BACKGROUND
mesenchymal stem cells  are multipotent progenitor cells characterized by their ability to both self-renew and differentiate into tissues of mesodermal origin   <cit> .
despite several tissues have been explored for the isolation of msc including adipose, umbilical and placental; animal bone marrow  is the most common source of msc for clinical and research uses.
msc are directly isolated from bone marrow aspirates based on their ability to adhere to plastic when plated in monolayer culture and thereafter replicate ex vivo to form a phenotypically homogeneous population of cells  <cit> .
plastic adherence under standard culture conditions is one of the criteria for defining msc by the international society for cellular therapy .
other requirements include trilineage differentiation potential and expression of msc surface antigens markers cd <dig> , cd <dig>  and cd <dig> , and lack of expression of hematopoietic markers cd <dig> , cd <dig>  and cd <dig>   <cit> .
despite the wide relevance of the bovine experimental model in both in vivo and in vitro experiments, limited information regarding bovine msc  is available.
similarities in organ size and physiology with humans and a longer life span in comparison with traditional laboratory animal models support the use of large animal models for long-term experiments in regenerative medicine  <cit> .
development of a bmsc model would be invaluable for testing the efficiency and safety of these cells for future cell therapies and for the creation of human disease models.
moreover, cattle can give advantages for clinical applications of msc to human and veterinary medicine especially in musculoskeletal disorders  <cit> .
previous studies reported isolation and mesenchymal differentiation of bmsc from calf bm  <cit>  and bovine umbilical cord  <cit> .
we have reported the isolation and mesenchymal multilineage differentiation of bmsc derived from bm of abattoir-derived bovine fetuses  <cit> .
studies performed on human msc isolated from fetal bm have shown that these cells possess higher proliferative capacity, trilineage differentiation potential and lower immunogenicity compared to msc from umbilical cord, adult bm or adipose tissue  <cit> .
moreover, human fetal msc isolated from bm had higher proliferative and osteogenic capacity than msc derived from other ontological and anatomical origins, suggesting that they are superior candidates for bone tissue engineering  <cit> .
the simplicity of isolation and the potential to differentiate into several cell types lays the foundation for fetal bmsc, as an interesting source for investigation of stem cell biology.
moreover, the development of large animal experimental models including cattle may open alternative strategies for investigating msc physiology and eventual applications for regenerative therapy in human and veterinary medicine.
as an example, recently it has been proposed the potential application of various stem/progenitor cells including mammary stem cells for the repair of post-mastitis structural defects in dairy animals  <cit> .
the plasticity or transdifferentiation potential of msc is not limited to mesenchymal derivatives, since under appropriate cell culture conditions and stimulation by certain exogenous or endogenous bioactive factors, msc have also been differentiated into endodermal  and neuroectodermal  cells  <cit> .
the potential for hepatogenic differentiation of msc has been evaluated by measuring the expression of endodermal or hepatocyte markers including α-fetoprotein , albumin , alpha <dig> antitrypsin , connexin  <dig> , tyrosine aminotransferase  and cytochrome p <dig>   <cit> .
the functional capacity has also been assessed by determining urea production and glycogen storage  <cit> .
moreover, a large body of evidence has established the neural differentiation potential of msc derived from bm.
treatment of bm-msc with different molecules and growth factors induced rapid morphological changes that are typical of neural cells together with the expression of neural markers such as neuroepithelial stem cell intermediate filament , microtubule associated protein  <dig> , nerve growth factor , tropomyosin-related kinase a  and cellular prion protein   <cit> .
thus, the potential for isolation and differentiation into hepatocyte- and neuron-like cells, as well as their capacity for transplantation, suggests that msc represent an attractive therapeutic candidate for treating degenerative diseases.
in the present study, we used abattoir-derived bovine fetuses as an available and plentiful source of bm with the aim to obtain an abundant supply of bmsc for in vitro differentiation experiments.
our main objective was to characterize the hepatogenic and neurogenic differentiation potential of bmsc isolated from abattoir-derived bovine fetuses by gene expression and functional analyses.
