BACKGROUND
during the odontogenesis, reciprocal epithelial-mesenchymal interactions are of paramount importance to the tooth initiation and subsequent dental morphogenesis  <cit> .
at the beginning of odontogenesis, the ectomesenchyme provide the initial inductive signals and bring about the formation of dental placode.
subsequent cell proliferation, condensation, polarization, and differentiation of the epithelium and mesenchyme contribute to the crown morphogenesis  <cit> .
at the cap stage, dental mesenchyme has acquired the dentinogenic capacity to instruct subsequent tooth development  <cit> .
when the first layer of dentin matrix is generated between dental epithelium and mesenchyme, the reciprocal interactions are interrupted because of the dentin barrier.
dental mesenchymal cells in this stage can still perform the regular dentinogenesis without the epithelial-mesenchymal interactions  <cit> .
at the following developmental stages, the primary and secondary dentinogenesis mediated by the dental mesenchyme are still going on around the dental pulp in the absence of epithelial components throughout the rest life of a healthy tooth.
our previous study has proved that induced dental pulp stem cells  can undergo the odontoblastic differentiation and dentinogenesis in the presence of epithelial signals  <cit> , however, whether naive dpscs can spontaneously give birth to the odontoblasts and solely carry out the dentinogenesis in the absence of inductive agents  has not been fully elucidated.
many studies have demonstrated that dpscs have the ability to undertake the self renewal and differentiate into the neurogenic, osteogenic, dentinogenic, and myogenic cell lineages in different inductive media  <cit> .
as a type of adult stem cells, dpscs usually perform the asymmetric cell division, which gives rise to one daughter cell with stem-cell fate and another which can pursue further cell divisions to generate differentiated progenies.
for the lack of specific cell surface markers, the identification of dpscs mainly relies on their biological features, including small cell volume, high proliferation potency, high clonogenicity, self-renewal, and multiple differentiation potential  <cit> .
dpscs in vivo usually remain quiescent within adult dental pulps, but respond during injury to produce progenies with high proliferative potential which can differentiate into terminally differentiated odontoblasts.
thus, the amount of dpscs in the normal dental pulp remains relatively constant.
when one dpsc divide  <dig> times, it will generate  <dig> lineage-specific progeny cells plus one unaltered daughter stem cell.
these lineage-specific progenies in vivo may bring about different cell types which contribute to the maintenance and homeostasis of dental pulp tissues.
however, little is known about the fate of these lineage-specific progenies after serial passaging.
to date, there is no optimal culture medium that can allow adult stem cell amplification without differentiation  <cit> , it is reasonable that naive dpscs in vitro can spontaneously differentiate  into mature cell lineages via asymmetric cell division.
for this purpose, this study was designed to evaluate the differentiation potential of dpscs at different passages in the routine culture system.
the findings presented in this study indicate that stro-1+ dpscs in vitro can spontaneously differentiate into several mature cell lineages with passage time, while the differentiation capacity of these stem cells changes during cell passaging and dpscs at the 9th passage in vivo restrict their differentiation potential to osteoblast lineages.
RESULTS
morphological and growth features of stro-1+ dpscs at the 1st and 9th passages
to investigate the proliferation features of stro-1+ dpscs at different passages, growth curve was plotted with culture time and population doubling time  was calculated according to the patterson formulation.
as compared with polymorphic dpscs  at the 1st passage , some dpscs at the 9th passage  displayed the enlarged cell bodies and elongated appearance with one or two long cellular processes .
as showed in the growth curve , cells began to grow exponentially at day  <dig> in dpsc-p <dig> group and at day  <dig> in dpsc-p <dig> group respectively after an initial lag phase.
in the logarithmic phase, population doubling time  in dpsc-p <dig> group  was longer than that in dpsc-p <dig>  group, indicating a downregulated proliferation potential in dpsc-p <dig> cells.
gene and protein expression of stro-1+ dpscs at the 1st and 9th passages
to evaluate the phenotype changes of in vitro dpscs during the long-term passage, the gene/protein expression of stro-1+ dpscs at the 1st passage and 9th passage was detected by means of real-time rt-pcr and western blot respectively.
the gene expression of alkaline phosphatase , osteopontin , osterix , bone sialoprotein , dentin sialophosphoprotein , and type ii collagen  was significantly upregulated in dpsc-p <dig> group, in comparison with dpsc-p <dig> group .
