BACKGROUND
obesity, which is characterized by an excess of adipose mass, is a major public health-problem.
hypertrophy, i.e.
increase in the adipocyte size and hyperplasia, i.e.
increase in the adipocyte numbers, are observed in severe obesity.
it is now well established that multipotent stem cells exist within adipose tissue throughout the life  <cit>  and that an excessive recruitment of these adipose precursor cells could lead to hyperplasia.
as opposed to hypertrophy, hypoplasia of adipose tissue is observed in lipodystrophy and is associated with diabetes and hyperlipidaemia.
adipocytes and osteoblasts share the same mesenchymal precursor cell  <cit> .
adipogenesis and osteogenesis are processes that respond to a balance in bone marrow and this balance can be disrupted under pathological conditions such as osteoporosis where adipocytes develop at the expense of osteoblasts  <cit> .
therefore, pharmacological molecules that control the pool of adipose stem cells are of great interest.
glycogen synthase kinase  <dig> , a serine/threonine kinase existing in two isoforms gsk3α and gsk3β, is a key regulator of numerous signalling pathways.
in particular, gsk <dig> has been involved in multiple cellular processes including wnt and hedgehog  pathways.
in the activation of the canonical wnt pathway, inhibition of gsk <dig> results in dephosphorylation of β-catenin leading to its nuclear accumulation.
inhibition of gsk <dig> also contributes to activation of the hh pathway by stabilisation of gli 2/ <dig> transcription factors, favouring their nuclear translocation and leading to transcription of target genes.
gli <dig> is one of them and induction of gli <dig> gene expression has been characterized as a reliable marker of hh signalling activity  <cit> .
the role of gsk <dig> in the differentiation of preadipose cells has been previously described.
it has been shown that activation of the wnt pathway via inhibition of gsk <dig> inhibits adipogenesis of murine preadipocytes and in mice  <cit> .
expression of hh target genes was reduced in fat depots of obese mice, suggesting anti-adipogenic properties of this pathway  <cit> .
gsk <dig> is also a key component of the circadian apparatus.
the circadian clock may play a role in adipocyte metabolism and it has been recently shown that inhibition of gsk <dig> in human adipocytes lengthened the period of expression of core circadian transcriptional apparatus  <cit> .
therefore, gsk <dig> could represent a potential therapeutic target to modulate proliferation and differentiation of adipocyte precursors.
however, the impact of gsk <dig> inhibition on human adipose-derived stem cells remained to be investigated.
to address this point we have studied the effects of two pharmacological inhibitors of gsk <dig>  lithium chloride   <cit>  and 6-bromoindirubin-3'-oxime   <cit> , on multipotent stem cells derived form human adipose tissue   <cit> .
thus, hmads cells represent a potent cellular model to investigate pathways regulating self-renewal, adipogenesis and osteogenesis  <cit> .
RESULTS
functional inhibition of gsk <dig> in human adipose-derived stem cells
in order to determine the effective concentrations of bio and licl, nuclear translocation of β-catenin and induction of gli <dig> gene expression have been analyzed in hmads <dig> and hmads <dig> cells, two stem cell populations isolated from separate donors.
as shown in fig 1a, stimulation of hmads <dig> cells with  <dig>  μm bio led to nuclear accumulation of β-catenin, whereas the inactive bio molecule  had no effect.
under our culture conditions,  <dig>  μm bio had no effect on β-catenin stabilization and  <dig> μm led to cell death .
treatment of cells with  <dig> mm licl had the same effect than  <dig>  μm bio whereas  <dig> mm licl and  <dig> mm sodium chloride  had no effect .
nucleofection of hmads <dig> cells with a reporter gene under the control of tcf/lef response-element showed that the β-catenin pathway was activated in hmads cells after inhibiting gsk <dig> .
gli <dig> gene expression was also stimulated in hmads <dig> and hmads <dig> cells that were maintained in the presence of  <dig>  μm bio or  <dig> mm licl .
effect of gsk <dig> inhibitors on proliferation of human adipose-derived stem cells
then, the functional consequence of gsk <dig> inhibition on proliferation of hmads cells has been investigated.
morphology of hmads cells changed after treatment with  <dig>  μm bio or  <dig> mm licl.