RESULTS
mesenchymal cell surface marker and multipotent profile of bm-bmsc from abattoir-derived fetuses
fetal bmsc were isolated from bm based on the capacity for plastic attachment under standard culture conditions that included dmem media supplemented with 10% fbs.
after  <dig> to 6 days of culture, colonies of fibroblast-like cells were visualized attached to plastic culture flasks.
isolated cells were cultured for several weeks in monolayer and used for differentiation experiments after  <dig> to  <dig> passages.
flow cytometric analysis of bmsc demonstrated that higher  percentage of these cells were positive for msc markers cd <dig>  and cd <dig>  and pluripotency markers oct <dig>  and nanog  .
in comparison, a higher  percentage of cells was negative for hematopoietic markers cd <dig> and cd <dig> .
gene expression profile in bmsc during hepatogenic differentiation
culture of bmsc in monolayer under hepatogenic conditions induced polygonal cell morphology and formation of cell aggregates at day  <dig> of culture .
afp protein was immunodetected in differentiated bmsc at day  <dig> of culture .
at this stage, levels of afp, alb, α1at, cnx <dig>  tat mrna levels were up-regulated  in differentiated bmsc  .
levels of cyp3a <dig> mrna were higher  in differentiated compared to control bmsc from day  <dig> until day  <dig> of culture .
in contrast, nanog mrna levels were lower  in differentiated bmsc at days  <dig>   <dig> and  <dig> of culture .
functional characterization of bmsc during hepatogenic differentiation
more intense pas staining associated to stored glycogen was observed in differentiated compared to control bmsc at day  <dig> of culture .
at days  <dig> and  <dig> of differentiation, higher  secretion of albumin  and urea concentration  were detected in differentiated bmsc cultures .
gene expression profile in bmsc during neurogenic differentiation
protocol  <dig> used for neurogenic differentiation induced formation of compact and spherical cell bodies with multiple extensions since 96 h of culture .
no significant differences in mrna levels of neural markers nestin, map <dig>  ngf and trka were detected between bmsc cultured under differentiating and control conditions .
protocol  <dig> induced cell body retraction and formation of multiple and complex cell extensions in differentiated bmsc since 96 h of culture .
these cells expressed nestin and map <dig> proteins at 0 h of culture; whereas, at 144 h also expressed trka and prpc .
differentiated bmsc expressed higher  levels of map <dig> mrna  at 144 h of culture .
similarly, levels of trka mrna were higher  in differentiated bmsc at 96 h and 144 h of culture .
conversely, differentiated bmsc expressed lower  mrna levels of nanog at 96 h and 144 h of culture .
no significant differences were detected for nestin, ngf and prp
c
mrna levels between treated and untreated bmsc; however, both cell populations expressed higher  levels of nestin at 24 h of culture.
discussion
in the present study, bmsc were isolated from bm collected from abattoir-derived fetuses based on the capacity to adhere to plastic substrate under monolayer culture conditions.
these cells expressed msc specific markers cd <dig> and cd <dig> and lacked expression of hematopoietic surface markers cd <dig> and cd <dig>  little information is available regarding expression of msc surface markers in bmsc.
analyses by flow cytometry in bmsc isolated from the wharton’s jelly detected high proportion of cells positive for cd <dig>  cd <dig>  cd <dig> and cd <dig> and negative for cd <dig> and cd <dig>  <cit> .
using q-pcr, we reported a similar msc marker profile in bmsc isolated from bm of abattoir-derived bovine fetuses  <cit> .
recently, these results have been confirmed by immunofluorescence and rt-pcr in bmsc isolated from fetal bm  <cit> .
thus, in vitro characterization presented in this study and in previous reports including adherence to plastic substrate, expression of mesenchymal markers and differentiation into mesodermal lineages suggests that bmsc isolated from fetal bm by plastic adherence fulfill the minimal criteria for definition of msc.