western blot results further showed that the expression of osteoblast-specific matrix proteins , odontoblast marker protein , and chondrocyte marker protein  was significantly elevated in dpsc-p <dig> cells, as compared with dpsc-p <dig> cells .
there was no significant difference between two groups in the gene/protein expression  for runt-related transcription factor  <dig> , type i collagen , and osteonectin .
since runx <dig> is an early osteoblastic marker  <cit> , the similar levels of runx <dig> expression in dpsc-p <dig> and dpsc-p <dig> cells may imply that these two groups share almost the same percentage of immature osteoblasts.
alp and calcium deposition of stro-1+ dpscs in the mineralization-inducing medium
to further study their differentiation pace towards osteo-/odontoblast lineages in vitro, stro-1+ dpscs at different passages were cocultured with the mineralization-inducing media.
dpsc-p <dig> group in the mineralization-inducing media presented a higher alp level than dpsc-p <dig> group at day  <dig> and day  <dig> respectively .
the results of alizarin red staining demonstrated that more mineralization nodules were detected in dpsc-p <dig> group than in dpsc-p <dig> group after 14-day induction in the mineralization-inducing media .
the calcium concentrations in induced dpsc-p <dig> group were much higher than those in induced dpsc-p <dig> group .
in vivo differentiation of stro-1+ dpscs at the 1st and 9th passages
to further assess the self-differentiation ability of stro-1+ dpscs in vivo, rat dpsc pellets at the 1st and 9th passages were transplanted into the renal capsules of adult rat hosts.
all implants were retrieved at day  <dig> post-transplantation and processed for hematoxylin and eosin staining.
in vivo transplantation results showed that all dpsc-p <dig> cell pellets gave birth to woven bone tissues , among which  <dig> samples  simultaneously produced dentin structures  with typical odontoblasts aligning along the inner side of the predentin and  <dig> samples  generated cartilage structures .
osteoblasts and osteocytes were distinct around or inside bone structures , while chondrocytes can be found in the cartilage tissues .
all dpsc-p <dig> cell pellets  in vivo developed into bone tissues with thicker matrix, less lacunae, and less osteocytes.
no osteoblast can be observed around bone tissues in dpsc-p <dig> group .
there was a statistically significant difference  in the formation rate of dentin tissues between dpsc-p <dig> and dpsc-p <dig> pellets, whereas the differences between two groups were not statistically significant in the formation rate of cartilage  or bone  tissues.
discussion
stem cell is a cell that can continuously give birth to unaltered daughters and also has the ability to generate progenitors that will differentiate into mature cell types.
in this study, dpsc-p <dig> cells presented the enlarged cell bodies, decreased proliferation ability, upregulated marker gene/protein expression of different matrix-forming cells, as compared with dpsc-p <dig> cells.
these findings indicate that stro-1+ dpscs are spontaneously undergoing the multi-differentiation along different matrix-forming cell lineages with passage time.
many studies have proved that dpscs can differentiate into odontoblast, osteoblast, and chondrocyte lineages in the presence of different inductive agents   <cit> .
our results revealed for the first time that dpscs at the 9th passage can spontaneously develop into odontoblasts, osteoblasts, and chondrocytes in the routine culture media, as indicated by the elevated gene/protein expression of dspp/dsp, alp, opn, osx, bsp, and col ii.
when transplanted into the renal capsules, dpsc-p <dig> cells can respectively generate bone, dentin, and cartilage structures after  <dig> days of in vivo incubation, further suggesting that these stem cells are composed of several interrelated subpopulations including the progenitors of odontoblasts, osteoblasts, and chondrocytes.
the multi-differentiation capacity of these stem cells in different inductive media, as proven in many studies  <cit> , may be attributed to these different subpopulations residing in dpscs.
when undifferentiated dpscs are cultured in the inductive media , only one subpopulation  can grow in a prevalent way and give birth to the differentiated mature cells .
stro- <dig> is thought to be a putative cell surface marker for the isolation of stem cells from both human and rat dental pulps  <cit> .
when recombined with the scaffold hydroxyapatite/tricalcium phosphate , stro-1+ dpscs can bring about the formation of abundant hard tissues in the subcutaneous area of nude mice  <cit> .
