BACKGROUND
increased adipose tissue mass associated with obesity is due to the increased number and size of adipocytes  <cit> .
adipocyte differentiation from mesenchymal stem cells plays an important role in the hyperplasia of adult adipose tissue.
a population of cells resident in the vascular stroma of adipose tissue can differentiate into adipocytes in vitro and in vivo  <cit> .
recent studies indicate that pericytes in blood vessel walls have adipogenic potential, express mesenchymal stem cell  markers and are multipotent  <cit> .
in addition to resident stem cells, non-resident stem cells can serve as a source of adipocyte precursors; bone marrow mscs can be recruited to adipose tissue and generate new adipocytes in response to treatment with thiazolidinediones  or high fat stimulation  <cit> .
the characteristics and molecular mechanism underlying adipocyte differentiation have been extensively investigated in the murine pre-adipocyte cell lines 3t3-l <dig> and 3t3-f442a  <cit> .
growth-arrested pre-adipocytes have been shown to re-enter the cell cycle synchronously and undergo mitotic clonal expansion in response to mdi  treatment, before exiting the cell cycle and terminally differentiating  <cit> .
the transcription factors c/ebpβ , c/ebpα  and pparγ  act sequentially during 3t3-l <dig> pre-adipocyte differentiation  <cit> .
c/ebpβ is induced immediately after exposure to the differentiation cocktail, resulting in phosphorylation and activation  <cit> , and it transactivates the expression of c/ebpα and pparγ  <cit> .
c/ebpα and pparγ, together or in isolation, can initiate differentiation without inducers  <cit> .
c/epbα is believed to be relevant to the acquisition of insulin sensitivity  <cit> .
mscs have been isolated and induced to differentiate into adipocytes in a variety of organs  <cit> .
however, the differentiation procedure and the roles of adipose-related genes in that procedure have not been characterized completely owing to the heterogeneity, low proliferation ability and ineffective ectopic gene transfection of hbmscs  <cit> .
human primary cells are of great interest because of their biological and therapeutic potential, therefore this study extends the research carried out in murine 3t3-l <dig> cells to hbmscs from bone marrow.
RESULTS
isolation and adipogenic differentiation of hbmscs
isolated hbmscs presented with a typical spindle-shape phenotype , and cells from passages 3- <dig> were used for the following studies.
in addition to fetal bovine serum , methyl-isobutyl-xanthine, dexamethasone and insulin  used to induce 3t3-l <dig> adipocyte differentiation, indomethacin , a pparγ agonist  <cit> , was added to the culture medium  to induce adipocyte differentiation from hbmscs  <cit> .
each cycle of mdi+indo threatment only induced a portion of hbmscs to go into adipocyte differentiation, and about 60%-70% hbmscs differentiated into adipocytes after three cycles of mdi+indo induction as indicated by oil red o staining .
consistent with the morphological changes, the expression of the adipose-specific gene fabp <dig>  was significantly induced throughout differentiation as determined by western blotting .
cell cycle alteration during adipocyte differentiation from hbmscs
hbmscs proliferated slowly, approximately <10% of cells were actively dividing revealed by dna content with flow cytometry .
about 90% of the cells in g0/g <dig> phase were at the dividing stage, and approximately 95% at the post-confluence stage .
contact inhibition was not apparent, as observed that if plated at a density of  <dig> cells/cm <dig> and cultured for five weeks, the cells locally grew into multi-layers .
cell cycle regulation is an important event in adipocyte differentiation of mouse 3t3-l <dig> pre-adipocytes  <cit> .
growth-arrested 3t3-l <dig> pre-adipocytes synchronously re-enter the cell cycle upon mdi induction and undergo two rounds of division before expression of adipocyte-specific genes and presenting with the mature adipocyte phenotype.
in order to investigate whether hbmscs undergo division during adipocyte differentiation, the number of cells was counted .
the cell number marginally increased  in the control group after a 21-day culture .
there was an increase in the cell number  in differentiation cultures but less than that in the control, and as the number of differentiated cells increased after repeated inductions, the rate of increase of cell numbers declined.
these results suggest that the proliferation of undifferentiated cells contributed to the increase in cell numbers.
brdu incorporation assays were performed to investigate whether dna synthesis occurs during adipocyte differentiation from hbmscs.
we found that differentiated hbmscs were brdu negative, while differentiated 3t3-l <dig> cells were brdu positive .
confocal microscopy verified the positional relationship between nuclei  and cells with lipid droplets in the cytoplasm .