the 2-step sequential hepatogenic differentiation protocol used in our study included early , middle , and late  differentiation factors  <cit> .
hgf is an endocrine or paracrine factor essential for liver development that promotes cell survival and regeneration  <cit> .
osm is a member of the interleukin- <dig> cytokine family that increases cell size of hepatocytes and induces cell differentiation and formation of bile canaliculi  <cit> .
the capacity to induce hepatocyte functions, albumin secretion and cytochrome activity may be enhanced when osm is combined with dmso  <cit> .
dexamethasone, a synthetic glucocorticoid that induces hepatic gluconeogenesis, promotes the expression of hepatocyte phenotype through the suppression of cell division  <cit> .
the hepatogenic differentiation potential of fetal bmsc was evaluated using quantitative and qualitative analyses.
spindle shape and polygonal cell morphology observed at day  <dig> in differentiated bmsc was not typical hepatocyte-like morphology described in previous studies.
however, during mesenchyme-to-hepatocyte differentiation, expression of specific transcription factors of early endoderm, bipotential cells, and finally hepatocytes were up-regulated.
it is recognized that afp and alb are abundant proteins synthesized from early fetal to adult mature hepatocytes  <cit> .
tat and α1at are liver-specific enzymes involved in degradation of tyrosine and inhibition of proteases, and are known to be late markers of the hepatocyte lineage  <cit> .
cnx <dig> is a major gap junction protein which account for the 90% of connexin proteins in the liver  <cit> .
expression of these genes at the latest stage of differentiation indicates that prolonged exposure to hepatogenic factors is required in order to trigger the mesenchymal-to-hepatic transition.
however, increasing levels of cyp3a <dig> mrna day  <dig> of culture suggest that activation of catalytic enzymes involved in the hepatic metabolism occurs at early stages of hepatogenic differentiation  <cit> .
moreover, production of albumin and urea at days  <dig> and  <dig> of hepatogenic differentiation demonstrate the acquisition of hepatocyte metabolic activity in differentiated bmsc  <cit> .
these results along with detection of glycogen storage indicate that bmsc underwent transdifferentiation into the endodermic hepatogenic lineage in agreement with the attainment of the hepatocyte gene expression profile.
in order to investigate the ectodermal differentiation potential, bmsc were induced toward neurogenic lineage using a previously reported bme-based protocol  <cit> .
bmsc morphology including formation of compact cell bodies with multiple extensions along with expression of neural stem cell marker nestin and mature neuron markers map <dig>  ngf and trka, initially suggested the acquisition of neural properties in treated bmsc.
however, absence of neural marker increment in bmsc after treatment with a bme indicates that this simple chemical neuronal induction protocol was not able to induce neural differentiation.
previous studies have reported that bme-based protocols may induce neuronal phenotype in other cell types lacking any stem cell characteristics   <cit> .
this neuron-like appearance was associated to disruption of cytoskeleton after cell stress due to toxic chemicals rather than cell differentiation.
a second protocol based on combination of growth factor pretreatment and induction with previously reported factors was assessed in fetal bmsc for neurogenic differentiation  <cit> .
egf-bfgf pretreatment have been associated to initiation of msc cycle exit and activation of neural/neuronal gene expression pattern  <cit> .
thereafter, culture in differentiation medium containing kcl, valproic acid, forskolin and hydrocortisone has showed to induce neuronal morphological and immunocytochemical changes in human and murine adipose-derived stem cells  <cit> .
in our study, morphology changes in bmsc during culture were characterized by cell body retraction and formation of multiple and complex cell extensions that were consistent with neuronal differentiation.
these cells expressed nestin and map <dig> at 24 h of culture, in addition with trka and prpc after 144 h of culture.
the capacity of msc to coexpress immature and mature neuronal or glial proteins has been previously associated to the plasticity to differentiate in various tissues  <cit> .
in our study, immunodetection of nestin, map <dig>  trka and prpc proteins, along with up-regulation of map <dig> and trka mrna in differentiated bmsc indicated that these cells were induced toward neural linage.
