BACKGROUND
in recent years mesenchymal stem cells  have generated a great deal of interest as a potential source for cell-based therapeutic strategies.
human msc are easy to isolate from small aspirate of bone marrow via their adherence ability.
these cells readily generate single-cell-derived colonies that can be highly expanded and differentiated into a variety of cell types, such as osteoblasts  <cit> , adipocytes  <cit> , myocytes  <cit> , astrocytes and neurons  <cit> .
further, human msc can improve cardiac function after infarction  <cit>  or symptoms of bone and cartilage defects  <cit> , as well as neurodegenerative diseases such as alzheimer's  <cit> .
their efficiency in multiple types of cellular therapeutic strategies has been demonstrated, including applications in treating children with osteogenesis imperfecta  <cit> , hematopoietic recovery  <cit> , and bone tissue regeneration  <cit> .
also first preclinical trails are in progress to test their capacity and toxicity in applications for human treatment  <cit> .
one great advantage of msc is that these cells may be directly obtained from individual patients, thereby eliminating the complications associated with immune rejection of allogenic tissue and infectious diseases.
however, for cell therapies msc have to be expanded and/or manipulated to obtain a sufficient amount of cells that can be subsequently used for treatment.
despite growing experience and knowledge concerning human msc and their use in cell-based strategies, the molecular mechanisms that govern msc self-renewal, expansion and multilineage differentiation are not well understood and remain an active area of investigation.
in this study we asked if human msc are developing in an aberrant or unwanted way during ex vivo long-term cultivation and if cultivation conditions exert any influence on their stem cell maintenance.
to address this question systematically and comprehensively we first developed human oligonucleotide microarrays with  <dig>  elements and then performed large-scale expression profiling of long-term expanded msc isolated from clinically relevant samples.
we monitored these cells during their expansion ex vivo with respect to proliferation kinetics, surface marker profile and differentiation potential.
finally we analyzed the gene expression profiles of msc during osteogenic differentiation.
our results showed that expansion of msc does not result in substantial genetic and morphological aberrations.
we illustrated for the first time in a human model the three main stages of osteogenic development, and we could show the diverse regulation of the smad pathways by tgf-β <dig> and bmps.
RESULTS
human msc maintain their undifferentiated phenotype during long-term expansion
the results of the ex vivo long-term expansion experiments showed that the undifferentiated phenotype of mscs is maintained with respect to differentiation potential, surface marker profile and gene expression profiles.
at confluence of 75–85% the cultivated cells were detached and flow cytometry analysis were performed to verify the purity of the cell population without any contaminations, such as haematopoietic cells.
following surface marker profile were detected: cd44+, cd90+, cd73+, cd105+, cd166+, cd11b-, cd34-, cd45-, cd117-, hla dr-.
for the subsequent experiments mscs were expanded until the end of the tenth passage.
at the end of each passage the cells were analyzed by flow cytometry and a cell aliquot was seeded out for testing their differentiation ability.
proliferation kinetics and differentiation potential
the growth kinetics of five donors was investigated from the primary culture through the tenth passage, corresponding to approximately  <dig> cell doublings.
for recalculating the starting number of msc in the mnc fraction, cfu assays were performed and the msc frequency was determined.
primary cultures reached their first confluence from around 80% in about  <dig> weeks and about  <dig> cell doublings.
during the following passages the proliferation rate slowed down.
during the whole expansion period – starting with passage two until passage ten – in every passage the osteogenic differentiation ability was tested.
the osteogenic differentiation was confirmed by alizarin red s staining and alkaline phosphatase assay.
throughout this examination period differentiation ability into osteoblasts was observed in low passages  as well as in a higher passage , .
msc expanded to approximately  <dig> cell doublings were able to differentiate into adipocytes .
surface marker profile
after the cultivation of msc from the mononuclear cell fraction  and at the end of each individual passage, the msc were analyzed by flow cytometry to monitor the surface marker profile during their ex vivo expansion.
each investigated passage depicted homogenous cell population demonstrating that msc did not alter their physical and morphological properties during high grade ex vivo expansion under the culture conditions tested .
in addition to the examination of the surface marker profile, we also performed real-time rt-pcr sybr™ green i assays for stem cell specific genes.
the examined stem cell markers, cd <dig>  cd <dig>  cd <dig>  cd <dig>  cd <dig> and stro <dig> showed stable expression level during the whole expansion period proving the results obtained by facs analyses.
as expected, cd <dig>  cd <dig> and tert could not be detected on any examined time point .
large-scale gene expression profiling of msc during ex vivo expansion
in order to monitor the molecular events that govern the ex vivo expansion of undifferentiated msc we performed large-scale gene expression profiling using oligonucleotide microarrays with  <dig> elements .
