BACKGROUND
mesenchymal stem cells  are attractive for the regeneration of damaged tissues in clinical applications, since they are characterized as undifferentiated cells, able to self-renew with a high proliferative capacity and possess mesodermal differentiation potential  <cit> .
bone marrow-derived mscs have great potential as therapeutic agents for neurological diseases, because they are easily obtained from bone marrow and expand rapidly in vitro.
moreover, there is a lower risk of rejection using mscs compared to other sources of stem cells as they can be autogenic.
it has been held that mscs can give rise to osteocytes, chondrocytes, adipocytes, and neural cells  <cit> .
however, current induction methods obtain a lower percentage of neural differentiation and consume long induction time.
cocaine- and amphetamine-regulated transcript  peptides have emerged as major neurotransmitters and hormones.
they are widely distributed in the cns and involved in regulating many processes, including food intake, the maintenance of body weight and endocrine functions.
moreover, previous study demonstrated that cart was up-regulated in the cerebral cortex following focal cerebral ischemia in vivo and in cultured cortical neurons subjected to oxygen-glucose deprivation  in vitro.
this regulation led to the reduction of infarct size and ogd-induced cell death  <cit> .
also, cart promoted the survival and differentiation of primary hippocampal neurons by up-regulating bdnf mrna expression and protein synthesis  <cit> .
ngf is critical for the survival and maintenance of sympathetic and sensory neurons.
without it, these neurons undergo apoptosis  <cit> .
nerve growth factor induces axonal growth including axonal branching and a bit of elongation  <cit> .
bdnf is the second neurotrophic factor to be characterized after ngf.
they help support the survival of existing neurons and encourage the growth and differentiation of new neurons and synapses  <cit> .
emerging evidence established that growth factors such as bdnf and ngf are physiological inductors for neural differentiation of mscs not only in the mscs, recipients but also in the mscs, co-cultured medium  <cit> .
this study tested the hypothesis that cart could promote the differentiation of mscs into neural cells through increasing neurofactors such as bndf and ngf.
RESULTS
<dig>  identification of mouse mscs
firstly, cell morphology was observed daily by phase contrast invert microscopy.
in the early days, individual adherent cells appeared.
among the adherent cells, some were fibroblastic in shape and the others were round with dark centers and transparent peripheries.
in the subsequent days, some fibroblastic cells proliferated.
they gave rise to clones of pure fibroblastic cells, each of which was composed of several cells.
finally, these cells were almost completely obscured by the fibroblastic cells .
fluorescent activated cell sorting  analysis demonstrated that the expanded plastic adherent cells were positive for the mesenchymal stem cell-associated surface markers cd <dig>  cd <dig> and cd <dig>  but negative for the hematopoietic progenitor's specific surface maker cd <dig> .
thus, the cells used in this study fulfilled all criterion to be defined as mscs.
<dig>  neuronal induction of mouse mscs treated by cart
<dig>  cell culture
48- <dig> h after induction, some cells became shorter.
in addition, the cytoplasm gathered towards the nucleus and formed the axons and dendrites .
another  <dig> days later, the majority of cells turned into neuron-like cells.
they had large and round cell bodies with longer axons similar to the shape of cells induced by endothelial growth factor  and basic fibroblast grow factor .
the uninduced group remained in fiber-like cells .
<dig>  immunofluorescence assay of neural marker proteins
mscs treated or not treated by cart were prepared for immunofluorescence analysis by antibodies against the neural precursor nestin , the markers of neurons map- <dig>  and neun , and the marker of astrocytes gfap .
quantification of immunofluorescence staining from three independent experiments revealed that nestin, map- <dig>  gfap and neun were  <dig>  ±  <dig> %,  <dig>  ±  <dig> %,  <dig>  ±  <dig> %,  <dig>  ±  <dig> % of all cells in  <dig> days and  <dig>  ±  <dig> %,  <dig>  ±  <dig> %,  <dig>  ±  <dig> %,  <dig>  ±  <dig> % in  <dig> days , respectively.
a similar rate of conversion was induced by egf/bfgf, with  <dig>  ±  <dig> %,  <dig>  ±  <dig> %,  <dig>  ±  <dig> %,  <dig>  ±  <dig> % in  <dig> days and  <dig>  ±  <dig> %,  <dig>  ±  <dig> %,  <dig>  ±  <dig> %,  <dig>  ±  <dig> % in  <dig> days.
only a trace amount of mscs in the untreated group expressed low levels of nestin  but no map- <dig>  gfap or neun.