however, a functional assay will be required before concluding that these cells have the potential to become neurons.
lack of significant differences in nestin, ngf and prp
c
mrna levels between bmsc treatments were probably due to reduced level of expression of these genes.
constituently expression of nestin and map <dig> in msc cultured without any previous induction, have been previously associated to the presence of a multidifferentiated state, in which msc may retain a native potential for differentiation  <cit> .
previously, it have been reported that after in utero transplantation of human msc in sheep or systemic infusion in baboons, msc are found distributed in several tissues including gastrointestinal, kidney, liver, and skin  <cit> .
these studies support the hypothesis  <cit>  that subpopulations of msc are in reality in a multidifferentiated state and participate in vivo in the turnover and replacement of a wide variety of tissues.
furthermore, these subpopulations may also explain the expression of lineage-specific markers in supposedly undifferentiated msc populations.
although expression of nestin has been associated to early acquisition of neuronal lineage, expression in other cell type including myogenic, endothelial and hepatic suggest that this protein may not play a unique role in neurogenic differentiation  <cit> .
considering the proposed role of prpc in neural differentiation of embryonic stem cells   <cit>  and its participation in bovine spongiform encephalopathy   <cit> , transdifferentiation of msc into neural cells could provide a valuable in vitro model for the study of prion diseases.
using co-localization analyses in the neuroepithelium of bovine fetuses and an in vitro differentiating esc model, it has been shown that prpc influences the differentiation of nestin-expressing cell lineages, including neural lineages  <cit> .
these data provides clues of the complex signaling pathways that mediate esc neural differentiation; however, further in vitro differentiation experiments would be required in order to elucidate if this interactions are similar in msc.
the expression of pluripotent markers oct <dig> and nanog was analyzed with the aim to evaluate the state of multipotency in bmsc during mesenchymal differentiation.
oct <dig> and nanog regulate the maintenance of pluripotent state in embryos and derived cells in most mammalian species  <cit> .
these transcription factors had been proposed to play a similar role in adult stem cells  <cit> .
however, recent reports have indicated that nanog expression is associated to adaptation to in vitro cell growth conditions in differentiating msc  <cit> .
in our study, the patterns of expression of oct <dig> and nanog in undifferentiated msc support the potential participation of these factors during the multipotent state.
moreover, our data suggest that the multipotent capacity of bmsc is reduced according to the acquisition of the hepatogenic phenotype and functional activity.
CONCLUSIONS
the expression patterns of lineage-specific markers and the production of functional metabolites demonstrate the acquisition of hepatocyte and neuronal profiles in bmsc after independent in vitro hepatogenic and neurogenic independent differentiations.
the simplicity of isolation and the potential to differentiate into several cell types lays the foundation for bm msc isolated from abattoir-derived bovine fetuses, as an alternative source of msc for investigation of biology and eventual applications for regenerative therapy in veterinary medicine.