after background correction, global mean and dye swap normalization, and replicate handling,  <dig> genes could be detected as differentially expressed with at least 2-fold change, between cells from passage  <dig> with cells from passage  <dig>  and  <dig> genes, when comparing passage  <dig> cells with passage  <dig> cells .
a modified version of the regularized t-test  <cit>  with the complete data set was performed to identify significantly differentially expressed genes in ten examined donors  and five examined donors .
the results showed that only  <dig> genes were significantly differentially expressed  during  <dig> cell doublings when comparing cells from passage  <dig> versus cells from passage  <dig> .
significantly differentially expressed genes represent proteins with diverse functions: extracellular matrix proteins , proteins involved in bone, fat and epithelial cell metabolism , pain perception and stress response , growth-promoting activity , and signaling and cell survival mechanisms .
the hypothetical protein xp_ <dig> is annotated as similar to keratinocyte growth factor-like protein, and the hypothetical protein dkfz434b <dig> is defined as a cysteine-rich secretory protein containing lccl domain  <dig>  the expression data from the above mentioned genes could be confirmed by independent real-time rt-pcr .
list of differentially expressed genes after statistical analysis by modified version of the regularized t-test  <cit>   for ten donors  and five donors .
in sum, these results show that long-term expansion of msc did not alter their differentiation potential, surface marker profiles, and expression profiles.
expression profiling of msc during osteogenic differentiation reveals distinct phases of osteogenic development
osteogenic differentiation
human msc from three different donors  were cultured until their fourth passage and were induced to osteogenic differentiation at a confluence of 70–80%.
the osteogenic differentiation was observed during the whole period by microscopy and was terminated on day  <dig> after induction.
first signs for calcification appeared as black regions within the cell monolayer after around seven days.
the maximum of calcified extracellular matrix was observed after  <dig> days of treatment .
the osteoblast phenotype was confirmed by alkaline phosphatase assay and alizarin red s staining .
expression of osteoblast specific marker genes
we performed real-time rt-pcr assays to detect the expression levels of osteoblast specific marker genes msx <dig> , vdr , col1a <dig> , alpl , sparc , spp <dig>  and bglap  to prove the osteoblast phenotype.
the log <dig> transformed results are shown in the supplementary material .
the data obtained by real-time rt-pcr showed the expected expression profiles of the osteoblast phenotype.
alpl showed the expected expression level progression.
its expression was increased starting with day four of the differentiation period and decreased after day  <dig> until day  <dig> during the mineralization phase.
the homeobox gene msx <dig>  which is implicated in osteoprogenitor cell function  <cit>  and an up-stream regulator of runx <dig>  which was described to be important for the osteogenic differentiation  <cit> , were up-regulated during the whole period of differentiation.
col1a <dig> is known to be an early marker of osteoprogenitor cells  <cit> .
its maximum of expression was reached on day  <dig>  also sparc showed a nearly constant, up-regulated expression level with its maximum on day  <dig> of differentiation.
spp <dig> is an extracellular matrix protein, known to peak twice in its expression  <cit> : around day  <dig>  during proliferation, and between days 14– <dig>  during mineralization.
bglap was described as a late marker of developing osteoblasts appearing with matrix mineralization  <cit>  and was maximally expressed on day  <dig>  interestingly, bglap was already significantly up-regulated on day four, whereas the mineralization could not be observed before day seven.
large-scale gene expression profiling of msc during osteogenesis
the rna was harvested at four time points during the differentiation period and hybridized on human oligonucleotide microarrays.
as reference, rna harvested from msc one day before the osteogenic induction was used .
after lowess-subgrid normalization and filtering missing values  <dig> genes were selected with two-fold up- or downregulated expression in at least one time point .
correspondence between co-expressed genes and phenotypic changes
differentially expressed genes were clustered according to their expression profiles using the k-means clustering method  <cit>  .
the cluster analysis was performed with at maximum  <dig> iterations and using  <dig> different clusters, each containing between  <dig> and  <dig> genes .
the number of clusters was estimated by fom analyses .
additionally, hierarchical clustering was performed and the results were comparable to k-means clustering .
by analyzing the genes of the different clusters and their expression profiles, we were able to reconstruct three main phases of osteogenic development : proliferation, matrix maturation, and mineralization.
additionally, novel candidate genes could be found which are not known to play a role during osteogenic differentiation: id <dig> , cryab  and sort <dig> .
the expression of these genes was subsequently confirmed by quantitative rt-pcr .
for gene ontology  analysis we categorized ests with available refseq annotation  according to go terms for biological process , cellular component, and molecular function .
in detail, cluster  <dig> and  <dig> represented proliferation phase describing genes, which were involved in signal transduction, regulation of cell cycle, metabolism and regulation of transcription.