from a fibroblastic-like morphology, gsk <dig> inhibitor-treated cells displayed a cuboidal shape .
we did not observe any change in morphology or any effect on proliferation when cells were treated with  <dig>  μm bio .
in contrast, at a higher concentration of  <dig>  μm bio, treatment of hmads <dig> cells for  <dig> days led to a 40–50% inhibition of proliferation.
effects of bio on cell morphology and proliferation were reversible indicating that the gsk <dig> inhibitor did not affect cell viability .
mebio-treated cells did not display any significant inhibition of proliferation.
the inhibitory effect of bio was also observed after treatment of mesenchymal stem cells isolated from human bone-marrow  .
bio-inhibitory effect was detected as early as  <dig> days after treatment and was reproducible on hmads <dig> cells .
inhibition on hmads <dig> cell proliferation was also observed to a similar extent after treatment with  <dig> mm licl but not with nacl .
as shown in fig.
<dig>  bio inhibited the ability of hmads cells and hmscs to proliferate at the single cell level, a crucial feature of stem cells.
this indicates that bio cannot be used to expand mesenchymal stem cells ex vivo.
this is in contrast with a previous report showing that bio was able to maintain pluripotent embryonic stem cells by activating wnt pathway  <cit>  and that inhibition of β-catenin promotes proliferation of uncharacterized stromal cells of adipose tissue  <cit> .
these discrepancies could be due to a differential effect of wnt pathway in embryonic versus human adult mesenchymal stem cells or/and to the fact that, as shown above, bio activated both wnt and hh pathways within the same cell population.
the effect of hh pathway on proliferation on hmads cells remains to be investigated.
in addition, as also shown above, gsk <dig> is involved in several pathways and we cannot rule out that bio could have other targets than gsk <dig> in hmads cells.
altogether, these data show that bio inhibited the proliferation of human stem cells, strongly suggesting that gsk <dig> inhibitors could be potent regulators of the pool of adipocyte precursors in adipose tissue.
effects of bio on differentiation of human adipose-derived stem cells
to investigate the effect of gsk <dig> inhibition on adipocyte differentiation, hmads cells were induced to differentiate in the presence of bio or mebio.
adipocytes and osteoblasts share the same mesenchymal precursor cell.
adipogenesis and osteogenesis are processes that respond to a balance in bone marrow and this balance can be disrupted under pathological conditions such as osteoporosis in which adipocytes develop at the expense of osteoblasts  <cit> .
we took advantage of hmads cells that were previously demonstrated to differentiate in vitro into functional adipocytes and osteoblasts  <cit> , to investigate the effect of gsk <dig> inhibition on both lineages.
adipogenic and osteogenic differentiations, as indicated by oil red o and alizarin staining respectively, were dramatically inhibited in the presence of bio, whereas mebio did not display any significant effect .
bio-inhibitory effect was quantified using enzymatic activities such as glycerol-3-dehydrogenase , which is expressed in adipocytes but not in osteoblasts, and alkaline phosphatase  activity, which is expressed in osteoblasts but not in adipocytes.
as shown in fig.
<dig>  gpdh activity was inhibited when hmads cells were maintained for  <dig> days in adipogenic medium or in a medium allowing simultaneously adipogenic and osteogenic differentiations supplemented with bio.
alp activity was also inhibited when cells were maintained in osteogenic or adipogenic/osteogenic media supplemented with the gsk <dig> inhibitor .
then, we investigated the impact of bio treatment during cell proliferation on subsequent differentiation.
quantification of gpdh enzymatic activity indicated that cells previously treated with bio during proliferation displayed a lower capacity to undergo adipocyte differentiation than untreated- or mebio-treated cells .
this result indicates that gsk <dig> plays a role in maintaining the differentiation potential of undifferentiated hmads cells.
this is reminiscent of our previous work showing the critical role of fgf <dig> pathway on self-renewal of hmads cells  <cit> .
finally, analysis of adipocyte- and osteoblast-gene expression confirmed that treatment of hmads cells with of bio during differentiation led to the inhibition of both differentiation programs.
as shown in fig.