* p <  <dig>  versus control group
<dig>  function of these differentiated cells
to investigate the function of differentiated neurons, chat  and th  were detected by immunofluorescence.
results showed that a few neun positive cells co-expressed chat  or th .
the mscs in the control and the growth factor-treated groups hardly expressed either of them.
also, we carried out the nissl stain to look for nissl bodies among the mscs with cart for  <dig> days.
we found that nissl bodies existed as dark blue particles in the cytoplasm of several differentiated mscs .
no similar structure was detected in control group.
what is more, excitable properties of msc-derived neural like cells were detected by whole cell recording.
voltage-gated potassium current and voltage-gated calcium current were elicited in cart treated mscs .
<dig>  cart up-regulated neurofactors bdnf and ngf
in addition to immunofluorescence assay, the neural specific rna transcripts bdnf and ngf were amplified by rt-pcr.
for each marker, the rna extracts of mscs were used as controls.
this study showed that bdnf and ngf were highly induced on  <dig> days after cart treatment.
compared to the control group, the amount of bdnf was  <dig>  folds  and  <dig>  folds  higher with the incubation of cart  <dig> days and  <dig> days.
ngf was also higher with the incubation of cart, with  <dig>  fold of control group in day  <dig>  and  <dig>  folds of control group in day  <dig> .
there were no significant differences between cart treated-group and growth factor treated-group .
discussion
in the present study, we thoroughly assessed for the first time the capacity of cart to enhance the neural differentiation of mscs.
we demonstrated that, when exposed to cart, map- <dig>  gfap or neun positive cells were traced in the cultured mscs.
furthermore, the proposed differentiated neurons not only have the potency to express chat and th, but also display the nissl bodies in the cytoplasm.
in addition, neurotrophic factors including ngf and bdnf were up-regulated in cart-treated group.
our results suggest that cart may promote the differentiation of mscs in vitro by enhancing the endogenous expression of ngf and bdnf.
mscs extracted from mouse bone marrow have great multiplication potency.
cell-doubling time is 48- <dig> h and cells can be expanded in culture for more than  <dig> doublings  <cit> .
herein, we isolated bm-mscs with the characteristics involving adherence to cell culture plastic, specific surface antigen expression, and multipotent differentiation which met the criterion defined by the mesenchymal and tissue stem cell committee of the international society for cellular therapy  <cit> .
also, bm-mscs possess pluripotency defined by their ability to differentiate into bone, fat, cartilage and muscle  <cit> .
compelling evidences unveiled that mscs are competent to break germ layer commitment and differentiate into cells expressing neuroectodermal properties  <cit> .
such a capacity opens extensive possibilities for autologous therapeutic treatments in a variety of neurological disorders.
also, as there are ethical issues related to isolating stem cells from fetal tissue and from adults, alternative sources of neuron-like cells for cell therapy are necessary.
indeed, a portion of studies indicate that, in contrast to the primary stem cells, the transplantation of pre-differentiated stem cells can diminish the risk of tumogenesis in the host patients  <cit> .
however, current inducing methods obtain a lower percentage of neuronal differentiation by a long inducing time  <cit> .
in  <dig>  sanchez-ramos et al used ra in combination with bdnf to introduce differentiation of bmsc to neural phenotypes.
neun and gfap positive cells only rank  <dig>  and 1% respectively  <cit> .
likewise, woodbury et al found β-mercaptoethanol  can rapidly induce the transition from mscs into neurons but not glial cells  <cit> .
there is also a debate about whether or not the converted neural phenotyped cells are able to work  <cit> .
this premise provided the impetus to investigate better and new reagent which can yield functional neuronal cells at a high rate.
here we extended our study on the neuroectodermal conversion of bm-mscs in vitro by exposure to the prospective peptide cart.
after treatment with cart, we found that cart could successfully differentiate mscs into neural cells in vitro.
these cells were morphologically similar to neural stem cells.
more importantly, they could express neural progenitor stem cell marker nestin and mature glial marker gfap as well as mature neuron markers like map- <dig> and neun.
it indicated that the protocol of using cart for three or six days induced significant changes in morphology and expression of markers of early and mature neural cell types.
similar with induction rate of bfgf plus egf, cart exposure obtained that map- <dig>  gfap and neun expressions were found in  <dig>  ±  <dig> %,  <dig>  ±  <dig> %,  <dig>  ±  <dig> % of all cells in  <dig> days, and  <dig>  ±  <dig> %,  <dig>  ±  <dig> %,  <dig>  ±  <dig> % in  <dig> days .