cluster  <dig> and  <dig> illustrated genes which were up-or down regulated during the whole period of differentiation.
they describe the phase matrix maturation and are involved in development, cell differentiation, metabolism, regulation of dna-dependent transcription.
the phase of mineralization is evident in cluster  <dig> and  <dig>  which include genes involved in signal transduction, transport, metabolism, and development.
the go analysis showed that genes found in cluster  <dig> and  <dig> were also involved in cell adhesion, signal transduction, cell cycle, and regulation of transcription, similar to cluster  <dig> and  <dig>  cluster  <dig> described genes of development and metabolism among the rest.
cluster  <dig> and  <dig> combined genes of cell adhesion, transport, cell proliferation and regulation of transcription.
cluster  <dig> represented genes associated with transport and regulation of transcription.
in summary, go annotation showed that nearly in each cluster genes of cell adhesion, signal transduction, transport, and regulation of transcription were present, illustrating the complex regulation of different events during osteogenesis.
pathway analysis of the expression data set
the data set of  <dig> genes with significant up or down regulation  was mapped with the pathway explorer on pathways derived from the biocarta and kegg database  <cit> .
in total  <dig> out of  <dig> genes could be mapped with their available refseq numbers .
only pathways where at least 10% and more than five mapped genes could be found were selected for further examination.
following these criteria the tgf-β signaling pathway  was ranked first, which summarized the different smad pathways regulated by the members of the tgf-β family: bmp, tgf-β and inhibin.
cell growth and proliferation is controlled by the members of the tgf-β superfamily which includes structurally related proteins like tgf-β, activins, and bone morphogenetic proteins   <cit> .
in the here presented tgf-β signaling pathway bmp <dig> and bmp <dig>  madh <dig> and madh <dig>  were up-regulated and the inhibitor of dna binding id <dig> was down-regulated .
bmp <dig> and bmp <dig> are able to bind to bmpr-type i and ii, whereas bmp <dig> is able to bind to actriia  <cit> .
due to the binding of these two molecules to the heterodimeric receptor complex the smad signaling pathway is activated.
our data illustrated further, that the inhibitor of dna binding, id <dig> was down regulated and the osteogenic differentiation could be induced.
in parallel, tgf-β <dig>  inhibin a and b were down-regulated during the differentiation period, whereby the following events were suppressed.
hence, under these experimental conditions the smad-signaling pathway activated by bmp <dig> was responsible for the osteogenic development.
in contrast, the activation of the smad pathway by tgf-β and inhibin was suppressed.
in vivo relevance of human msc as in vitro differentiation model
the in vitro differentiation of msc into osteoblasts is often used model to examine the regulation mechanisms during osteoblast development.
to evaluate the in vivo relevance of this model, we compared the rna obtained at the diverse time points during osteogenic differentiation with rna isolated from primary cultures of normal non-mineralized human osteoblasts derived from hipbone using our microarrays  .
the results showed that msc on day seven of their differentiation were most similar to human osteoblasts .
this observation could be confirmed by the comparison of the morphology of the two cell types: human osteoblasts isolated from hipbone represented non-mineralized osteoblasts with fibroblast-like phenotype, morphologically similar to induced msc on day seven after induction.
discussion
in this study we could show for the first time that msc maintain their undifferentiated phenotype after ex vivo long-term expansion.
the results of the microarray assays showed that mscs did not change significantly their gene expression profiles during long-term expansion since only nine out of almost  <dig>  genes were differentially expressed between passage  <dig> and passage  <dig> in ten donors.
in addition, no changes in the expression profiles after additional  <dig> passages could be observed in five of the ten donors.
these surprising results can only be partially explained by the limitations of the microarray technology.
due to the lower sensitivity and uncomplete coverage of the transcriptome, some genes required for stem cell maintenance might have been missed.
additionally, individual differences between the donors and slightly different conditions during tissue extraction and sample treatment could increase the number identified genes which were otherwise filtered out during averaging.
however, the microarray results were supported by the analyses of the effects of extensive long-term ex vivo cultivation of msc on their proliferation capacity, their morphology, surface marker profile, and differentiation ability.
during the long cultivation time we could observe, in concordance with other groups  <cit> , a progressive lost of proliferation ability and a slight shift in the cell-morphology of msc of higher passage.
however, their surface marker profile determined by flow cytometry  and by real-time rt-pcr was not altered.
over and above, the stem cell markers like cd <dig> and stro <dig> showed stable expression levels, which further supports our observation that msc maintained their undifferentiated phenotype and remained capable of osteogenic differentiation during ex vivo expansion at all tested passages .
additionally to this clinically relevant evidence, we provide novel biological insights derived from the global view of the molecular processes during osteogenic development.
first, we were able to illustrate the different phases of osteogenic development in a human model.
temporal expression of cell growth and osteoblast phenotype-related genes in a rodent model were first described by stein et al.