<dig>  all of the genes known to be expressed during adipocyte differentiation that we have analyzed were inhibited in the presence of bio.
genes expressed during osteogenesis were also inhibited by bio, although at a lower extent compared to adipogenesis-related genes.
altogether, these results are in agreement with previous reports showing that activation of wnt or hh pathways precludes differentiation of murine 3t3-l <dig> preadipose cells into adipocytes  <cit> .
our data indicate that the gsk <dig> inhibitor was a potent inhibitor of commitment of human preadipocyte precursors into the adipogenic lineage.
the effect of wnt pathway on osteogenesis remains more controversial.
wnt has been reported to enhance osteogenic differentiation of murine mesenchymal stem cells  <cit> .
more recently, cho et al.
have shown that wnt signalling suppresses osteogenic differentiation of stroma cells isolated from human adipose tissue  <cit> .
therefore, the effect of wnt signalling on bone formation seems different in human versus mouse and our data with bio-treated hmads cells are in agreement with a negative effect of wnt on osteogenesis of human cells.
it has been observed that patients treated with licl for bipolar disorder display weight gain and reduced risk of fractures, suggesting that licl promotes both adipogenesis and osteogenesis in humans in vivo  <cit> .
however, a central effect of licl and an indirect effect on adipocytes and osteoblasts in these patients cannot be excluded.
differential effects of bio treatment during early steps of adipogenesis and osteogenesis
then, we analyzed bio-inhibition on adipogenic and osteogenic differentiations of hmads cells more precisely in order to address whether a chronic treatment with the inhibitor was required.
for that purpose, we treated hmads cells during the first  <dig> days of differentiation and analyzed the impact of this treatment on gpdh and alp enzymatic activities as well as on the expression of specific markers.
in regards to adipogenesis, treatment of hmads cells for the first  <dig> days only was sufficient to inhibit gpdh activity measured at day  <dig> .
analysis of adipocyte gene expression confirmed that the inhibitory effect of bio after  <dig> days of treatment persisted at day  <dig> for most of the genes analyzed .
in contrast, a treatment restricted to the first  <dig> days had no effect on both osteogenic markers such as alp enzymatic activity and expression of several genes known to play a role in osteogenesis.
altogether, data indicate that treatment of hmads cells with bio during the early step of differentiation only led to inhibition of late steps of adipogenesis, whereas it had no effect on osteogenesis.
this strongly suggests that the molecular mechanisms underlying inhibition of adipogenic and osteogenic differentiation by bio are different.
identification of mechanisms leading to the differential effects on adipogenesis and osteogenesis of hmads cells could lead to a preferential use of the gsk <dig> inhibitors on adipocyte differentiation in vivo.
in conclusion, our data strongly suggest that gsk <dig> is a promising pharmacological target to regulate both the number and differentiation of adipocyte precursors in human adipose tissue.
however, we have to keep in mind that potential adverse effects could be observed due to the fact that gsk <dig> is implicated in numerous signalling pathways.
therefore, it would be important in the future to identify the signalling pathway mediating the adipogenic effect of gsk <dig>
CONCLUSIONS
we have shown that bio and licl, two inhibitors of gsk <dig>  inhibited proliferation as well as adipogenic and osteogenic differentiations of stem cells isolated from human adipose tissue.
data demonstrate the feasibility of a pharmacological approach to regulate adipose-derived stem cell function.