these results demonstrate that hbmscs did not undergo mitotic clonal expansion during adipogenic differentiation under culture conditions.
role of c/ebpβ in adipocyte differentiation of hbmscs
in order to define the role of c/ebpβ in adipocyte differentiation of hbmscs, the expression profile was determined.
expression of c/ebpβ in hbmscs could be detected at the start of induction by real-time pcr; the expression level did not change significantly during the early stages of induction  but declined after  <dig> days when most of the cells had differentiated.
regarding the expression difference between 3t3-l <dig> and hbmscs, c/ebpβ expression was knocked down by sirna to determine whether c/ebpβ is essential during adipocyte differentiation from hbmscs; and knocked-down expression of c/ebpβ was confirmed by real-time pcr .
hbmscs failed to differentiate into adipocytes after c/ebpβ was knocked down .
c/ebpβ was over-expressed in hbmscs using an adenovirus expression system  to investigate its function during differentiation.
control cells expressing lac z didn't differentiate, while expression of exogenous c/ebpβ alone induced adipogenesis , and some cells presented with small intracellular fat droplets that could not be adequately stained using oil red o. however, fabp <dig> expression was detected by western blotting  and was significantly up-regulated by the addition of inducers, the highest levels of expression being evident when indomethacin  was included .
role of c/ebpα in adipocyte differentiation from hbmscs
expression of c/ebpα increased one day after induction, reached a maximum level after three days and decreased by day  <dig> when adipocyte differentiation had occurred .
as figure 5b demonstrates, knocked-down expression of c/ebpα  with an adenovirus carrying c/ebpα shrna impaired the differentiation of hbmscs, while over-expression of c/ebpα  in hbmscs did not induce adipocyte differentiation.
however, c/ebpα together with indomethacin or mdi induced a small proportion of the cells to differentiate, and when both indomethatin and mdi were added with c/ebpα, the number of differentiated adipocytes increased as demonstrated by oil red o staining  and western blotting of fabp <dig> expression .
role of pparγ in adipocyte differentiation from hbmscs
the induction of pparγ expression was similar to that of c/ebpα .
knock-down of pparγ expression in hbmscs  prevented adipocyte differentiation , while over-expression of pparγ  induced adipogenic differentiation , resulting in fat droplet accumulation in the vast majority of cells.
addition of an exogenous pparγ agonist  enhanced the function of pparγ as determined by oil red o staining  and fabp <dig> expression .
fat droplets appeared three days after adenoviral infection but were smaller than those induced by mdi+indo .
the expression ratio of glut <dig> to fabp <dig> in adipocytes induced by pparγ over-expression was lower than that in cells induced by three cycles of mdi+indo .
discussion
hbmscs are more difficult to handle than mouse stem cell lines but their importance and therapeutic potential necessitate their use in research of the type outlined herein.
the previous studies are focused on the mouse stem cell lines but the regulation of them could be different in some aspects, and results of murine cells would be less convincing in interpreting the onset of human disease.
on the other hand, adipocytes differentiated from hbmscs would be of better immuno-compatibility in autograft for plastic purpose.
so, in this study, a comprehensive analysis of adipocyte differentiation from multipotent human stem cells was carried out.
hbmscs were isolated from bone marrow and induced to differentiate into adipocytes under culture conditions.
the pparγ agonist, indomethacin, was added as well as the conventional inducers used in adipocyte differentiation protocols for murine pre-adipocytes.
hbmscs behaved differently from 3t3-l <dig> pre-adipocytes, with only a small number of cells differentiating into adipocytes after one cycle of treatment; approximately 60%~70% of hbmscs differentiated into adipocytes after three cycles of treatment .
a long g <dig> phase and a lack of contact inhibition  meant that hbmscs did not synchronize at the time when differentiation was initiated .
growth arrest is a prerequisite for adipocyte differentiation  <cit> , so it was concluded that only a minority of hbmscs were growth arrested when differentiation was induced.
mce  is an essential event associated with adipocyte differentiation from mouse pre-adipocyte cell lines  <cit> .
however, it is not known whether mce is required for adipocyte differentiation from all cell types.
we have previously demonstrated that committed c3h10t1/ <dig> cells treated with bmp <dig> divide when induced to differentiate  <cit> , and primary cultures of mouse embryonic fibroblasts  undergo mce when differentiating into adipocytes  <cit> .
in this study, hbmscs from bone marrow did not undergo division during differentiation , which is in agreement with other reports showing that adipose precursor cells prepared from human adipose tissue  did not divide during differentiation under culture conditions  <cit> .