<cit> .
in our study, using large-scale expression profiling of clinical samples we could associate gene clusters to the three distinct phases of the development of osteoblast phenotype: proliferation, matrix maturation and mineralization.
second, we could identify markers of late osteogenesis: id <dig>  cryab and sort1:
a) id <dig> is one of the four members of the id  transcription factor family, which belongs to the basic-helix-loop-helix  family.
the id members play a key role in differentiation processes and are involved in cell cycle control and cellular senescence  <cit> .
it has been shown that id proteins, by heterodimerization with bhlh proteins , inhibit their binding to dna, thus acting as dominant negative regulators, and have to be down-regulated to continue the differentiation process  <cit> .
to the best of our knowledge only id <dig>  id <dig> and id <dig> have been identified as most significantly up-regulated early targets of the osteogenic bmp <dig>  bmp <dig> and bmp <dig>  <cit> .
in our study we provide evidence that also id <dig> exhibits a specific role during the osteogenic differentiation.
b) the second identified gene is crystallin-αb .
alpha crystallins are small heat shock proteins and composed of two gene products: alpha-a and alpha-b, for acidic and basic.
alpha-a is preferentially restricted to the vertebrate eye lens where it maintains the transparency and refractive index of the lens, whereas alpha-b is widely expressed in many tissues and organs  <cit> .
the here observed up-regulation of cryab suggests an involvement in osteogenic differentiation but its specific role is still unknown.
this involvement is supported by furushima et al.
<cit> , who performed a linkage study with knowledge-based candidate genes to detect genetic determinants associated with opll , a predominant myelopathy among japanese.
the candidate genes for examination were obtained by cdna microarray analysis of gene expression profiles during osteoblastic differentiation of msc.
among the  <dig> genes identified in cdna microarray analysis which could be associated with bone metabolism, cryab was the only gene which showed significant evidence of linkage.
c) further, we could show the role of sortilin <dig> during osteogenic differentiation.
sortilin  <dig>  represents a multi-ligand type- <dig> receptor which binds a number of unrelated ligands that participate in a wide range of cellular processes.
the expression of sort <dig> was increasing until day seven and then slowly decreasing.
sortilin <dig>  also known as neurotensin receptor- <dig>  is a glycoprotein originally purified from human brain  <cit> .
the expression of sortilin <dig> is ubiquitous; it has been observed in heart, brain, placenta, lung, skeletal, muscle, pancreas, prostate, testis, small intestine, thyroid, and spinal cord, whereas its expression in bone marrow could not be detected  <cit> .
the function of srotilin <dig> in bone metabolism is poorly understood.
maeda et al.
<cit>  reported first differential expression during osteogenic differentiation and performed further experiments, which showed that sortilin  <dig> is promoting extracellular matrix maturation.
our results presented here strengthen this hypothesis, since sortilin  <dig> shows a similar expression progression as other well known osteogenic genes, such as alpl or bmps.
in summary, we have identified by expression profiling and confirmed by quantitative rt-pcr three markers of late osteogenesis.
further studies are necessary to quantify the expression of id <dig>  sort- <dig> and cryab in primary human osteoblasts and other cell types and to identify the role of these genes in osteogenesis.
third, by mapping the obtained expression data on curated biomolecular pathways, we were able to illustrate the distinct activation of the different smad pathways induced by bmps, tgf-β and inhibin, which were summarized as tgf-β pathways.
the mapping of the expression data demonstrated that the fate of cell differentiation was simultaneously controlled at three different points: 1) bmp <dig>  bmp <dig> and bmp <dig> activated the smad-signaling pathway, consisting of the main components smad  <dig>  smad  <dig> and smad  <dig> resulting in osteoblast differentiation; 2) tgf-β <dig>  regulating smad  <dig> and smad  <dig>  was down regulated and the subsequent mechanisms were suppressed; and 3) inhibin was, like tgf-β <dig>  down regulated.
these findings have been confirmed by recent reports describing the diverse smad-signaling pathways, but have never been illustrated in a general overview before this study  <cit> .
finally, the comparison of in vitro differentiated osteoblasts with in vivo developed osteoblasts on morphological and genetic level proved the usefulness of msc as in vitro model for the investigation of the osteogenic development.
CONCLUSIONS
the here presented results demonstrate that msc represent a cell population which can be expanded ex vivo and differentiated into osteoblasts.
the genomic approach presented here represents a powerful tool to systematically and comprehensively investigate therapeutic approaches and hence, facilitate translational research on stem cells.
however, it should be pointed out that new quality and safety standards have to be developed to guarantee risk free usage of ex vivo manipulated cell material for future therapeutic applications.

