher concentrations by wj-msc, which did not secrete mmp- <dig>  we could not detect mmp- <dig> in at-msc supernatants, but these cells where the best secreting-cells of mmp- <dig>  bm-msc were the best secreting cells regarding mmp- <dig>  mmp- <dig> and mmp- <dig>
discussion
the present study was performed in order to compare the msc derived from bone marrow, adipose tissue and wharton’s jelly along the in vitro differentiation into the adipocyte-like phenotype, in terms of gene expression and protein secretion into the culture supernatant.
the major objective was improving the potential protocols for the “cell-assisted lipotransfer”, obtaining a more efficient and durable gain in volume of the transplanted tissue.
in general terms, we wanted also to check differences among msc from three standard sources, often used in regenerative therapies, when submitted to the same biological induction of differentiation.
within these tissues, the msc populations have different compositions and specific functions, which may be partially maintained during in vitro expansion.
bone marrow is a traditional source of msc, in which the first mesenchymal stem cells giving rise to fibroblastoid colonies were described  <cit> .
bm-msc have multiple functions, notably the maintenance of the osteogenic cell pool  <cit>  and the provision of the stroma that sustains hematopoiesis  <cit> .
in the latter group  are found bone marrow fat-storing cells, which accumulate lipids in multilocular droplets, but have several physiological differences as compared to the typical adipose tissue adipocytes.
bone marrow also harbors multipotent mesenchymal stem cells that can be mobilized to the peripheral circulation by chemokines  <cit> , and participate in repair and regeneration in distant organs  <cit> .
adipose tissue msc are associated with the vascular stroma, where they participate in the maintenance of blood and lymphatic vessels  <cit> .
in the same location, they can give rise to preadipocytes, already engaged in adipocyte differentiation and expressing the major master genes of this pathway, but without accumulation of fat  <cit> .
wharton’s jelly contains msc that are not necessarily associated with vessels and have essentially a mechanical function  <cit> .
due to the easy access to this normally discarded tissue, the broad capacity of proliferation and the capacity of wj-msc to respond to differentiation induction  <cit> , these cells are considered to be a rich source of cells for allogeneic transplantation protocols.
as expected, the present study has shown broad differences among the msc behavior during the induction of the adipocyte-like differentiation.
regarding gene expression, at-msc showed the higher shift in the expression of adipogenic-related genes , when comparing expression after culturing cells in adipogenic media against baseline expression .
the lipid accumulation was more intense and rapid in at-msc as compared to other msc.
bm-msc were able to deposit lipids in droplets  of similar size as those found in at-msc, but the amount of lipid deposits was lower .
in contrast, wj-msc showed a more immature differentiation state at the end of the induction period , with few lipid vacuoles and little lipid accumulation.
similar results regarding lower adipogenic potential of wj-msc were reported previously by other authors  <cit> .
secretion profile of the adipocyte-like cells isolated from the three tissues would be expected to be similar, considering that they were differentiated into the same cell type, but behaviors were very different among all cells.
chemokines ip- <dig> and mcp- <dig> were found in adipocyte-like supernatants, and secretion rates were different among the three msc sources.
gerhardt and colleagues described in the year  <dig> mcp- <dig> secretion by pre-adipocytes and correlated it with macrophage recruitment  <cit> ; they demonstrated that mcp- <dig> secretion is up-regulated under inflammatory stimulation  and that secretion is reduced when pre-adypocytes are fully differentiated into adipocytes.
therefore, lower mcp- <dig> secretion by adipocyte-like cells derived from at-msc would indicate a more advanced differentiation stage.
xu and colleagues performed in vivo studies in obese mice and hypothesized that after a certain threshold of obesity, macrophage recruitment  is improved, resulting in a feedback loop where insulin signaling is impaired, leading to adipocyte lipolysis and necrosis  <cit> .
human adipocyte secretion of ip- <dig> was also confirmed and was positively correlated with body mass index  <cit> .