 obesity, which is characterized by an excess of adipose mass, is a major public health-problem. hypertrophy, i.e. increase in the adipocyte size and hyperplasia, i.e. increase in the adipocyte numbers, are observed in severe obesity. it is now well established that multipotent stem cells exist within adipose tissue throughout the life and that an excessive recruitment of these adipose precursor cells could lead to hyperplasia. as opposed to hypertrophy, hypoplasia of adipose tissue is observed in lipodystrophy and is associated with diabetes and hyperlipidaemia. adipocytes and osteoblasts share the same mesenchymal precursor cell. adipogenesis and osteogenesis are processes that respond to a balance in bone marrow and this balance can be disrupted under pathological conditions such as osteoporosis where adipocytes develop at the expense of osteoblasts. therefore, pharmacological molecules that control the pool of adipose stem cells are of great interest. glycogen synthase kinase, a serine/threonine kinase existing in two isoforms gsk3α and gsk3β, is a key regulator of numerous signalling pathways. in particular, gsk has been involved in multiple cellular processes including wnt and hedgehog pathways. in the activation of the canonical wnt pathway, inhibition of gsk results in dephosphorylation of β-catenin leading to its nuclear accumulation. inhibition of gsk also contributes to activation of the hh pathway by stabilisation of gli 2/ transcription factors, favouring their nuclear translocation and leading to transcription of target genes. gli is one of them and induction of gli gene expression has been characterized as a reliable marker of hh signalling activity. the role of gsk in the differentiation of preadipose cells has been previously described. it has been shown that activation of the wnt pathway via inhibition of gsk inhibits adipogenesis of murine preadipocytes and in mice. expression of hh target genes was reduced in fat depots of obese mice, suggesting anti-adipogenic properties of this pathway. gsk is also a key component of the circadian apparatus. the circadian clock may play a role in adipocyte metabolism and it has been recently shown that inhibition of gsk in human adipocytes lengthened the period of expression of core circadian transcriptional apparatus. therefore, gsk could represent a potential therapeutic target to modulate proliferation and differentiation of adipocyte precursors. however, the impact of gsk inhibition on human adipose-derived stem cells remained to be investigated. to address this point we have studied the effects of two pharmacological inhibitors of gsk lithium chloride and 6-bromoindirubin-3'-oxime, on multipotent stem cells derived form human adipose tissue. thus, hmads cells represent a potent cellular model to investigate pathways regulating self-renewal, adipogenesis and osteogenesis.  functional inhibition of gsk in human adipose-derived stem cells in order to determine the effective concentrations of bio and licl, nuclear translocation of β-catenin and induction of gli gene expression have been analyzed in hmads and hmads cells, two stem cell populations isolated from separate donors. as shown in fig 1a, stimulation of hmads cells with μm bio led to nuclear accumulation of β-catenin, whereas the inactive bio molecule had no effect. under our culture conditions, μm bio had no effect on β-catenin stabilization and μm led to cell death. treatment of cells with mm licl had the same effect than μm bio whereas mm licl and mm sodium chloride had no effect. nucleofection of hmads cells with a reporter gene under the control of tcf/lef response-element showed that the β-catenin pathway was activated in hmads cells after inhibiting gsk. gli gene expression was also stimulated in hmads and hmads cells that were maintained in the presence of μm bio or mm licl. effect of gsk inhibitors on proliferation of human adipose-derived stem cells then, the functional consequence of gsk inhibition on proliferation of hmads cells has been investigated. morphology of hmads cells changed after treatment with μm bio or mm licl. from a fibroblastic-like morphology, gsk inhibitor-treated cells displayed a cuboidal shape. we did not observe any change in morphology or any effect on proliferation when cells were treated with μm bio. in contrast, at a higher concentration of μm bio, treatment of hmads cells for days led to a 40–50% inhibition of proliferation. effects of bio on cell morphology and proliferation were reversible indicating that the gsk inhibitor did not affect cell viability. mebio-treated cells did not display any significant inhibition of proliferation. the inhibitory effect of bio was also observed after treatment of mesenchymal stem cells isolated from human bone-marrow. bio-inhibitory effect was detected as early as days after treatment and was reproducible on hmads cells. inhibition on hmads cell proliferation was also observed to a similar extent after treatment with mm licl but not with nacl. as shown in fig. bio inhibited the ability of hmads cells and hmscs to proliferate at the single cell level, a crucial feature of stem cells. this indicates that bio cannot be used to expand mesenchymal stem cells ex vivo. this is in contrast with a previous report showing that bio was able to maintain pluripotent embryonic stem cells by activating wnt pathway and that inhibition of β-catenin promotes proliferation of uncharacterized stromal cells of adipose tissue. these discrepancies could be due to a differential effect of wnt pathway in embryonic versus human adult mesenchymal stem cells or/and to the fact that, as shown above, bio activated both wnt and hh pathways within the same cell population. the effect of hh pathway on proliferation on hmads cells remains to be investigated. in addition, as also shown above, gsk is involved in several pathways and we cannot rule out that bio could have other targets than gsk in hmads cells. altogether, these data show that bio inhibited the proliferation of human stem cells, strongly suggesting that gsk inhibitors could