 in recent years mesenchymal stem cells have generated a great deal of interest as a potential source for cell-based therapeutic strategies. human msc are easy to isolate from small aspirate of bone marrow via their adherence ability. these cells readily generate single-cell-derived colonies that can be highly expanded and differentiated into a variety of cell types, such as osteoblasts, adipocytes, myocytes, astrocytes and neurons. further, human msc can improve cardiac function after infarction or symptoms of bone and cartilage defects, as well as neurodegenerative diseases such as alzheimer's. their efficiency in multiple types of cellular therapeutic strategies has been demonstrated, including applications in treating children with osteogenesis imperfecta, hematopoietic recovery, and bone tissue regeneration. also first preclinical trails are in progress to test their capacity and toxicity in applications for human treatment. one great advantage of msc is that these cells may be directly obtained from individual patients, thereby eliminating the complications associated with immune rejection of allogenic tissue and infectious diseases. however, for cell therapies msc have to be expanded and/or manipulated to obtain a sufficient amount of cells that can be subsequently used for treatment. despite growing experience and knowledge concerning human msc and their use in cell-based strategies, the molecular mechanisms that govern msc self-renewal, expansion and multilineage differentiation are not well understood and remain an active area of investigation. in this study we asked if human msc are developing in an aberrant or unwanted way during ex vivo long-term cultivation and if cultivation conditions exert any influence on their stem cell maintenance. to address this question systematically and comprehensively we first developed human oligonucleotide microarrays with elements and then performed large-scale expression profiling of long-term expanded msc isolated from clinically relevant samples. we monitored these cells during their expansion ex vivo with respect to proliferation kinetics, surface marker profile and differentiation potential. finally we analyzed the gene expression profiles of msc during osteogenic differentiation. our results showed that expansion of msc does not result in substantial genetic and morphological aberrations. we illustrated for the first time in a human model the three main stages of osteogenic development, and we could show the diverse regulation of the smad pathways by tgf-β and bmps.  human msc maintain their undifferentiated phenotype during long-term expansion the results of the ex vivo long-term expansion experiments showed that the undifferentiated phenotype of mscs is maintained with respect to differentiation potential, surface marker profile and gene expression profiles. at confluence of 75–85% the cultivated cells were detached and flow cytometry analysis were performed to verify the purity of the cell population without any contaminations, such as haematopoietic cells. following surface marker profile were detected: cd44+, cd90+, cd73+, cd105+, cd166+, cd11b-, cd34-, cd45-, cd117-, hla dr-. for the subsequent experiments mscs were expanded until the end of the tenth passage. at the end of each passage the cells were analyzed by flow cytometry and a cell aliquot was seeded out for testing their differentiation ability. proliferation kinetics and differentiation potential the growth kinetics of five donors was investigated from the primary culture through the tenth passage, corresponding to approximately cell doublings. for recalculating the starting number of msc in the mnc fraction, cfu assays were performed and the msc frequency was determined. primary cultures reached their first confluence from around 80% in about weeks and about cell doublings. during the following passages the proliferation rate slowed down. during the whole expansion period – starting with passage two until passage ten – in every passage the osteogenic differentiation ability was tested. the osteogenic differentiation was confirmed by alizarin red s staining and alkaline phosphatase assay. throughout this examination period differentiation ability into osteoblasts was observed in low passages as well as in a higher passage,. msc expanded to approximately cell doublings were able to differentiate into adipocytes. surface marker profile after the cultivation of msc from the mononuclear cell fraction and at the end of each individual passage, the msc were analyzed by flow cytometry to monitor the surface marker profile during their ex vivo expansion. each investigated passage depicted homogenous cell population demonstrating that msc did not alter their physical and morphological properties during high grade ex vivo expansion under the culture conditions tested. in addition to the examination of the surface marker profile, we also performed real-time rt-pcr sybr™ green i assays for stem cell specific genes. the examined stem cell markers, cd cd cd cd cd and stro showed stable expression level during the whole expansion period proving the results obtained by facs analyses. as expected, cd cd and tert could not be detected on any examined time point. large-scale gene expression profiling of msc during ex vivo expansion in order to monitor the molecular events that govern the ex vivo expansion of undifferentiated msc we performed large-scale gene expression profiling using oligonucleotide microarrays with elements. after background correction, global mean and dye swap normalization, and replicate handling, genes could be detected as differentially expressed with at least 2-fold change, between cells from passage with cells from passage and genes, when comparing passage cells with passage cells. a modified version of the regularized t-test with the complete data set was performed to identify significantly differentially expressed genes in ten examined donors and five examined donors. the results showed that only genes were significantly differentially expressed during cell doublings when comparing cells from