il- <dig> and il- <dig> secretion by adipocytes was confirmed in  <dig> and  <dig>  respectively, and plasmatic concentrations of both cytokines were positively correlated with obesity  <cit>  regarding il- <dig>  results are not conclusive: okada and coworkers reported that il- <dig> secretion was augmented after bm-msc adipogenesis  <cit> , similar to what we obtained, if we compare secretion of adipocyte-like cells derived from bm-msc and basal secretion .
on the other side, fain reported in  <dig> that non-fat cells of adipose tissue secrete higher concentrations of il- <dig>  when compared with adipocytes  <cit> .
these results are in agreement with what we are reporting here: undifferentiated at-msc showed a higher il- <dig> secretion rate than adipocyte-like cells derived from these cells  <cit> .
when looking at il- <dig>  blaber and coworkers found that il- <dig> secretion was higher in at-msc than in adipocytes, results that agree with what we found for at- and wj-msc, but not for bm-msc  <cit> .
as we reported here, il- <dig> was not detected in supernatants of human adipocytes  <cit> .
while we did not detected ifn-γ, il1β, il <dig>  tnfα, il <dig>  il <dig>  il <dig>  mip1α, bfgf, gm-csf and il <dig> in adipocyte-like cell supernatant, blaber and colleagues also reported low concentrations of these cytokines in adipocyte supernatants, suggesting a difference in secretome composition when comparing mature adipocytes with adipocyte-like cells obtained by differentiation induction of msc  <cit> .
all angiogenic factors were expressed by adipocyte-like cells and by the msc as well  <cit> .
already during embyogenesis, angiogenesis and adipogenesis are strongly related and it was previously demonstrated that at-msc, pre-adipocytes and adipocytes secrete a variety of angiongenic factors .
pro-inflammatory cytokines are also involved in angiogenesis, since they attract cells that also are able to secrete pro-angiogenic factors.
angiogenesis is highly desirable during fat transplantation, since new vasculature would allow tissue survival and less reabsortion.
therefore, adipocyte-like cells derived from bm-msc would be a good supplement for adipose tissue during transplantation.
metalloproteinases  are essential during angiogenesis and consequently during adipose tissue development.
adipose tissue secretes several mmps, like mmp- <dig> and − <dig>  and downregulation was observed for other mmps, like mmp- <dig> .
neither wj-msc nor adipocyte-like cells from wj-msc secreted mmp- <dig> in detectable concentrations; the same for mmp- <dig> in at-msc and adipocytes differentiated from them  <cit> .
bm-msc augmented mmp- <dig> and − <dig> secretion after differentiation, again indicating suitability for fat transplantation supplementation.
mmp- <dig> degrades several types of extracellular matrix proteins, like collagen types ii, iii, iv, ix, and x, proteoglycans, fibronectin, laminin, and elastin and can also activate other mmps, such as mmp- <dig>  mmp- <dig>  and mmp- <dig>
growth factors found in adipocyte-like cells were g-csf, hgf, pdgf-aa and tgf-β <dig> and β <dig>  rahimi and colleagues reported in  <dig> that pre-adipocytes and adipocytes secreted both tgf-β <dig> and β <dig> and that these growth factors negatively affected adipocyte differentiation  <cit> .
our results showed an increased tgf secretion rate for all msc differentiating into adipocyte-like cells, except for tgf-β <dig> in adipocyte-like cells derived from wj-msc.
no information regarding pdgf-aa secretion by adipocytes was found, but since this molecule is a potent mitogen for msc, its secretion is considered positive for fat transplantation.
results reported before by our group showed that non-induced bm-, at- and wj-msc secreted pdgf-bb.
in the present study, after adipogenic differentiation pdgf-bb was not detected in any supernatant, indicating a downregulation when cells enter the adipogenic differentiation pathway  <cit> .
collagen type iv is the most abundant collagen type in adipose tissue and its absence had been linked with obesity  <cit> .
considering our results, wj-msc would not be appropriate cells for fat transplantation, since neither the undifferentiated cells nor the adipocyte-like cells derived from them secreted detectable concentrations of collagen type iv.
on the other side, bm-msc did not secreted detectable concentrations of collagen type iv while undifferentiated but adipogenic media stimulated its secretion at a rate of  <dig>  ±  <dig>  pg/ <dig> cells/day.