the authors argued that hadscs had completed division before being isolated; however, hadscs are multipotent and can differentiate into other cell lineages including adipocytes ex vivo  <cit> .
hadscs could behave similarly to hbmscs from bone marrow under culture conditions and remain uncommitted.
the diversity of cell cycle alterations during adipocyte differentiation could be species-specific.
murine proteins and comparable human proteins can function differently in the same context.
in this study, c/ebpβ expression in hbmscs did not alter significantly during the early stages of induction whereas expression was up-regulated immediately following induction and declined after two days in 3t3-l <dig> pre-adipocytes  <cit> .
the decline of c/ebpβ at  <dig> day might result from most of cells being terminal differentiated.
however, c/ebpβ was required for adipocyte differentiation in hbmscs as its knock-down expression impaired differentiation .
c/ebpβ has important roles in mitosis and terminal adipocyte differentiation  <cit> , but mitosis did not occur during differentiation of hbmscs  and that could possibly explain the lack of differential expression of c/ebpβ upon induction.
it is likely that the role of c/ebpβ during adipocyte differentiation from hbmscs relates to its modification and not its expression levels, although the importance of c/ebpβ phosphorylation requires further investigation.
c/ebpβ or c/ebpα is sufficient to induce 3t3-l <dig> pre-adipocytes to differentiate into mature adipocytes without using inducers  <cit> .
over-expression of c/ebpβ alone stimulated differentiation of hbmscs, as evidenced by fabp <dig> expression .
c/ebpα was less effective than c/ebpβ as expression of c/ebpα alone did not stimulate differentiation .
c/ebpβ and c/ebpα individually enhanced adipocyte differentiation of hbmscs dependent on exogenous hormone agent treatment, particularly in the presence of a pparγ activator .
hbmscs may lack endogenous pparγ ligands; however, this cannot be determined at this time because the results concerning the study of natural pparγ ligands are indecisive  <cit> .
pparγ plays pivotal roles in adipocyte differentiation as it induces adipogenesis in cultured mouse fibroblasts  <cit> .
with the use of high affinity, selective pparγ agonists, pparγ activation stimulates 3t3-f442a cells to develop into mature fat cells with a phenotype that includes morphological changes, lipid accumulation, and the acquisition of insulin sensitivity  <cit> .
in addition, ectopic expression of pparγ in hbmscs initiates adipocyte differentiation.
however, these cells were immature adipocytes, as demonstrated by morphological observations and the expression of some adipocyte-specific genes .
in humans, pparγ functions to regulate a part of genes required for adipocyte maturation, as demonstrated by its ability to induce fabp <dig> but not glut <dig> expression .
in addition, pparγ could play a role in cytoskeletal alterations associated with the morphological changes during differentiation, as the cells rounded up when pparγ was over-expressed and elongated when expression of pparγ was knocked down.
CONCLUSIONS
this study demonstrates that the characteristics of hbmscs during adipogenic differentiation are different from those of mouse cells.
hbmscs do not undergo mitotic clonal expansion during adipocyte differentiation.
c/ebpβ, c/ebpα, and pparγ are all required but not sufficient for adipocyte differentiation from hbmscs.
the ability of the transcription factors to stimulate adipocyte differentiation differed between human and murine cells.
further studies concerning on how c/ebpβ, c/ebpα and pparγ regulating human adipocyte differentiation could help to elucidate the molecular mechanism of adipocyte differentiation from human stem cells, help to elucidate the mechanisms underlying human obesity and identify therapeutic targets.