be potent regulators of the pool of adipocyte precursors in adipose tissue. effects of bio on differentiation of human adipose-derived stem cells to investigate the effect of gsk inhibition on adipocyte differentiation, hmads cells were induced to differentiate in the presence of bio or mebio. adipocytes and osteoblasts share the same mesenchymal precursor cell. adipogenesis and osteogenesis are processes that respond to a balance in bone marrow and this balance can be disrupted under pathological conditions such as osteoporosis in which adipocytes develop at the expense of osteoblasts. we took advantage of hmads cells that were previously demonstrated to differentiate in vitro into functional adipocytes and osteoblasts, to investigate the effect of gsk inhibition on both lineages. adipogenic and osteogenic differentiations, as indicated by oil red o and alizarin staining respectively, were dramatically inhibited in the presence of bio, whereas mebio did not display any significant effect. bio-inhibitory effect was quantified using enzymatic activities such as glycerol-3-dehydrogenase, which is expressed in adipocytes but not in osteoblasts, and alkaline phosphatase activity, which is expressed in osteoblasts but not in adipocytes. as shown in fig. gpdh activity was inhibited when hmads cells were maintained for days in adipogenic medium or in a medium allowing simultaneously adipogenic and osteogenic differentiations supplemented with bio. alp activity was also inhibited when cells were maintained in osteogenic or adipogenic/osteogenic media supplemented with the gsk inhibitor. then, we investigated the impact of bio treatment during cell proliferation on subsequent differentiation. quantification of gpdh enzymatic activity indicated that cells previously treated with bio during proliferation displayed a lower capacity to undergo adipocyte differentiation than untreated- or mebio-treated cells. this result indicates that gsk plays a role in maintaining the differentiation potential of undifferentiated hmads cells. this is reminiscent of our previous work showing the critical role of fgf pathway on self-renewal of hmads cells. finally, analysis of adipocyte- and osteoblast-gene expression confirmed that treatment of hmads cells with of bio during differentiation led to the inhibition of both differentiation programs. as shown in fig. all of the genes known to be expressed during adipocyte differentiation that we have analyzed were inhibited in the presence of bio. genes expressed during osteogenesis were also inhibited by bio, although at a lower extent compared to adipogenesis-related genes. altogether, these results are in agreement with previous reports showing that activation of wnt or hh pathways precludes differentiation of murine 3t3-l preadipose cells into adipocytes. our data indicate that the gsk inhibitor was a potent inhibitor of commitment of human preadipocyte precursors into the adipogenic lineage. the effect of wnt pathway on osteogenesis remains more controversial. wnt has been reported to enhance osteogenic differentiation of murine mesenchymal stem cells. more recently, cho et al. have shown that wnt signalling suppresses osteogenic differentiation of stroma cells isolated from human adipose tissue. therefore, the effect of wnt signalling on bone formation seems different in human versus mouse and our data with bio-treated hmads cells are in agreement with a negative effect of wnt on osteogenesis of human cells. it has been observed that patients treated with licl for bipolar disorder display weight gain and reduced risk of fractures, suggesting that licl promotes both adipogenesis and osteogenesis in humans in vivo. however, a central effect of licl and an indirect effect on adipocytes and osteoblasts in these patients cannot be excluded. differential effects of bio treatment during early steps of adipogenesis and osteogenesis then, we analyzed bio-inhibition on adipogenic and osteogenic differentiations of hmads cells more precisely in order to address whether a chronic treatment with the inhibitor was required. for that purpose, we treated hmads cells during the first days of differentiation and analyzed the impact of this treatment on gpdh and alp enzymatic activities as well as on the expression of specific markers. in regards to adipogenesis, treatment of hmads cells for the first days only was sufficient to inhibit gpdh activity measured at day. analysis of adipocyte gene expression confirmed that the inhibitory effect of bio after days of treatment persisted at day for most of the genes analyzed. in contrast, a treatment restricted to the first days had no effect on both osteogenic markers such as alp enzymatic activity and expression of several genes known to play a role in osteogenesis. altogether, data indicate that treatment of hmads cells with bio during the early step of differentiation only led to inhibition of late steps of adipogenesis, whereas it had no effect on osteogenesis. this strongly suggests that the molecular mechanisms underlying inhibition of adipogenic and osteogenic differentiation by bio are different. identification of mechanisms leading to the differential effects on adipogenesis and osteogenesis of hmads cells could lead to a preferential use of the gsk inhibitors on adipocyte differentiation in vivo. in conclusion, our data strongly suggest that gsk is a promising pharmacological target to regulate both the number and differentiation of adipocyte precursors in human adipose tissue. however, we have to keep in mind that potential adverse effects could be observed due to the fact that gsk is implicated in numerous signalling pathways. therefore, it would be important in the future to identify the signalling pathway mediating the adipogenic effect of gsk  we have shown that bio and licl, two inhibitors of gsk inhibited proliferation as well as adipogenic and osteogenic differentiations of stem cells isolated from human adipose tissue. data demonstrate the feasibility of a pharmacological approach to regulate adipose-derived stem cell function. 