nakajima and coworkers reported that laminin and fibronectin are also important components of adipose tissue extracellular matrix, while collagen type ii was found in an extremely low amount  <cit> .
fibronectin was not detected in any msc supernatant  <cit>  and after adipogenic differentiation, only adipocyte-like cells derived from at-msc secreted it in detectable concentrations.
laminin was secreted by bm- and at-msc in both, differentiated and undifferentiated states, but no laminin was detected in wj-msc supernatants.
collagen type ii was only quantified in adipocytes derived from bm-msc.
another important result we obtained here is that adipocyte-like cells derived from wj-msc were poor producers of extracellular matrix components and this correlated with results reported before by our group where undifferentiated wj-msc secretion was quantified  <cit> .
CONCLUSIONS
at-msc, bm-msc and wj-msc were successfully differentiated into adipogenic-like cells under appropriate stimulation by the culture media.
considering the behavior described in vitro for all three adipocyte-like cells obtained in the present work, at- and bm-msc would be good candidates for fat supplementation during transplantation.

 adipose tissue has gained intense interest in the last decades. early in the 19 s, adipose tissue was considered an agglomeration of fat-storing adipocytes surrounded by connective tissue. only in shapiro and wertheimer suggested that adipose tissue was metabolically active. since then, several studies have improved our knowledge of adipose tissue metabolic, endocrine and immunologic functions. fully differentiated adipocytes were first isolated using collagenase and flotation in by rodbell. early in the 1990s, the first evidence of adipose tissue secretory function was reported. adipocytes secrete a wide variety of proteins, including adipokines, a family of proteins exclusively secreted by adipocytes. the first adipokine described in was leptin, which confirmed that fat tissue has an important endocrine function. in zuk and colleagues described the procedure to process lipoaspirates. the same group published subsequently biochemical and molecular analysis of the adipose tissue-derived stem cells, confirming their differentiation capacity into different mesodermal cell types and the expression of specific genes. the possibility of stem cell isolation from the easily accessible and abundant adipose tissue was relevant in the stem cell and regenerative medicine field. before researchers completely understood adipose tissue biology, fat was already used in cosmetic, plastic, and reconstructive surgeries. in neuber performed the first clinical fat transplantation, using small pieces of autologous material for filling depressed scars. problems related to necrosis, cyst formation and reabsorption were usual, due to low revascularization, especially when large fat pieces were transplanted. autologous fat transplantation has several advantages: it results in a satisfactory tissue repair, avoids large incision scars, causes no immune host responses, and avoids use of prosthesis. its major disadvantage is the apparently unpredictable survival of the transplanted fat, since variable resorption rates have been reported. in order to obtain the desired final volume of implant, multiple interventions may be required. mature adipocytes are fragile cells, and a large part may suffer extensive lesions with a loss of membrane integrity and release of fat. sydney coleman developed in an improved method for fat transplantation that minimizes the stress during tissue collection, by recommending transplantation of small fat tissue fragments in multiple steps, in order to facilitate revascularization and, therefore, tissue survival. this tissue processing, called coleman’s method, resulted in reduced graft resorption. the discovery of stem cells in adipose tissue by zuk and colleagues in complemented the understanding of the durability of fat transfer. matsumoto and coworkers developed in the year a new technique to reduce tissue resorption, using the “cell-assisted lipotransfer”. the technique consisted in separating a part of the aspirated fat in a stromal vascular fraction enriched in msc. the lipotransfer containing these cells allowed a higher survival and vascularization of the graft in pre-clinical and clinical trials. further studies have proposed alternative procedures to manipulate lipoaspirates, and overall recommendations for handling of samples and for characterization of transferred cells. the msc content in primary cell suspensions collected from lipoaspirates is however low. immunophenotyping of cells in the stromal fraction indicates presence of blood-vessel components and associated elements, as well as pre-adipocytes and msc. the latter two are characterized as cd45neg, cd34pos and/or cd105pos, and their content in the stromal fraction was reported to be below 5%. rigotti and coworkers reported in the year that the content of msc in the adipose tissue stromal fraction was in order of 1%, but