 mesenchymal stem cells are multipotent progenitor cells characterized by their ability to both self-renew and differentiate into tissues of mesodermal origin. despite several tissues have been explored for the isolation of msc including adipose, umbilical and placental; animal bone marrow is the most common source of msc for clinical and research uses. msc are directly isolated from bone marrow aspirates based on their ability to adhere to plastic when plated in monolayer culture and thereafter replicate ex vivo to form a phenotypically homogeneous population of cells. plastic adherence under standard culture conditions is one of the criteria for defining msc by the international society for cellular therapy. other requirements include trilineage differentiation potential and expression of msc surface antigens markers cd, cd and cd, and lack of expression of hematopoietic markers cd, cd and cd. despite the wide relevance of the bovine experimental model in both in vivo and in vitro experiments, limited information regarding bovine msc is available. similarities in organ size and physiology with humans and a longer life span in comparison with traditional laboratory animal models support the use of large animal models for long-term experiments in regenerative medicine. development of a bmsc model would be invaluable for testing the efficiency and safety of these cells for future cell therapies and for the creation of human disease models. moreover, cattle can give advantages for clinical applications of msc to human and veterinary medicine especially in musculoskeletal disorders. previous studies reported isolation and mesenchymal differentiation of bmsc from calf bm and bovine umbilical cord. we have reported the isolation and mesenchymal multilineage differentiation of bmsc derived from bm of abattoir-derived bovine fetuses. studies performed on human msc isolated from fetal bm have shown that these cells possess higher proliferative capacity, trilineage differentiation potential and lower immunogenicity compared to msc from umbilical cord, adult bm or adipose tissue. moreover, human fetal msc isolated from bm had higher proliferative and osteogenic capacity than msc derived from other ontological and anatomical origins, suggesting that they are superior candidates for bone tissue engineering. the simplicity of isolation and the potential to differentiate into several cell types lays the foundation for fetal bmsc, as an interesting source for investigation of stem cell biology. moreover, the development of large animal experimental models including cattle may open alternative strategies for investigating msc physiology and eventual applications for regenerative therapy in human and veterinary medicine. as an example, recently it has been proposed the potential application of various stem/progenitor cells including mammary stem cells for the repair of post-mastitis structural defects in dairy animals. the plasticity or transdifferentiation potential of msc is not limited to mesenchymal derivatives, since under appropriate cell culture conditions and stimulation by certain exogenous or endogenous bioactive factors, msc have also been differentiated into endodermal and neuroectodermal cells. the potential for hepatogenic differentiation of msc has been evaluated by measuring the expression of endodermal or hepatocyte markers including α-fetoprotein, albumin, alpha antitrypsin, connexin, tyrosine aminotransferase and cytochrome p. the functional capacity has also been assessed by determining urea production and glycogen storage. moreover, a large body of evidence has established the neural differentiation potential of msc derived from bm. treatment of bm-msc with different molecules and growth factors induced rapid morphological changes that are typical of neural cells together with the expression of neural markers such as neuroepithelial stem cell intermediate filament, microtubule associated protein, nerve growth factor, tropomyosin-related kinase a and cellular prion protein. thus, the potential for isolation and differentiation into hepatocyte- and neuron-like cells, as well as their capacity for transplantation, suggests that msc represent an attractive therapeutic candidate for treating degenerative diseases. in the present study, we used abattoir-derived bovine fetuses as an available and plentiful source of bm with the aim to obtain an abundant supply of bmsc for in vitro differentiation experiments. our main objective was to characterize the hepatogenic and neurogenic differentiation potential of bmsc isolated from abattoir-derived bovine fetuses by gene expression and functional analyses.  