the recombinations between stro-1+ dpscs and ha/tcp in vitro can trigger the upregulation of several odontoblast-related gene expression including bsp, dspp, and dentin matrix protein  <dig> .
in the present study, the most striking feature of stro-1+ dpscs is their ability to self-differentiate into odontoblasts with passage time, as indicated by the expression of dspp gene and dsp protein in dpsc-p <dig> cells.
when transplanted in vivo, stro-1+ dpscs at the 1st passage can give rise to the formation of dentin-pulp-like complex containing typical columnar odontoblasts.
these data strongly imply that in situ odontoblasts are derived from dpscs in adult dental pulps.
the terminal differentiation of odontoblasts in the adult stages thus may contain the following successive stages: dpsc, odontoblast precursor, preodontoblast, young odontoblast, mature  odontoblast, and aging odontoblast  <cit> .
after the asymmetric cell division, dpscs can exit from the self-renewal, narrow their differentiation potential to the odontoblast lineage and give rise to the differentiated daughter cells including odontoblast precursors, pre-odontoblasts, and terminally differentiated odontoblasts  <cit> .
although the marker genes/proteins of osteoblasts, odontoblasts, and chondrocytes were upregulated in stro-1+ dpscs at the 9th passage in vitro, these stem cells can only produce bone tissues in vivo, due to the following reasons.
firstly, dpsc-p <dig> cells presented a much stronger osteo-dentinogenic differentiation capacity than dpsc-p <dig> cells as indicated by elevated alp levels and calcium deposition in the inductive medium.
these differentiated stem cells at the 9th passage have a stronger matrix-secreting ability, trap themselves in the fast-deposited matrix and ultimately result in the formation of a big mineralized mass with few cells inside.
secondly, to date, there is no optimal medium that can keep adult stem cells in an undifferentiated state during in vitro cell amplification.
long-term in vitro passage of stem cells may induce the cell differentiation, loss of self renewal ability  <cit> , and decreased percentage of undifferentiated stem cells due to the asymmetric cell division.
when these stem cells at the 9th passage are implanted into the renal capsules, most stem cells are in a differentiated state, which conversely leads to the decrease of stem cell number, results in the loss of balance between stem cell proliferation and differentiation, and finally brings about the irregular tissue development  <cit> .
since the difference between dentin and bone is largely due to the different arrangement of matrix  <cit> , irregular array of dentin and bone matrix secreted by odontoblasts and osteoblasts respectively may bring about the same appearance of bone-like calcification.
finally, the cell number of differentiated chondrocytes may be too limited  to perform the sustainable morphogenesis of cartilage in dpsc-p <dig> group.
together, dpsc-p <dig> cells in vivo can not perform the dentinogenesis or chondrogenesis but restrict their differentiation potential to the osteogenesis and generate the mineralized mass with few cells scattered inside.
CONCLUSIONS
our observations suggest that stro-1+ dpscs consist of at least three interrelated subpopulations including the progenitors of odontoblasts, osteoblasts, and chondrocytes, which can form dentin, bone, and cartilage tissues respectively.
the differentiation potential of these stem cells changes during serial cell passaging, and there is a restriction in the differentiation capacity of in vivo dpscs at the 9th passage, i.e., they can only differentiate along the osteoblast lineages.
further study is necessary to trace the mechanism of the restriction as well as the fate of other subpopulations residing in dpscs.