importantly, the level of clonogenic colony-forming units that possibly better reflects the quantity of cells able to participate in long-term tissue regeneration was tenfold lower. in contrast, when selected by plastic adhesion and expanded in vitro, msc are selected and can represent 70-90% of the harvested cells. the use of these cells offers new cues in regenerative procedures using much higher levels of msc in the “cell-assisted lipotransfers”. the msc used in this procedure can be autologous, requiring a first harvest of fat tissue to establish cultures, and a second one to provide the tissue for the graft. when the donor adipose tissue is limited, alternative sources of msc may be used, associated with autologous fat graft, in order to grant a long-term restoration of fat tissue under controlled conditions, maintaining the volume and the implant’s form. among others, this may be the case of facial fat restoration in lipodystrophies or in chronic hiv infection. we hypothesized that msc from different origins other than adipose tissue can be used in assisting fat transplantation, especially if they show a high adipogenic differentiation potential. msc obtained from the wharton’s jelly of the umbilical cord, a convenient source of cells with high proliferation capacity, may be proposed for allogeneic transplantation. therefore, we characterized msc obtained from different sources regarding their ability to differentiate into adipocytes. characterization of msc and their ability to differentiate into adipocytes may be relevant in many other fields of regenerative medicine. novel proposals for tendon and muscle regeneration deal with the use of msc for repair of such lesions of shoulder, which are particularly sensitive to the fatty degeneration of muscles. this is generally an irreversible process, in which adipocytes invade and replace the muscle tissue. the origin of these adipocytes is a differentiation from resident msc, which in the absence of mechanical stimulation by muscle contraction suffer a decrease of wnt signaling, leading to transcription of adipogenic factors such as pparγ, c/ebpα and leptin. knowledge of which msc already express, or easily activate expression of adipogenic factors, and readily differentiate into adipocytes, may be relevant for the choice of msc to be used in such a therapy. in the present study, we compared adipogenic differentiation in vitro of msc derived from bone marrow, adipose tissue and wharton’s jelly. our main goal is to determine which source of msc is more appropriate for allogeneic transplantation, so they can be use together with adipose tissue for long-lasting effects after fat transplantation. we quantified the gene expression of key pluripotent and differentiation markers, and the secretion of different proteins, and we compared these results with those recently obtained by our group regarding protein secretion of undifferentiated bm-msc, at-msc and wj-msc.  msc obtained from human bone marrow, adipose tissue and wharton’s jelly identity was confirmed: they adhered to the plastic surfaces of cell culture flasks with a fibroblast–like morphology, expressed the cell surface markers cd cd and cd and negative for hematopoietic markers. msc also differentiated into all three mesodermal lineages: adipocytes, osteoblasts and chondroblasts. all msc cultured in dmem supplemented with fbs and with the compounds recommended for adipocyte induction differentiated into adipocyte-like cells. differentiation controls maintained in the standard medium were run in parallel for all conditions, and they were all negative, not presenting any visible lipid accumulation. gene expression during differentiation into adipocyte-like cells gene expression of pluripotent and adipogenic markers were studied, evaluating cell differentiation effects on gene expression. appropriate reference genes were rpl13a for wj-msc and at-msc, and hprt for bm-msc. tert expression was not detected in any sample. published data are contradictory regarding tert expression: some authors described tert mrna as a pluripotent marker in bm-msc but many others detected neither tert mrna nor tert activity in msc primary cultures. data on mrna expression during adipogenic differentiation are shown in table  a statistical analysis was performed comparing the expression of one gene for all cells and time points evaluated. for all the genes, at-msc at day expressed statistically significant higher amounts of the corresponding mrna. regarding pluripotent markers, only pou5f was downregulated in bm-msc cultures.table relative gene expression for human adipose tissue-, bone marrow- and wharton’s jelly-mesenchymal stromal cells when grown in low glucose media supplemented with 10% fetal bovine serum bm-msc at-msc wj-msc d10 d21 d10 d21 d10 d21 pparg adipoq cebpa sox2 pou5f1 adipoq: adiponectin; at-msc: adipose tissue-derived msc; bm-msc: bone marrow-derived msc; cepba: ccaat/enhancer-binding protein alpha; d10: day 10; d21: day 21; msc: mesenchymal stromal cells; pou5f1: pou domain, class transcription