passage versus cells from passage. significantly differentially expressed genes represent proteins with diverse functions: extracellular matrix proteins, proteins involved in bone, fat and epithelial cell metabolism, pain perception and stress response, growth-promoting activity, and signaling and cell survival mechanisms. the hypothetical protein xp_ is annotated as similar to keratinocyte growth factor-like protein, and the hypothetical protein dkfz434b is defined as a cysteine-rich secretory protein containing lccl domain the expression data from the above mentioned genes could be confirmed by independent real-time rt-pcr. list of differentially expressed genes after statistical analysis by modified version of the regularized t-test for ten donors and five donors. in sum, these results show that long-term expansion of msc did not alter their differentiation potential, surface marker profiles, and expression profiles. expression profiling of msc during osteogenic differentiation reveals distinct phases of osteogenic development osteogenic differentiation human msc from three different donors were cultured until their fourth passage and were induced to osteogenic differentiation at a confluence of 70–80%. the osteogenic differentiation was observed during the whole period by microscopy and was terminated on day after induction. first signs for calcification appeared as black regions within the cell monolayer after around seven days. the maximum of calcified extracellular matrix was observed after days of treatment. the osteoblast phenotype was confirmed by alkaline phosphatase assay and alizarin red s staining. expression of osteoblast specific marker genes we performed real-time rt-pcr assays to detect the expression levels of osteoblast specific marker genes msx, vdr, col1a, alpl, sparc, spp and bglap to prove the osteoblast phenotype. the log transformed results are shown in the supplementary material. the data obtained by real-time rt-pcr showed the expected expression profiles of the osteoblast phenotype. alpl showed the expected expression level progression. its expression was increased starting with day four of the differentiation period and decreased after day until day during the mineralization phase. the homeobox gene msx which is implicated in osteoprogenitor cell function and an up-stream regulator of runx which was described to be important for the osteogenic differentiation, were up-regulated during the whole period of differentiation. col1a is known to be an early marker of osteoprogenitor cells. its maximum of expression was reached on day also sparc showed a nearly constant, up-regulated expression level with its maximum on day of differentiation. spp is an extracellular matrix protein, known to peak twice in its expression: around day during proliferation, and between days 14– during mineralization. bglap was described as a late marker of developing osteoblasts appearing with matrix mineralization and was maximally expressed on day interestingly, bglap was already significantly up-regulated on day four, whereas the mineralization could not be observed before day seven. large-scale gene expression profiling of msc during osteogenesis the rna was harvested at four time points during the differentiation period and hybridized on human oligonucleotide microarrays. as reference, rna harvested from msc one day before the osteogenic induction was used. after lowess-subgrid normalization and filtering missing values genes were selected with two-fold up- or downregulated expression in at least one time point. correspondence between co-expressed genes and phenotypic changes differentially expressed genes were clustered according to their expression profiles using the k-means clustering method. the cluster analysis was performed with at maximum iterations and using different clusters, each containing between and genes. the number of clusters was estimated by fom analyses. additionally, hierarchical clustering was performed and the results were comparable to k-means clustering. by analyzing the genes of the different clusters and their expression profiles, we were able to reconstruct three main phases of osteogenic development: proliferation, matrix maturation, and mineralization. additionally, novel candidate genes could be found which are not known to play a role during osteogenic differentiation: id, cryab and sort. the expression of these genes was subsequently confirmed by quantitative rt-pcr. for gene ontology analysis we categorized ests with available refseq annotation according to go terms for biological process, cellular component, and molecular function. in detail, cluster and represented proliferation phase describing genes, which were involved in signal transduction, regulation of cell cycle, metabolism and regulation of transcription. cluster and illustrated genes which were up-or down regulated during the whole period of differentiation. they describe the phase matrix maturation and are involved in development, cell differentiation, metabolism, regulation of dna-dependent transcription. the phase of mineralization is evident in cluster and which include genes involved in signal transduction, transport, metabolism, and development. the go analysis showed that genes found in cluster and were also involved in cell adhesion, signal transduction, cell cycle, and regulation of transcription, similar to cluster and cluster described genes of development and metabolism among the rest. cluster and combined genes of cell adhesion, transport, cell proliferation and regulation of transcription. cluster represented genes associated with transport and regulation of transcription. in summary, go annotation showed that nearly in each cluster genes of cell adhesion, signal transduction, transport, and regulation of transcription were present, illustrating the complex regulation of different events during osteogenesis. pathway analysis of the expression data set the data set of genes with significant up or down regulation was mapped with the pathway explorer on pathways derived from the biocarta and kegg