 during the odontogenesis, reciprocal epithelial-mesenchymal interactions are of paramount importance to the tooth initiation and subsequent dental morphogenesis. at the beginning of odontogenesis, the ectomesenchyme provide the initial inductive signals and bring about the formation of dental placode. subsequent cell proliferation, condensation, polarization, and differentiation of the epithelium and mesenchyme contribute to the crown morphogenesis. at the cap stage, dental mesenchyme has acquired the dentinogenic capacity to instruct subsequent tooth development. when the first layer of dentin matrix is generated between dental epithelium and mesenchyme, the reciprocal interactions are interrupted because of the dentin barrier. dental mesenchymal cells in this stage can still perform the regular dentinogenesis without the epithelial-mesenchymal interactions. at the following developmental stages, the primary and secondary dentinogenesis mediated by the dental mesenchyme are still going on around the dental pulp in the absence of epithelial components throughout the rest life of a healthy tooth. our previous study has proved that induced dental pulp stem cells can undergo the odontoblastic differentiation and dentinogenesis in the presence of epithelial signals, however, whether naive dpscs can spontaneously give birth to the odontoblasts and solely carry out the dentinogenesis in the absence of inductive agents has not been fully elucidated. many studies have demonstrated that dpscs have the ability to undertake the self renewal and differentiate into the neurogenic, osteogenic, dentinogenic, and myogenic cell lineages in different inductive media. as a type of adult stem cells, dpscs usually perform the asymmetric cell division, which gives rise to one daughter cell with stem-cell fate and another which can pursue further cell divisions to generate differentiated progenies. for the lack of specific cell surface markers, the identification of dpscs mainly relies on their biological features, including small cell volume, high proliferation potency, high clonogenicity, self-renewal, and multiple differentiation potential. dpscs in vivo usually remain quiescent within adult dental pulps, but respond during injury to produce progenies with high proliferative potential which can differentiate into terminally differentiated odontoblasts. thus, the amount of dpscs in the normal dental pulp remains relatively constant. when one dpsc divide times, it will generate lineage-specific progeny cells plus one unaltered daughter stem cell. these lineage-specific progenies in vivo may bring about different cell types which contribute to the maintenance and homeostasis of dental pulp tissues. however, little is known about the fate of these lineage-specific progenies after serial passaging. to date, there is no optimal culture medium that can allow adult stem cell amplification without differentiation, it is reasonable that naive dpscs in vitro can spontaneously differentiate into mature cell lineages via asymmetric cell division. for this purpose, this study was designed to evaluate the differentiation potential of dpscs at different passages in the routine culture system. the findings presented in this study indicate that stro-1+ dpscs in vitro can spontaneously differentiate into several mature cell lineages with passage time, while the differentiation capacity of these stem cells changes during cell passaging and dpscs at the 9th passage in vivo restrict their differentiation potential to osteoblast lineages.  morphological and growth features of stro-1+ dpscs at the 1st and 9th passages to investigate the proliferation features of stro-1+ dpscs at different passages, growth curve was plotted with culture time and population doubling time was calculated according to the patterson formulation. as compared with polymorphic dpscs at the 1st passage, some dpscs at the 9th passage displayed the enlarged cell bodies and elongated appearance with one or two long cellular processes. as showed in the growth curve, cells began to grow exponentially at day in dpsc-p group and at day in dpsc-p group respectively after an initial lag phase. in the logarithmic phase, population doubling time in dpsc-p group was longer than that in dpsc-p group, indicating a downregulated proliferation potential in dpsc-p cells. gene and protein expression of stro-1+ dpscs at the 1st and 9th passages to evaluate the phenotype changes of in vitro dpscs during the long-term passage, the gene/protein expression of stro-1+ dpscs at the 1st passage and 9th passage was detected by means of real-time rt-pcr and western blot respectively. the gene expression of alkaline phosphatase, osteopontin, osterix, bone sialoprotein, dentin sialophosphoprotein, and type ii collagen was significantly upregulated in dpsc-p group, in comparison with dpsc-p group. western blot results further showed that the expression of osteoblast-specific matrix proteins, odontoblast marker protein, and chondrocyte marker protein was significantly elevated in dpsc-p cells, as compared with dpsc-p cells. there was no significant difference between two groups in the gene/protein expression for runt-related transcription factor, type i collagen, and osteonectin. since runx is an early osteoblastic marker, the similar levels of runx expression in dpsc-p and dpsc-p cells may imply that these two groups share almost the same percentage of immature