factor 1; pparg: peroxisome proliferator-activated receptor gamma; sox2: sry -box 2; wj-msc: wharton’s jelly-derived msc; *not detected in initial sample; **not detected in that particular sample. negative values mean downregulation on mrna expression when compared to non-induced cultured cells, and positive values, upregulation. statistical significance was assessed by one-way non-parametric anova followed by bonferroni post-hoc test to compare all pairs of data for a determined gene. in a same row, a same letter means that both conditions are not significantly different; different letters indicate statistically different values. regarding adipogenic markers, adipoq was detected only at day in wj-msc, but it showed the highest increase in at- and bm-msc. highest upregulation of adipogenic markers in bm-msc and at-msc correlated with highest lipid accumulation in differentiation assays and with a lower expression levels of wj-msc. protein secretion by adipocyte-like cells results from cell culture supernatant quantification of secreted chemokines, pro- and anti-inflammatory cytokines, angiogenic and growth factors, extracellular matrix components and matrix metalloproteinases are shown in table  in order to make results comparable, supernatant concentration was expressed in pg secreted per million cells and per day, since msc from different sources proliferated in significantly different rates when differentiated into the adipogenic phenotype. final cell concentration was. cells/ml for bm-msc,. cells/ml for at-msc and. cells/ml for wj-msc.table protein secretion of adipose tissue-, bone marrow- and wharton’s jelly-mesenchymal stromal cells when grown in low glucose media supplemented with 10% fetal bovine serum bm-msc at-msc wj-msc chemokines ip-10 mcp-1 pro-inflammatory cytokines il-6 il-7 il-8 il-12 anti-inflammatory cytokines il-1ra angiogenic factors angiogenin thrombospondin-2 plgf afgf vegf-d endostatin angiopoietin-1 vegf growth factors tgf-β1 tgf-β2 hgf g-csf pdgf-aa extracellular matrix proteins collagen i collagen ii collagen iii collagen iv elastin fibronectin heparan sulfate laminin aggrecan matrix metalloproteinases mmp1 mmp3 mmp7 mmp8 mmp13 afgf: acidic fibroblast growth factor; at-msc: adipose tissue-derived msc; bm-msc: bone marrow-derived msc; gcs-f: granulocyte-colony stimulating factor; hgf: hepatocyte growth facctor; il: interleukin; ip-10: interferon-gamma induced protein 10; mcp-1: monocyte chemotactic protein 1; mmp: metalloproteinase; nd: not determined; pdgf-aa: platelet derived growth factor aa; pg: picogram; plgf: placental growth factor; tgf: transforming growth factor; vegf: vascular endothelial growth factor; wj-msc: wharton’s jelly-derived msc. protein concentration is expressed in pg secreted per million cells and per day. anova and bonferroni tests were applied for each quantified factor in order to determine significant differences between all three msc. for a specific protein; different letters mean statistically different expression levels. only ip- and mcp- were detected in supernatants of cells induced to differentiate into adipocytes. ip- secretion was higher for wj-msc, and mcp- was higher for bm-msc. at-msc secreted the lowest concentration of both chemokines. pro-inflammatory cytokines gm-csf, il-1β, tnf-α, ifn-γ, il- il-2r, il- and il- were not detected in any of the supernatants of differentiated cells. only il- il- il- and il- were detected in all supernatants. again, at-msc secreted the lowest concentration of all detected cytokines. bm-msc secreted higher amounts of il- and il- with concentration rates of  ±  and  ±   pg/ cells/day, respectively. on the other side, wj-msc secreted  ±   pg il-6/ cells/day and.3 ±   pg il-8/ cells/day, higher secretion rates when compared to bm- and at-msc. in the group of anti-inflammatory cytokines, only il-1ra was quantified in cell culture supernatants: the highest secretion rate was  ±   pg/ cells/day in wj-msc, but il-1ra was undetectable in at-msc supernatants. il- il- il- il- and ifn-α were not detected in any supernatant. all the studied angiogenic factors were found in culture supernatants. bm-msc secreted the highest concentrations of angiogenin, plgf, afgf, vegf-d, angiopoietin and vegf. wj-msc showed the highest secretion rates for thrombospondin- and endostatin. plgf was not detected in wj-msc supernatants and vegf-d was only detected in bm-msc cell cultures. egf, bfgf, igf- tgf-β pdgf-ab and pdgf-bb were the only growth factors that were not detected in adipocyte-like cell supernatants. g-csf was only detected in wj-msc, which also secreted the highest amounts of hgf, as compared to secretion rates of at-msc and bm-msc. bm-msc showed the highest secretion rates of pdgf-aa and both detected tgf forms, β and β. decorin and perlecan were not detected in any cell culture supernatant. only bm-msc secreted collagen type ii and only at-msc secreted fibronectin in detectable concentrations. wj-msc showed a poor extracellular matrix production, since several proteins were not detected in their supernatants. wj-sc