 increased adipose tissue mass associated with obesity is due to the increased number and size of adipocytes. adipocyte differentiation from mesenchymal stem cells plays an important role in the hyperplasia of adult adipose tissue. a population of cells resident in the vascular stroma of adipose tissue can differentiate into adipocytes in vitro and in vivo. recent studies indicate that pericytes in blood vessel walls have adipogenic potential, express mesenchymal stem cell markers and are multipotent. in addition to resident stem cells, non-resident stem cells can serve as a source of adipocyte precursors; bone marrow mscs can be recruited to adipose tissue and generate new adipocytes in response to treatment with thiazolidinediones or high fat stimulation. the characteristics and molecular mechanism underlying adipocyte differentiation have been extensively investigated in the murine pre-adipocyte cell lines 3t3-l and 3t3-f442a. growth-arrested pre-adipocytes have been shown to re-enter the cell cycle synchronously and undergo mitotic clonal expansion in response to mdi treatment, before exiting the cell cycle and terminally differentiating. the transcription factors c/ebpβ, c/ebpα and pparγ act sequentially during 3t3-l pre-adipocyte differentiation. c/ebpβ is induced immediately after exposure to the differentiation cocktail, resulting in phosphorylation and activation, and it transactivates the expression of c/ebpα and pparγ. c/ebpα and pparγ, together or in isolation, can initiate differentiation without inducers. c/epbα is believed to be relevant to the acquisition of insulin sensitivity. mscs have been isolated and induced to differentiate into adipocytes in a variety of organs. however, the differentiation procedure and the roles of adipose-related genes in that procedure have not been characterized completely owing to the heterogeneity, low proliferation ability and ineffective ectopic gene transfection of hbmscs. human primary cells are of great interest because of their biological and therapeutic potential, therefore this study extends the research carried out in murine 3t3-l cells to hbmscs from bone marrow.  isolation and adipogenic differentiation of hbmscs isolated hbmscs presented with a typical spindle-shape phenotype, and cells from passages 3- were used for the following studies. in addition to fetal bovine serum, methyl-isobutyl-xanthine, dexamethasone and insulin used to induce 3t3-l adipocyte differentiation, indomethacin, a pparγ agonist, was added to the culture medium to induce adipocyte differentiation from hbmscs. each cycle of mdi+indo threatment only induced a portion of hbmscs to go into adipocyte differentiation, and about 60%-70% hbmscs differentiated into adipocytes after three cycles of mdi+indo induction as indicated by oil red o staining. consistent with the morphological changes, the expression of the adipose-specific gene fabp was significantly induced throughout differentiation as determined by western blotting. cell cycle alteration during adipocyte differentiation from hbmscs hbmscs proliferated slowly, approximately <10% of cells were actively dividing revealed by dna content with flow cytometry. about 90% of the cells in g0/g phase were at the dividing stage, and approximately 95% at the post-confluence stage. contact inhibition was not apparent, as observed that if plated at a density of cells/cm and cultured for five weeks, the cells locally grew into multi-layers. cell cycle regulation is an important event in adipocyte differentiation of mouse 3t3-l pre-adipocytes. growth-arrested 3t3-l pre-adipocytes synchronously re-enter the cell cycle upon mdi induction and undergo two rounds of division before expression of adipocyte-specific genes and presenting with the mature adipocyte phenotype. in order to investigate whether hbmscs undergo division during adipocyte differentiation, the number of cells was counted. the cell number marginally increased in the control group after a 21-day culture. there was an increase in the cell number in differentiation cultures but less than that in the control, and as the number of differentiated cells increased after repeated inductions, the rate of increase of cell numbers declined. these results suggest that the proliferation of undifferentiated cells contributed to the increase in cell numbers. brdu incorporation assays were performed to investigate whether dna synthesis occurs during adipocyte differentiation from hbmscs. we found that differentiated hbmscs were brdu negative, while differentiated 3t3-l cells were brdu positive. confocal microscopy verified the positional relationship between nuclei and cells with lipid droplets in the cytoplasm. these results demonstrate that hbmscs did not undergo mitotic clonal expansion during adipogenic differentiation under culture conditions. role of c/ebpβ in adipocyte differentiation of hbmscs in order to define the role of c/ebpβ in adipocyte differentiation of hbmscs, the expression profile was determined. expression of c/ebpβ in hbmscs could be detected at the start of induction by real-time pcr; the expression level did not change significantly during the early stages of induction but declined after days when most of the cells had differentiated. regarding the expression difference between 3t3-l and hbmscs, c/ebpβ expression was knocked down by sirna to determine whether c/ebpβ is essential during adipocyte differentiation from hbmscs; and knocked-down expression of c/ebpβ was confirmed by real-time pcr. hbmscs failed to differentiate into adipocytes after c/ebpβ was knocked down. c/ebpβ was over-expressed in hbmscs using an adenovirus