osteoblasts. alp and calcium deposition of stro-1+ dpscs in the mineralization-inducing medium to further study their differentiation pace towards osteo-/odontoblast lineages in vitro, stro-1+ dpscs at different passages were cocultured with the mineralization-inducing media. dpsc-p group in the mineralization-inducing media presented a higher alp level than dpsc-p group at day and day respectively. the results of alizarin red staining demonstrated that more mineralization nodules were detected in dpsc-p group than in dpsc-p group after 14-day induction in the mineralization-inducing media. the calcium concentrations in induced dpsc-p group were much higher than those in induced dpsc-p group. in vivo differentiation of stro-1+ dpscs at the 1st and 9th passages to further assess the self-differentiation ability of stro-1+ dpscs in vivo, rat dpsc pellets at the 1st and 9th passages were transplanted into the renal capsules of adult rat hosts. all implants were retrieved at day post-transplantation and processed for hematoxylin and eosin staining. in vivo transplantation results showed that all dpsc-p cell pellets gave birth to woven bone tissues, among which samples simultaneously produced dentin structures with typical odontoblasts aligning along the inner side of the predentin and samples generated cartilage structures. osteoblasts and osteocytes were distinct around or inside bone structures, while chondrocytes can be found in the cartilage tissues. all dpsc-p cell pellets in vivo developed into bone tissues with thicker matrix, less lacunae, and less osteocytes. no osteoblast can be observed around bone tissues in dpsc-p group. there was a statistically significant difference in the formation rate of dentin tissues between dpsc-p and dpsc-p pellets, whereas the differences between two groups were not statistically significant in the formation rate of cartilage or bone tissues. discussion stem cell is a cell that can continuously give birth to unaltered daughters and also has the ability to generate progenitors that will differentiate into mature cell types. in this study, dpsc-p cells presented the enlarged cell bodies, decreased proliferation ability, upregulated marker gene/protein expression of different matrix-forming cells, as compared with dpsc-p cells. these findings indicate that stro-1+ dpscs are spontaneously undergoing the multi-differentiation along different matrix-forming cell lineages with passage time. many studies have proved that dpscs can differentiate into odontoblast, osteoblast, and chondrocyte lineages in the presence of different inductive agents. our results revealed for the first time that dpscs at the 9th passage can spontaneously develop into odontoblasts, osteoblasts, and chondrocytes in the routine culture media, as indicated by the elevated gene/protein expression of dspp/dsp, alp, opn, osx, bsp, and col ii. when transplanted into the renal capsules, dpsc-p cells can respectively generate bone, dentin, and cartilage structures after days of in vivo incubation, further suggesting that these stem cells are composed of several interrelated subpopulations including the progenitors of odontoblasts, osteoblasts, and chondrocytes. the multi-differentiation capacity of these stem cells in different inductive media, as proven in many studies, may be attributed to these different subpopulations residing in dpscs. when undifferentiated dpscs are cultured in the inductive media, only one subpopulation can grow in a prevalent way and give birth to the differentiated mature cells. stro- is thought to be a putative cell surface marker for the isolation of stem cells from both human and rat dental pulps. when recombined with the scaffold hydroxyapatite/tricalcium phosphate, stro-1+ dpscs can bring about the formation of abundant hard tissues in the subcutaneous area of nude mice. the recombinations between stro-1+ dpscs and ha/tcp in vitro can trigger the upregulation of several odontoblast-related gene expression including bsp, dspp, and dentin matrix protein. in the present study, the most striking feature of stro-1+ dpscs is their ability to self-differentiate into odontoblasts with passage time, as indicated by the expression of dspp gene and dsp protein in dpsc-p cells. when transplanted in vivo, stro-1+ dpscs at the 1st passage can give rise to the formation of dentin-pulp-like complex containing typical columnar odontoblasts. these data strongly imply that in situ odontoblasts are derived from dpscs in adult dental pulps. the terminal differentiation of odontoblasts in the adult stages thus may contain the following successive stages: dpsc, odontoblast precursor, preodontoblast, young odontoblast, mature odontoblast, and aging odontoblast. after the asymmetric cell division, dpscs can exit from the self-renewal, narrow their differentiation potential to the odontoblast lineage and give rise to the differentiated daughter cells including odontoblast precursors, pre-odontoblasts, and terminally differentiated odontoblasts. although the marker genes/proteins of osteoblasts, odontoblasts, and chondrocytes were upregulated in stro-1+ dpscs at the 9th passage in vitro, these stem cells can only produce bone tissues in vivo, due to the following reasons. firstly, dpsc-p cells presented a much stronger osteo-dentinogenic differentiation capacity than dpsc-p cells as indicated by elevated alp levels and calcium deposition in the inductive medium. these