secreted the highest amounts of collagen type iii, at-msc secreted the highest amounts of collagen type i, and bm-msc collagen type iv. bm-msc secreted highest heparan sulfate, laminin, aggrecan and elastin. all the matrix metalloproteinases were detected in adipocyte-like cell supernatants, except for mmp in wj-msc and mmp- in at-msc. mmp- was secreted in higher concentrations by wj-msc, which did not secrete mmp- we could not detect mmp- in at-msc supernatants, but these cells where the best secreting-cells of mmp- bm-msc were the best secreting cells regarding mmp- mmp- and mmp- discussion the present study was performed in order to compare the msc derived from bone marrow, adipose tissue and wharton’s jelly along the in vitro differentiation into the adipocyte-like phenotype, in terms of gene expression and protein secretion into the culture supernatant. the major objective was improving the potential protocols for the “cell-assisted lipotransfer”, obtaining a more efficient and durable gain in volume of the transplanted tissue. in general terms, we wanted also to check differences among msc from three standard sources, often used in regenerative therapies, when submitted to the same biological induction of differentiation. within these tissues, the msc populations have different compositions and specific functions, which may be partially maintained during in vitro expansion. bone marrow is a traditional source of msc, in which the first mesenchymal stem cells giving rise to fibroblastoid colonies were described. bm-msc have multiple functions, notably the maintenance of the osteogenic cell pool and the provision of the stroma that sustains hematopoiesis. in the latter group are found bone marrow fat-storing cells, which accumulate lipids in multilocular droplets, but have several physiological differences as compared to the typical adipose tissue adipocytes. bone marrow also harbors multipotent mesenchymal stem cells that can be mobilized to the peripheral circulation by chemokines, and participate in repair and regeneration in distant organs. adipose tissue msc are associated with the vascular stroma, where they participate in the maintenance of blood and lymphatic vessels. in the same location, they can give rise to preadipocytes, already engaged in adipocyte differentiation and expressing the major master genes of this pathway, but without accumulation of fat. wharton’s jelly contains msc that are not necessarily associated with vessels and have essentially a mechanical function. due to the easy access to this normally discarded tissue, the broad capacity of proliferation and the capacity of wj-msc to respond to differentiation induction, these cells are considered to be a rich source of cells for allogeneic transplantation protocols. as expected, the present study has shown broad differences among the msc behavior during the induction of the adipocyte-like differentiation. regarding gene expression, at-msc showed the higher shift in the expression of adipogenic-related genes, when comparing expression after culturing cells in adipogenic media against baseline expression. the lipid accumulation was more intense and rapid in at-msc as compared to other msc. bm-msc were able to deposit lipids in droplets of similar size as those found in at-msc, but the amount of lipid deposits was lower. in contrast, wj-msc showed a more immature differentiation state at the end of the induction period, with few lipid vacuoles and little lipid accumulation. similar results regarding lower adipogenic potential of wj-msc were reported previously by other authors. secretion profile of the adipocyte-like cells isolated from the three tissues would be expected to be similar, considering that they were differentiated into the same cell type, but behaviors were very different among all cells. chemokines ip- and mcp- were found in adipocyte-like supernatants, and secretion rates were different among the three msc sources. gerhardt and colleagues described in the year mcp- secretion by pre-adipocytes and correlated it with macrophage recruitment; they demonstrated that mcp- secretion is up-regulated under inflammatory stimulation and that secretion is reduced when pre-adypocytes are fully differentiated into adipocytes. therefore, lower mcp- secretion by adipocyte-like cells derived from at-msc would indicate a more advanced differentiation stage. xu and colleagues performed in vivo studies in obese mice and hypothesized that after a certain threshold of obesity, macrophage recruitment is improved, resulting in a feedback loop where insulin signaling is impaired, leading to adipocyte lipolysis and necrosis. human adipocyte secretion of ip- was also confirmed and was positively correlated with body mass index. il- and il- secretion by adipocytes was confirmed in and respectively, and plasmatic concentrations of both cytokines were positively correlated with obesity regarding il- results are not conclusive: okada and coworkers reported that il- secretion was augmented after bm-msc adipogenesis, similar to what