mesenchymal cell surface marker and multipotent profile of bm-bmsc from abattoir-derived fetuses fetal bmsc were isolated from bm based on the capacity for plastic attachment under standard culture conditions that included dmem media supplemented with 10% fbs. after to 6 days of culture, colonies of fibroblast-like cells were visualized attached to plastic culture flasks. isolated cells were cultured for several weeks in monolayer and used for differentiation experiments after to passages. flow cytometric analysis of bmsc demonstrated that higher percentage of these cells were positive for msc markers cd and cd and pluripotency markers oct and nanog. in comparison, a higher percentage of cells was negative for hematopoietic markers cd and cd. gene expression profile in bmsc during hepatogenic differentiation culture of bmsc in monolayer under hepatogenic conditions induced polygonal cell morphology and formation of cell aggregates at day of culture. afp protein was immunodetected in differentiated bmsc at day of culture. at this stage, levels of afp, alb, α1at, cnx tat mrna levels were up-regulated in differentiated bmsc. levels of cyp3a mrna were higher in differentiated compared to control bmsc from day until day of culture. in contrast, nanog mrna levels were lower in differentiated bmsc at days and of culture. functional characterization of bmsc during hepatogenic differentiation more intense pas staining associated to stored glycogen was observed in differentiated compared to control bmsc at day of culture. at days and of differentiation, higher secretion of albumin and urea concentration were detected in differentiated bmsc cultures. gene expression profile in bmsc during neurogenic differentiation protocol used for neurogenic differentiation induced formation of compact and spherical cell bodies with multiple extensions since 96 h of culture. no significant differences in mrna levels of neural markers nestin, map ngf and trka were detected between bmsc cultured under differentiating and control conditions. protocol induced cell body retraction and formation of multiple and complex cell extensions in differentiated bmsc since 96 h of culture. these cells expressed nestin and map proteins at 0 h of culture; whereas, at 144 h also expressed trka and prpc. differentiated bmsc expressed higher levels of map mrna at 144 h of culture. similarly, levels of trka mrna were higher in differentiated bmsc at 96 h and 144 h of culture. conversely, differentiated bmsc expressed lower mrna levels of nanog at 96 h and 144 h of culture. no significant differences were detected for nestin, ngf and prp c mrna levels between treated and untreated bmsc; however, both cell populations expressed higher levels of nestin at 24 h of culture. discussion in the present study, bmsc were isolated from bm collected from abattoir-derived fetuses based on the capacity to adhere to plastic substrate under monolayer culture conditions. these cells expressed msc specific markers cd and cd and lacked expression of hematopoietic surface markers cd and cd little information is available regarding expression of msc surface markers in bmsc. analyses by flow cytometry in bmsc isolated from the wharton’s jelly detected high proportion of cells positive for cd cd cd and cd and negative for cd and cd. using q-pcr, we reported a similar msc marker profile in bmsc isolated from bm of abattoir-derived bovine fetuses. recently, these results have been confirmed by immunofluorescence and rt-pcr in bmsc isolated from fetal bm. thus, in vitro characterization presented in this study and in previous reports including adherence to plastic substrate, expression of mesenchymal markers and differentiation into mesodermal lineages suggests that bmsc isolated from fetal bm by plastic adherence fulfill the minimal criteria for definition of msc. the 2-step sequential hepatogenic differentiation protocol used in our study included early, middle, and late differentiation factors. hgf is an endocrine or paracrine factor essential for liver development that promotes cell survival and regeneration. osm is a member of the interleukin- cytokine family that increases cell size of hepatocytes and induces cell differentiation and formation of bile canaliculi. the capacity to induce hepatocyte functions, albumin secretion and cytochrome activity may be enhanced when osm is combined with dmso. dexamethasone, a synthetic glucocorticoid that induces hepatic gluconeogenesis, promotes the expression of hepatocyte phenotype through the suppression of cell division. the hepatogenic differentiation potential of fetal bmsc was evaluated using quantitative and qualitative analyses. spindle shape and polygonal cell morphology observed at day in differentiated bmsc was not typical hepatocyte-like morphology described in previous studies. however, during mesenchyme-to-hepatocyte differentiation, expression of specific transcription factors of early endoderm, bipotential cells, and finally hepatocytes were up-regulated. it is recognized that afp and alb are abundant proteins synthesized from early fetal to adult mature hepatocytes. tat and α1at are liver-specific enzymes involved in degradation of tyrosine and inhibition of proteases, and are known to be late markers of the hepatocyte lineage. cnx is a major gap junction protein which account for the 90% of connexin proteins in the liver. expression of these genes at the latest stage of differentiation indicates that prolonged exposure to hepatogenic factors is required in order to trigger the mesenchymal-to-hepatic transition. however, increasing levels of cyp3a mrna day of culture suggest that activation of catalytic enzymes involved in the hepatic metabolism occurs at early stages of hepatogenic differentiation. moreover, production of albumin and urea at days and of hepatogenic differentiation demonstrate the acquisition of hepatocyte metabolic activity in differentiated bmsc. these results along with detection of glycogen storage indicate that bmsc underwent transdifferentiation into the endodermic hepatogenic lineage in agreement with the attainment of the hepatocyte gene expression profile. in order to investigate the ectodermal differentiation potential, bmsc were induced toward