differentiated stem cells at the 9th passage have a stronger matrix-secreting ability, trap themselves in the fast-deposited matrix and ultimately result in the formation of a big mineralized mass with few cells inside. secondly, to date, there is no optimal medium that can keep adult stem cells in an undifferentiated state during in vitro cell amplification. long-term in vitro passage of stem cells may induce the cell differentiation, loss of self renewal ability, and decreased percentage of undifferentiated stem cells due to the asymmetric cell division. when these stem cells at the 9th passage are implanted into the renal capsules, most stem cells are in a differentiated state, which conversely leads to the decrease of stem cell number, results in the loss of balance between stem cell proliferation and differentiation, and finally brings about the irregular tissue development. since the difference between dentin and bone is largely due to the different arrangement of matrix, irregular array of dentin and bone matrix secreted by odontoblasts and osteoblasts respectively may bring about the same appearance of bone-like calcification. finally, the cell number of differentiated chondrocytes may be too limited to perform the sustainable morphogenesis of cartilage in dpsc-p group. together, dpsc-p cells in vivo can not perform the dentinogenesis or chondrogenesis but restrict their differentiation potential to the osteogenesis and generate the mineralized mass with few cells scattered inside.  our observations suggest that stro-1+ dpscs consist of at least three interrelated subpopulations including the progenitors of odontoblasts, osteoblasts, and chondrocytes, which can form dentin, bone, and cartilage tissues respectively. the differentiation potential of these stem cells changes during serial cell passaging, and there is a restriction in the differentiation capacity of in vivo dpscs at the 9th passage, i.e., they can only differentiate along the osteoblast lineages. further study is necessary to trace the mechanism of the restriction as well as the fate of other subpopulations residing in dpscs. 
dental pulp stem cells can be driven into odontoblast, osteoblast, and chondrocyte lineages in different inductive media. during the long-term passage, the proliferation ability of human stro-1+ dpscs was downregulated as indicated by the growth kinetics. furthermore, human dpsc-p cells in the mineralization-inducing media presented higher levels of alkaline phosphatase at day and day respectively, and produced more mineralized matrix than dpsc-p cells at day in vivo transplantation results showed that rat dpsc-p cell pellets developed into dentin, bone and cartilage structures respectively, while dpsc-p cells can only generate bone tissues. the biological features of stro-1+ dpscs at the 1st and 9th passages were investigated. when compared with stro-1+ dpscs at the 1st passage, the expression of mature osteoblast-specific genes/proteins, odontoblast-specific gene/protein, and chondrocyte-specific gene/protein was significantly upregulated in human stro-1+ dpscs at the 9th passage. these findings suggest that stro-1+ dpscs consist of several interrelated subpopulations which can spontaneously differentiate into odontoblasts, osteoblasts, and chondrocytes. dpscs were isolated from human/rat dental pulps by the magnetic activated cell sorting based on stro- expression, cultured and passaged in the conventional culture media. however, the differentiation potential of naive dpscs after serial passaging in the routine culture system has not been fully elucidated. the differentiation capacity of these dpscs changes during cell passaging, and dpscs at the 9th passage restrict their differentiation potential to the osteoblast lineage in vivo.
 dental pulp stem cells can be driven into odontoblast, osteoblast, and chondrocyte lineages in different inductive media. however, the differentiation potential of naive dpscs after serial passaging in the routine culture system has not been fully elucidated.  dpscs were isolated from human/rat dental pulps by the magnetic activated cell sorting based on stro- expression, cultured and passaged in the conventional culture media. the biological features of stro-1+ dpscs at the 1st and 9th passages were investigated. during the long-term passage, the proliferation ability of human stro-1+ dpscs was downregulated as indicated by the growth kinetics. when compared with stro-1+ dpscs at the 1st passage, the expression of mature osteoblast-specific genes/proteins, odontoblast-specific gene/protein, and chondrocyte-specific gene/protein was significantly upregulated in human stro-1+ dpscs at the 9th passage. furthermore, human dpsc-p cells in the mineralization-inducing media presented higher levels of alkaline phosphatase at day and day respectively, and produced more mineralized matrix than dpsc-p cells at day in vivo transplantation results showed that rat dpsc-p cell pellets developed into dentin, bone and cartilage structures respectively, while dpsc-p cells can only generate bone tissues.  these findings suggest that stro-1+ dpscs consist of several interrelated subpopulations which can spontaneously differentiate into odontoblasts, osteoblasts, and chondrocytes. the differentiation capacity of these dpscs changes during cell passaging, and dpscs at the 9th passage restrict their differentiation potential to the osteoblast lineage in vivo. 