we obtained, if we compare secretion of adipocyte-like cells derived from bm-msc and basal secretion. on the other side, fain reported in that non-fat cells of adipose tissue secrete higher concentrations of il- when compared with adipocytes. these results are in agreement with what we are reporting here: undifferentiated at-msc showed a higher il- secretion rate than adipocyte-like cells derived from these cells. when looking at il- blaber and coworkers found that il- secretion was higher in at-msc than in adipocytes, results that agree with what we found for at- and wj-msc, but not for bm-msc. as we reported here, il- was not detected in supernatants of human adipocytes. while we did not detected ifn-γ, il1β, il tnfα, il il il mip1α, bfgf, gm-csf and il in adipocyte-like cell supernatant, blaber and colleagues also reported low concentrations of these cytokines in adipocyte supernatants, suggesting a difference in secretome composition when comparing mature adipocytes with adipocyte-like cells obtained by differentiation induction of msc. all angiogenic factors were expressed by adipocyte-like cells and by the msc as well. already during embyogenesis, angiogenesis and adipogenesis are strongly related and it was previously demonstrated that at-msc, pre-adipocytes and adipocytes secrete a variety of angiongenic factors. pro-inflammatory cytokines are also involved in angiogenesis, since they attract cells that also are able to secrete pro-angiogenic factors. angiogenesis is highly desirable during fat transplantation, since new vasculature would allow tissue survival and less reabsortion. therefore, adipocyte-like cells derived from bm-msc would be a good supplement for adipose tissue during transplantation. metalloproteinases are essential during angiogenesis and consequently during adipose tissue development. adipose tissue secretes several mmps, like mmp- and − and downregulation was observed for other mmps, like mmp-. neither wj-msc nor adipocyte-like cells from wj-msc secreted mmp- in detectable concentrations; the same for mmp- in at-msc and adipocytes differentiated from them. bm-msc augmented mmp- and − secretion after differentiation, again indicating suitability for fat transplantation supplementation. mmp- degrades several types of extracellular matrix proteins, like collagen types ii, iii, iv, ix, and x, proteoglycans, fibronectin, laminin, and elastin and can also activate other mmps, such as mmp- mmp- and mmp- growth factors found in adipocyte-like cells were g-csf, hgf, pdgf-aa and tgf-β and β rahimi and colleagues reported in that pre-adipocytes and adipocytes secreted both tgf-β and β and that these growth factors negatively affected adipocyte differentiation. our results showed an increased tgf secretion rate for all msc differentiating into adipocyte-like cells, except for tgf-β in adipocyte-like cells derived from wj-msc. no information regarding pdgf-aa secretion by adipocytes was found, but since this molecule is a potent mitogen for msc, its secretion is considered positive for fat transplantation. results reported before by our group showed that non-induced bm-, at- and wj-msc secreted pdgf-bb. in the present study, after adipogenic differentiation pdgf-bb was not detected in any supernatant, indicating a downregulation when cells enter the adipogenic differentiation pathway. collagen type iv is the most abundant collagen type in adipose tissue and its absence had been linked with obesity. considering our results, wj-msc would not be appropriate cells for fat transplantation, since neither the undifferentiated cells nor the adipocyte-like cells derived from them secreted detectable concentrations of collagen type iv. on the other side, bm-msc did not secreted detectable concentrations of collagen type iv while undifferentiated but adipogenic media stimulated its secretion at a rate of  ±   pg/ cells/day. nakajima and coworkers reported that laminin and fibronectin are also important components of adipose tissue extracellular matrix, while collagen type ii was found in an extremely low amount. fibronectin was not detected in any msc supernatant and after adipogenic differentiation, only adipocyte-like cells derived from at-msc secreted it in detectable concentrations. laminin was secreted by bm- and at-msc in both, differentiated and undifferentiated states, but no laminin was detected in wj-msc supernatants. collagen type ii was only quantified in adipocytes derived from bm-msc. another important result we obtained here is that adipocyte-like cells derived from wj-msc were poor producers of extracellular matrix components and this correlated with results reported before by our group where undifferentiated wj-msc secretion was quantified.  at-msc, bm-msc and wj-msc were successfully differentiated into adipogenic-like cells under appropriate stimulation by the culture media. considering the behavior described in vitro for all three adipocyte-like cells obtained in the present work, at- and bm-msc would be good candidates for fat supplementation during transplantation. 