database. in total out of genes could be mapped with their available refseq numbers. only pathways where at least 10% and more than five mapped genes could be found were selected for further examination. following these criteria the tgf-β signaling pathway was ranked first, which summarized the different smad pathways regulated by the members of the tgf-β family: bmp, tgf-β and inhibin. cell growth and proliferation is controlled by the members of the tgf-β superfamily which includes structurally related proteins like tgf-β, activins, and bone morphogenetic proteins. in the here presented tgf-β signaling pathway bmp and bmp madh and madh were up-regulated and the inhibitor of dna binding id was down-regulated. bmp and bmp are able to bind to bmpr-type i and ii, whereas bmp is able to bind to actriia. due to the binding of these two molecules to the heterodimeric receptor complex the smad signaling pathway is activated. our data illustrated further, that the inhibitor of dna binding, id was down regulated and the osteogenic differentiation could be induced. in parallel, tgf-β inhibin a and b were down-regulated during the differentiation period, whereby the following events were suppressed. hence, under these experimental conditions the smad-signaling pathway activated by bmp was responsible for the osteogenic development. in contrast, the activation of the smad pathway by tgf-β and inhibin was suppressed. in vivo relevance of human msc as in vitro differentiation model the in vitro differentiation of msc into osteoblasts is often used model to examine the regulation mechanisms during osteoblast development. to evaluate the in vivo relevance of this model, we compared the rna obtained at the diverse time points during osteogenic differentiation with rna isolated from primary cultures of normal non-mineralized human osteoblasts derived from hipbone using our microarrays. the results showed that msc on day seven of their differentiation were most similar to human osteoblasts. this observation could be confirmed by the comparison of the morphology of the two cell types: human osteoblasts isolated from hipbone represented non-mineralized osteoblasts with fibroblast-like phenotype, morphologically similar to induced msc on day seven after induction. discussion in this study we could show for the first time that msc maintain their undifferentiated phenotype after ex vivo long-term expansion. the results of the microarray assays showed that mscs did not change significantly their gene expression profiles during long-term expansion since only nine out of almost genes were differentially expressed between passage and passage in ten donors. in addition, no changes in the expression profiles after additional passages could be observed in five of the ten donors. these surprising results can only be partially explained by the limitations of the microarray technology. due to the lower sensitivity and uncomplete coverage of the transcriptome, some genes required for stem cell maintenance might have been missed. additionally, individual differences between the donors and slightly different conditions during tissue extraction and sample treatment could increase the number identified genes which were otherwise filtered out during averaging. however, the microarray results were supported by the analyses of the effects of extensive long-term ex vivo cultivation of msc on their proliferation capacity, their morphology, surface marker profile, and differentiation ability. during the long cultivation time we could observe, in concordance with other groups, a progressive lost of proliferation ability and a slight shift in the cell-morphology of msc of higher passage. however, their surface marker profile determined by flow cytometry and by real-time rt-pcr was not altered. over and above, the stem cell markers like cd and stro showed stable expression levels, which further supports our observation that msc maintained their undifferentiated phenotype and remained capable of osteogenic differentiation during ex vivo expansion at all tested passages. additionally to this clinically relevant evidence, we provide novel biological insights derived from the global view of the molecular processes during osteogenic development. first, we were able to illustrate the different phases of osteogenic development in a human model. temporal expression of cell growth and osteoblast phenotype-related genes in a rodent model were first described by stein et al.. in our study, using large-scale expression profiling of clinical samples we could associate gene clusters to the three distinct phases of the development of osteoblast phenotype: proliferation, matrix maturation and mineralization. second, we could identify markers of late osteogenesis: id cryab and sort1: a) id is one of the four members of the id transcription factor family, which belongs to the basic-helix-loop-helix family. the id members play a key role in differentiation processes and are involved in cell cycle control and cellular senescence. it has been shown that id proteins, by heterodimerization with bhlh proteins, inhibit their binding to dna, thus acting as dominant negative regulators, and have to be down-regulated to continue the differentiation process. to the best of our knowledge only id id and id have been identified as most significantly up-regulated early targets of the osteogenic bmp bmp and bmp. in our study we provide evidence that also id exhibits a specific role during the osteogenic differentiation. b) the second identified gene is crystallin-αb. alpha crystallins are small heat shock proteins and composed of two gene products: alpha-a and alpha-b, for acidic and basic. alpha-a is preferentially restricted to the vertebrate eye lens where it maintains the transparency and refractive index of the lens, whereas alpha-b is widely expressed in many tissues and organs. the here observed up-regulation of cryab suggests an involvement in osteogenic differentiation but its specific role is still unknown. this involvement is supported by furushima et al., who performed a linkage study with