expression system to investigate its function during differentiation. control cells expressing lac z didn't differentiate, while expression of exogenous c/ebpβ alone induced adipogenesis, and some cells presented with small intracellular fat droplets that could not be adequately stained using oil red o. however, fabp expression was detected by western blotting and was significantly up-regulated by the addition of inducers, the highest levels of expression being evident when indomethacin was included. role of c/ebpα in adipocyte differentiation from hbmscs expression of c/ebpα increased one day after induction, reached a maximum level after three days and decreased by day when adipocyte differentiation had occurred. as figure 5b demonstrates, knocked-down expression of c/ebpα with an adenovirus carrying c/ebpα shrna impaired the differentiation of hbmscs, while over-expression of c/ebpα in hbmscs did not induce adipocyte differentiation. however, c/ebpα together with indomethacin or mdi induced a small proportion of the cells to differentiate, and when both indomethatin and mdi were added with c/ebpα, the number of differentiated adipocytes increased as demonstrated by oil red o staining and western blotting of fabp expression. role of pparγ in adipocyte differentiation from hbmscs the induction of pparγ expression was similar to that of c/ebpα. knock-down of pparγ expression in hbmscs prevented adipocyte differentiation, while over-expression of pparγ induced adipogenic differentiation, resulting in fat droplet accumulation in the vast majority of cells. addition of an exogenous pparγ agonist enhanced the function of pparγ as determined by oil red o staining and fabp expression. fat droplets appeared three days after adenoviral infection but were smaller than those induced by mdi+indo. the expression ratio of glut to fabp in adipocytes induced by pparγ over-expression was lower than that in cells induced by three cycles of mdi+indo. discussion hbmscs are more difficult to handle than mouse stem cell lines but their importance and therapeutic potential necessitate their use in research of the type outlined herein. the previous studies are focused on the mouse stem cell lines but the regulation of them could be different in some aspects, and results of murine cells would be less convincing in interpreting the onset of human disease. on the other hand, adipocytes differentiated from hbmscs would be of better immuno-compatibility in autograft for plastic purpose. so, in this study, a comprehensive analysis of adipocyte differentiation from multipotent human stem cells was carried out. hbmscs were isolated from bone marrow and induced to differentiate into adipocytes under culture conditions. the pparγ agonist, indomethacin, was added as well as the conventional inducers used in adipocyte differentiation protocols for murine pre-adipocytes. hbmscs behaved differently from 3t3-l pre-adipocytes, with only a small number of cells differentiating into adipocytes after one cycle of treatment; approximately 60%~70% of hbmscs differentiated into adipocytes after three cycles of treatment. a long g phase and a lack of contact inhibition meant that hbmscs did not synchronize at the time when differentiation was initiated. growth arrest is a prerequisite for adipocyte differentiation, so it was concluded that only a minority of hbmscs were growth arrested when differentiation was induced. mce is an essential event associated with adipocyte differentiation from mouse pre-adipocyte cell lines. however, it is not known whether mce is required for adipocyte differentiation from all cell types. we have previously demonstrated that committed c3h10t1/ cells treated with bmp divide when induced to differentiate, and primary cultures of mouse embryonic fibroblasts undergo mce when differentiating into adipocytes. in this study, hbmscs from bone marrow did not undergo division during differentiation, which is in agreement with other reports showing that adipose precursor cells prepared from human adipose tissue did not divide during differentiation under culture conditions. the authors argued that hadscs had completed division before being isolated; however, hadscs are multipotent and can differentiate into other cell lineages including adipocytes ex vivo. hadscs could behave similarly to hbmscs from bone marrow under culture conditions and remain uncommitted. the diversity of cell cycle alterations during adipocyte differentiation could be species-specific. murine proteins and comparable human proteins can function differently in the same context. in this study, c/ebpβ expression in hbmscs did not alter significantly during the early stages of induction whereas expression was up-regulated immediately following induction and declined after two days in 3t3-l pre-adipocytes. the decline of c/ebpβ at day might result from most of cells being terminal differentiated. however, c/ebpβ was required for adipocyte differentiation in hbmscs as its knock-down expression impaired differentiation. c/ebpβ has important roles in mitosis and terminal adipocyte differentiation, but mitosis did not occur during differentiation of hbmscs and that could possibly explain the lack of differential expression of c/ebpβ upon induction. it is likely that the role of c/ebpβ during adipocyte differentiation from hbmscs relates to its modification and not its expression levels, although the importance of c/ebpβ phosphorylation requires further investigation. c/ebpβ or c/ebpα is sufficient to induce 3t3-l pre-adipocytes to differentiate into mature adipocytes without using inducers. over-expression of c/ebpβ alone