we quantified the expression of stem cell and adipocyte genetic markers using quantitative real time pcr, as well as the secretion of cytokines, extracellular matrix components and growth factors using luminex and elisa. adipocyte-like cells obtained from bm-msc secreted high amounts of most angiogenic factors, growth factors tgf-β and tgf-β collagens type ii and iv, heparan sulfate, laminin and aggrecan. mesenchymal stromal cells can be obtained from potentially any tissue from the human body, but cells purified from different sources are undoubtedly different, and for each medical application, the msc with the best regenerative potential should be chosen. bone marrow-derived mesenchymal stromal cells, adipose tissue-derived mesenchymal stromal cells and wharton’s jelly-derived mesenchymal stromal cells were isolated from human tissues and were cultured under differentiation media supplemented with fetal bovine serum. mesenchymal stromal cells purified from different tissues have a different behavior when induced to differentiate into adipocyte-like cells. human mesenchymal stromal cellsadipogenesisqpcrprotein quantificationissue-copyright-statement© the author 2014 regarding protein secretion, adipocyte-like cells generated from wj-msc secreted the highest chemokine levels. at-msc-derived adipocyte-like cells secreted the lowest cytokine amounts and the highest quantity of collagen types i and iii. at-msc showed the highest shift in adipoq, cebpa and pparg mrna expression. bm-msc kept high expression levels of stem-cell markers sox and pou5f wj-msc showed the lowest increase in mrna expression when cells were induced to differentiate into adipocytes.
 mesenchymal stromal cells can be obtained from potentially any tissue from the human body, but cells purified from different sources are undoubtedly different, and for each medical application, the msc with the best regenerative potential should be chosen.  bone marrow-derived mesenchymal stromal cells, adipose tissue-derived mesenchymal stromal cells and wharton’s jelly-derived mesenchymal stromal cells were isolated from human tissues and were cultured under differentiation media supplemented with fetal bovine serum. we quantified the expression of stem cell and adipocyte genetic markers using quantitative real time pcr, as well as the secretion of cytokines, extracellular matrix components and growth factors using luminex and elisa. all three msc differentiated into adipogenic cells. at-msc showed the highest shift in adipoq, cebpa and pparg mrna expression. bm-msc kept high expression levels of stem-cell markers sox and pou5f wj-msc showed the lowest increase in mrna expression when cells were induced to differentiate into adipocytes. regarding protein secretion, adipocyte-like cells generated from wj-msc secreted the highest chemokine levels. at-msc-derived adipocyte-like cells secreted the lowest cytokine amounts and the highest quantity of collagen types i and iii. adipocyte-like cells obtained from bm-msc secreted high amounts of most angiogenic factors, growth factors tgf-β and tgf-β collagens type ii and iv, heparan sulfate, laminin and aggrecan.  mesenchymal stromal cells purified from different tissues have a different behavior when induced to differentiate into adipocyte-like cells. keywords human mesenchymal stromal cellsadipogenesisqpcrprotein quantificationissue-copyright-statement© the author 2014 