neurogenic lineage using a previously reported bme-based protocol. bmsc morphology including formation of compact cell bodies with multiple extensions along with expression of neural stem cell marker nestin and mature neuron markers map ngf and trka, initially suggested the acquisition of neural properties in treated bmsc. however, absence of neural marker increment in bmsc after treatment with a bme indicates that this simple chemical neuronal induction protocol was not able to induce neural differentiation. previous studies have reported that bme-based protocols may induce neuronal phenotype in other cell types lacking any stem cell characteristics. this neuron-like appearance was associated to disruption of cytoskeleton after cell stress due to toxic chemicals rather than cell differentiation. a second protocol based on combination of growth factor pretreatment and induction with previously reported factors was assessed in fetal bmsc for neurogenic differentiation. egf-bfgf pretreatment have been associated to initiation of msc cycle exit and activation of neural/neuronal gene expression pattern. thereafter, culture in differentiation medium containing kcl, valproic acid, forskolin and hydrocortisone has showed to induce neuronal morphological and immunocytochemical changes in human and murine adipose-derived stem cells. in our study, morphology changes in bmsc during culture were characterized by cell body retraction and formation of multiple and complex cell extensions that were consistent with neuronal differentiation. these cells expressed nestin and map at 24 h of culture, in addition with trka and prpc after 144 h of culture. the capacity of msc to coexpress immature and mature neuronal or glial proteins has been previously associated to the plasticity to differentiate in various tissues. in our study, immunodetection of nestin, map trka and prpc proteins, along with up-regulation of map and trka mrna in differentiated bmsc indicated that these cells were induced toward neural linage. however, a functional assay will be required before concluding that these cells have the potential to become neurons. lack of significant differences in nestin, ngf and prp c mrna levels between bmsc treatments were probably due to reduced level of expression of these genes. constituently expression of nestin and map in msc cultured without any previous induction, have been previously associated to the presence of a multidifferentiated state, in which msc may retain a native potential for differentiation. previously, it have been reported that after in utero transplantation of human msc in sheep or systemic infusion in baboons, msc are found distributed in several tissues including gastrointestinal, kidney, liver, and skin. these studies support the hypothesis that subpopulations of msc are in reality in a multidifferentiated state and participate in vivo in the turnover and replacement of a wide variety of tissues. furthermore, these subpopulations may also explain the expression of lineage-specific markers in supposedly undifferentiated msc populations. although expression of nestin has been associated to early acquisition of neuronal lineage, expression in other cell type including myogenic, endothelial and hepatic suggest that this protein may not play a unique role in neurogenic differentiation. considering the proposed role of prpc in neural differentiation of embryonic stem cells and its participation in bovine spongiform encephalopathy, transdifferentiation of msc into neural cells could provide a valuable in vitro model for the study of prion diseases. using co-localization analyses in the neuroepithelium of bovine fetuses and an in vitro differentiating esc model, it has been shown that prpc influences the differentiation of nestin-expressing cell lineages, including neural lineages. these data provides clues of the complex signaling pathways that mediate esc neural differentiation; however, further in vitro differentiation experiments would be required in order to elucidate if this interactions are similar in msc. the expression of pluripotent markers oct and nanog was analyzed with the aim to evaluate the state of multipotency in bmsc during mesenchymal differentiation. oct and nanog regulate the maintenance of pluripotent state in embryos and derived cells in most mammalian species. these transcription factors had been proposed to play a similar role in adult stem cells. however, recent reports have indicated that nanog expression is associated to adaptation to in vitro cell growth conditions in differentiating msc. in our study, the patterns of expression of oct and nanog in undifferentiated msc support the potential participation of these factors during the multipotent state. moreover, our data suggest that the multipotent capacity of bmsc is reduced according to the acquisition of the hepatogenic phenotype and functional activity.  the expression patterns of lineage-specific markers and the production of functional metabolites demonstrate the acquisition of hepatocyte and neuronal profiles in bmsc after independent in vitro hepatogenic and neurogenic independent differentiations. the simplicity of isolation and the potential to differentiate into several cell types lays the foundation for bm msc isolated from abattoir-derived bovine fetuses, as an alternative source of msc for investigation of biology and eventual applications for regenerative therapy in veterinary medicine. 