glycogen synthase kinase has been previously described as involved in differentiation of preadipose cells and might be a potential therapeutic target to modulate proliferation and differentiation of adipocyte precursors. the impact of gsk inhibition on differentiation of hmads cells was also investigated. our results showed that gsk inhibitors inhibited proliferation and clonogenicity of human stem cells, strongly suggesting that gsk inhibitors could be potent regulators of the pool of adipocyte precursors in adipose tissue. these results demonstrated the feasibility of a pharmacological approach to regulate adipose-derived stem cell function and that gsk could represent a potential target for controlling adipocyte precursor pool under conditions where fat tissue formation is impaired. whereas a chronic treatment was required to inhibit osteogenesis, a treatment that was strictly restricted to the early step of differentiation was sufficient to inhibit adipogenesis. lithium chloride and bio on proliferation and adipocyte differentiation of multipotent stem cells derived from human adipose tissue. an abnormal recruitment of these adipose precursor cells could participate to hyperplasia of adipose tissue observed in severe obesity or to hypoplasia of adipose tissue observed in lipodystrophy. multipotent stem cells exist within adipose tissue throughout life. therefore, pharmacological molecules that control the pool of stem cells in adipose tissue are of great interest. adipogenic and osteogenic differentiations were inhibited upon hmads treatment with bio. to reach this goal, we studied the effects of pharmacological inhibitors of gsk i.e. however, the impact of gsk inhibition on human adipose-derived stem cells remained to be investigated.
 multipotent stem cells exist within adipose tissue throughout life. an abnormal recruitment of these adipose precursor cells could participate to hyperplasia of adipose tissue observed in severe obesity or to hypoplasia of adipose tissue observed in lipodystrophy. therefore, pharmacological molecules that control the pool of stem cells in adipose tissue are of great interest. glycogen synthase kinase has been previously described as involved in differentiation of preadipose cells and might be a potential therapeutic target to modulate proliferation and differentiation of adipocyte precursors. however, the impact of gsk inhibition on human adipose-derived stem cells remained to be investigated. the aim of this study was to investigate gsk as a possible target for pharmacological inhibition of stem cell adipogenesis. to reach this goal, we studied the effects of pharmacological inhibitors of gsk i.e. lithium chloride and bio on proliferation and adipocyte differentiation of multipotent stem cells derived from human adipose tissue.  our results showed that gsk inhibitors inhibited proliferation and clonogenicity of human stem cells, strongly suggesting that gsk inhibitors could be potent regulators of the pool of adipocyte precursors in adipose tissue. the impact of gsk inhibition on differentiation of hmads cells was also investigated. adipogenic and osteogenic differentiations were inhibited upon hmads treatment with bio. whereas a chronic treatment was required to inhibit osteogenesis, a treatment that was strictly restricted to the early step of differentiation was sufficient to inhibit adipogenesis.  these results demonstrated the feasibility of a pharmacological approach to regulate adipose-derived stem cell function and that gsk could represent a potential target for controlling adipocyte precursor pool under conditions where fat tissue formation is impaired. 