knowledge-based candidate genes to detect genetic determinants associated with opll, a predominant myelopathy among japanese. the candidate genes for examination were obtained by cdna microarray analysis of gene expression profiles during osteoblastic differentiation of msc. among the genes identified in cdna microarray analysis which could be associated with bone metabolism, cryab was the only gene which showed significant evidence of linkage. c) further, we could show the role of sortilin during osteogenic differentiation. sortilin represents a multi-ligand type- receptor which binds a number of unrelated ligands that participate in a wide range of cellular processes. the expression of sort was increasing until day seven and then slowly decreasing. sortilin also known as neurotensin receptor- is a glycoprotein originally purified from human brain. the expression of sortilin is ubiquitous; it has been observed in heart, brain, placenta, lung, skeletal, muscle, pancreas, prostate, testis, small intestine, thyroid, and spinal cord, whereas its expression in bone marrow could not be detected. the function of srotilin in bone metabolism is poorly understood. maeda et al. reported first differential expression during osteogenic differentiation and performed further experiments, which showed that sortilin is promoting extracellular matrix maturation. our results presented here strengthen this hypothesis, since sortilin shows a similar expression progression as other well known osteogenic genes, such as alpl or bmps. in summary, we have identified by expression profiling and confirmed by quantitative rt-pcr three markers of late osteogenesis. further studies are necessary to quantify the expression of id sort- and cryab in primary human osteoblasts and other cell types and to identify the role of these genes in osteogenesis. third, by mapping the obtained expression data on curated biomolecular pathways, we were able to illustrate the distinct activation of the different smad pathways induced by bmps, tgf-β and inhibin, which were summarized as tgf-β pathways. the mapping of the expression data demonstrated that the fate of cell differentiation was simultaneously controlled at three different points: 1) bmp bmp and bmp activated the smad-signaling pathway, consisting of the main components smad smad and smad resulting in osteoblast differentiation; 2) tgf-β regulating smad and smad was down regulated and the subsequent mechanisms were suppressed; and 3) inhibin was, like tgf-β down regulated. these findings have been confirmed by recent reports describing the diverse smad-signaling pathways, but have never been illustrated in a general overview before this study. finally, the comparison of in vitro differentiated osteoblasts with in vivo developed osteoblasts on morphological and genetic level proved the usefulness of msc as in vitro model for the investigation of the osteogenic development.  the here presented results demonstrate that msc represent a cell population which can be expanded ex vivo and differentiated into osteoblasts. the genomic approach presented here represents a powerful tool to systematically and comprehensively investigate therapeutic approaches and hence, facilitate translational research on stem cells. however, it should be pointed out that new quality and safety standards have to be developed to guarantee risk free usage of ex vivo manipulated cell material for future therapeutic applications. 
the results showed that msc did not alter their osteogenic differentiation capacity, surface marker profile, and the expression profiles of msc during expansion. microarray analysis of msc during osteogenic differentiation identified three candidate genes for further examination and functional analysis: id cryab, and sort additionally, we were able to reconstruct the three developmental phases during osteoblast differentiation: proliferation, matrix maturation, and mineralization, and illustrate the activation of the smad signaling pathways by tgf-β and bmps. to address this question we first developed human oligonucleotide microarrays with elements and then performed large-scale expression profiling of long-term expanded msc and msc during differentiation into osteoblasts. however, their low frequency in bone marrow necessitate ex vivo expansion for further clinical application. in this study we asked if msc are developing in an aberrant or unwanted way during ex vivo long-term cultivation and if artificial cultivation conditions exert any influence on their stem cell maintenance. with a variety of assays we could show that msc represent a cell population which can be expanded for therapeutic applications.
 human mesenchymal stem cells with the capacity to differentiate into osteoblasts provide potential for the development of novel treatment strategies, such as improved healing of large bone defects. however, their low frequency in bone marrow necessitate ex vivo expansion for further clinical application. in this study we asked if msc are developing in an aberrant or unwanted way during ex vivo long-term cultivation and if artificial cultivation conditions exert any influence on their stem cell maintenance. to address this question we first developed human oligonucleotide microarrays with elements and then performed large-scale expression profiling of long-term expanded msc and msc during differentiation into osteoblasts.  the results showed that msc did not alter their osteogenic differentiation capacity, surface marker profile, and the expression profiles of msc during expansion. microarray analysis of msc during osteogenic differentiation identified three candidate genes for further examination and functional analysis: id cryab, and sort additionally, we were able to reconstruct the three developmental phases during osteoblast differentiation: proliferation, matrix maturation, and mineralization, and illustrate the activation of the smad signaling pathways by tgf-β and bmps.  with a variety of assays we could show that msc represent a cell population which can be expanded for therapeutic applications. 