stimulated differentiation of hbmscs, as evidenced by fabp expression. c/ebpα was less effective than c/ebpβ as expression of c/ebpα alone did not stimulate differentiation. c/ebpβ and c/ebpα individually enhanced adipocyte differentiation of hbmscs dependent on exogenous hormone agent treatment, particularly in the presence of a pparγ activator. hbmscs may lack endogenous pparγ ligands; however, this cannot be determined at this time because the results concerning the study of natural pparγ ligands are indecisive. pparγ plays pivotal roles in adipocyte differentiation as it induces adipogenesis in cultured mouse fibroblasts. with the use of high affinity, selective pparγ agonists, pparγ activation stimulates 3t3-f442a cells to develop into mature fat cells with a phenotype that includes morphological changes, lipid accumulation, and the acquisition of insulin sensitivity. in addition, ectopic expression of pparγ in hbmscs initiates adipocyte differentiation. however, these cells were immature adipocytes, as demonstrated by morphological observations and the expression of some adipocyte-specific genes. in humans, pparγ functions to regulate a part of genes required for adipocyte maturation, as demonstrated by its ability to induce fabp but not glut expression. in addition, pparγ could play a role in cytoskeletal alterations associated with the morphological changes during differentiation, as the cells rounded up when pparγ was over-expressed and elongated when expression of pparγ was knocked down.  this study demonstrates that the characteristics of hbmscs during adipogenic differentiation are different from those of mouse cells. hbmscs do not undergo mitotic clonal expansion during adipocyte differentiation. c/ebpβ, c/ebpα, and pparγ are all required but not sufficient for adipocyte differentiation from hbmscs. the ability of the transcription factors to stimulate adipocyte differentiation differed between human and murine cells. further studies concerning on how c/ebpβ, c/ebpα and pparγ regulating human adipocyte differentiation could help to elucidate the molecular mechanism of adipocyte differentiation from human stem cells, help to elucidate the mechanisms underlying human obesity and identify therapeutic targets. 
the characteristics of hbmscs during adipogenic differentiation are different from those of murine cells. adipocyte hyperplasia is associated with obesity and arises due to adipogenic differentiation of resident multipotent stem cells in the vascular stroma of adipose tissue and remote stem cells of other organs. the mechanistic characterization of adipocyte differentiation has been researched in murine pre-adipocyte models, revealing that growth-arrest pre-adipocytes undergo mitotic clonal expansion and that regulation of the differentiation process relies on the sequential expression of three key transcription factors. however, the mechanisms underlying adipocyte differentiation from multipotent stem cells, particularly human mesenchymal stem cells, remain poorly understood. this study investigated cell cycle regulation and the roles of c/ebpβ, c/ebpα and pparγ during adipocyte differentiation from hbmscs. utilising a brdu incorporation assay and manual cell counting it was demonstrated that induction of adipocyte differentiation in culture resulted in 3t3-l pre-adipocytes but not hbmscs undergoing mitotic clonal expansion. knock-down and over-expression assays revealed that c/ebpβ, c/ebpα and pparγ were required for adipocyte differentiation from hbmscs. c/ebpβ and c/ebpα individually induced adipocyte differentiation in the presence of inducers; pparγ alone initiated adipocyte differentiation but the cells failed to differentiate fully. therefore, the roles of these transcription factors during human adipocyte differentiation are different from their respective roles in mouse. these findings could be important in elucidating the mechanisms underlying human obesity further.
 adipocyte hyperplasia is associated with obesity and arises due to adipogenic differentiation of resident multipotent stem cells in the vascular stroma of adipose tissue and remote stem cells of other organs. the mechanistic characterization of adipocyte differentiation has been researched in murine pre-adipocyte models, revealing that growth-arrest pre-adipocytes undergo mitotic clonal expansion and that regulation of the differentiation process relies on the sequential expression of three key transcription factors. however, the mechanisms underlying adipocyte differentiation from multipotent stem cells, particularly human mesenchymal stem cells, remain poorly understood. this study investigated cell cycle regulation and the roles of c/ebpβ, c/ebpα and pparγ during adipocyte differentiation from hbmscs.  utilising a brdu incorporation assay and manual cell counting it was demonstrated that induction of adipocyte differentiation in culture resulted in 3t3-l pre-adipocytes but not hbmscs undergoing mitotic clonal expansion. knock-down and over-expression assays revealed that c/ebpβ, c/ebpα and pparγ were required for adipocyte differentiation from hbmscs. c/ebpβ and c/ebpα individually induced adipocyte differentiation in the presence of inducers; pparγ alone initiated adipocyte differentiation but the cells failed to differentiate fully. therefore, the roles of these transcription factors during human adipocyte differentiation are different from their respective roles in mouse.  the characteristics of hbmscs during adipogenic differentiation are different from those of murine cells. these findings could be important in elucidating the mechanisms underlying human obesity further. 