levels of map and trka mrna were up-regulated at the end of the differentiation period. the expression patterns of linage-specific markers and the production of functional metabolites support the potential for hepatogenic and neurogenic differentiation of bmsc isolated from bm of abattoir-derived fetuses. the plasticity or transdifferentiation potential of msc is not limited to mesodermal derivatives, since under appropriate cell culture conditions and stimulation by bioactive factors, msc have also been differentiated into endodermal and neuroectodermal cells. mesenchymal stem cells are multipotent progenitor cells characterized by their ability to both self-renew and differentiate into tissues of mesodermal origin. functional analyses in differentiated bmsc cultures evidenced an increase in albumin and urea production and glycogen storage. flow cytometric analysis demonstrated that bmsc populations were positive for msc markers cd and cd and pluripotency markers oct and nanog; whereas, were negative for hematopoietic markers cd and cd levels of mrna of hepatic genes α-fetoprotein, albumin, alpha antitrypsin, connexin, tyrosine aminotransferase and cytochrome p were up-regulated in bmsc during a 28-day period of hepatogenic differentiation. in the present study we sought to characterize the hepatogenic and neurogenic differentiation and multipotent potential of bovine msc isolated from bone marrow of abattoir-derived fetuses. the simplicity of isolation and the potential to differentiate into a wide variety of cell lineages lays the foundation for bmsc as an interesting alternative for investigation in msc biology and eventual applications for regenerative therapy in veterinary medicine. the potential of msc for hepatogenic and neurogenic differentiation has been well documented in different animal models; however, few reports are currently available on large animal models. conversely, bmsc expressed lower levels of nanog mrna during both hepatogenic and neurogenic differentiation processes. plastic-adherent bmsc isolated from fetal bm maintained a fibroblast-like morphology under monolayer culture conditions.
 mesenchymal stem cells are multipotent progenitor cells characterized by their ability to both self-renew and differentiate into tissues of mesodermal origin. the plasticity or transdifferentiation potential of msc is not limited to mesodermal derivatives, since under appropriate cell culture conditions and stimulation by bioactive factors, msc have also been differentiated into endodermal and neuroectodermal cells. the potential of msc for hepatogenic and neurogenic differentiation has been well documented in different animal models; however, few reports are currently available on large animal models. in the present study we sought to characterize the hepatogenic and neurogenic differentiation and multipotent potential of bovine msc isolated from bone marrow of abattoir-derived fetuses.  plastic-adherent bmsc isolated from fetal bm maintained a fibroblast-like morphology under monolayer culture conditions. flow cytometric analysis demonstrated that bmsc populations were positive for msc markers cd and cd and pluripotency markers oct and nanog; whereas, were negative for hematopoietic markers cd and cd levels of mrna of hepatic genes α-fetoprotein, albumin, alpha antitrypsin, connexin, tyrosine aminotransferase and cytochrome p were up-regulated in bmsc during a 28-day period of hepatogenic differentiation. functional analyses in differentiated bmsc cultures evidenced an increase in albumin and urea production and glycogen storage. bmsc cultured under neurogenic conditions expressed nestin and map proteins at 24 h of culture; whereas, at 144 h also expressed trka and prpc. levels of map and trka mrna were up-regulated at the end of the differentiation period. conversely, bmsc expressed lower levels of nanog mrna during both hepatogenic and neurogenic differentiation processes.  the expression patterns of linage-specific markers and the production of functional metabolites support the potential for hepatogenic and neurogenic differentiation of bmsc isolated from bm of abattoir-derived fetuses. the simplicity of isolation and the potential to differentiate into a wide variety of cell lineages lays the foundation for bmsc as an interesting alternative for investigation in msc biology and eventual applications for regenerative therapy in veterinary medicine. bovine fetusesmesenchymal stem celldifferentiation potentialhepatocyte-like cellneuron-like cell 