however, the expression of one or even two neuronal proteins does not prove that the cell bearing these "neuronal markers" is capable of all the complex functions of a neuron.
it will be important to determine whether these cells possess functional characteristics of neurons.
subsequently, double-labeled immunofluorescence was performed to calculate the function of the converted cells.
it was intriguing that the differentiated neun positive cells co-expressed chat or th.
our study on excitable property of msc-derived neural like cells demonstrated that the differentiated cells pocess founctional patassium and cacium channels .
recent research discovered that the neuron-like cells differentiated from bm-mscs resemble the endogenous neural progenitors in morphological, immunocytochemical, and functional characteristics  <cit> .
another independent investigator also reported that the transformed bm-mscs exhibit both neuron-like biochemical function and some corresponding electrophysiological properties  <cit> .
cart is an endogenous neuropeptide which is widely distributed in the brain and peripheral nervous system.
it has been well-established that cart can promote the survival and differentiation of neurons.
some investigators disclosed that cart has the capability to modulate the expression of growth factors and neurotrophic factors, which probably participated in the underlying effect of cart  <cit> .
based on this theory, we made an assumption that neurotrophic factors could be the bridge between cart and stem cell differentiation.
in order to verify this hypothesis, the amount of ngf and bdnf was determined by rt-pcr.
the result that neurotrophic factors are significant higher in cart treated group implied cart could promote the endogenous production of neurotrophic factors.
neurotrophic factors in cell and tissue culture have been shown to promote neuronal survival and differentiation and also regulate the growth of axons and dendrites  <cit> .
the presence of growth factors such as bdnf or ngf in the transplantation or the co-culture experiment are considered as physiological inductors for neural differentiation of mscs  <cit> .
mscs can induce a variety of neuro-regulatory proteins in addition to bdnf and β-ngf  <cit> .
further study demonstrated that the ngf and bdnf receptor trka and trkb do exist on the surface of mscs  <cit> .
moreover, the trkb receptor modified by bdnf may trigger the mapk signal pathway.
this process contributes to the maturation and neural differentiation of mscs  <cit> .
early studies confirmed that bdnf+ra  adult bone marrow stromal cells can be induced to differentiate into neural cells in vitro  <cit> .
however, cart stimulated the endogenous bdnf and ngf expression of mscs.
this stimulation may imitate the natural physiological pattern.
as a result, we obtained a higher yield rate than in previous studies.
in the hippocampus, bdnf and other neurotrophins are expressed from early stage of neuron development  <cit> .
this process is a prerequisite for the differentiation of neurons derived from hippocampal precursor cells in vitro  <cit> .
overall, findings suggest that the bdnf and ngf enhanced by cart may be the foundation of stem cell differentiation and maturation.
CONCLUSIONS
in summary, we identified cart as a potential neurotrophic factor.
cart can promote the neural differentiation of bm-mscs, probably through enhancing the endogeneous expression of neurotrophic factors.
combined our previous studies  <cit> , this study further confirmed the benefitial role of cart in the central nervous system.
besides the protective effect on neurons through complicated interaction with immunology and inflammation, cart also contributes to neuronal cells differentiation and generation.
in the future, cart pre-treatment may become an attractive and implemental method in stem cell therapy for neurological disease.

 mesenchymal stem cells are attractive for the regeneration of damaged tissues in clinical applications, since they are characterized as undifferentiated cells, able to self-renew with a high proliferative capacity and possess mesodermal differentiation potential. bone marrow-derived mscs have great potential as therapeutic agents for neurological diseases, because they are easily obtained from bone marrow and expand rapidly in vitro. moreover, there is a lower risk of rejection using mscs compared to other sources of stem cells as they can be autogenic. it has been held that mscs can give rise to osteocytes, chondrocytes, adipocytes, and neural cells. however, current induction methods obtain a lower percentage of neural differentiation and consume long induction time. cocaine- and amphetamine-regulated transcript peptides have emerged as major neurotransmitters and hormones. they are widely distributed in the cns and involved in regulating many processes, including food intake, the maintenance of body weight and endocrine functions. moreover, previous study demonstrated that cart was up-regulated in the cerebral cortex following focal cerebral ischemia in vivo and in cultured cortical neurons subjected to oxygen-glucose deprivation in vitro. this regulation led to the reduction of infarct size and ogd-induced cell death. also, cart promoted the survival and differentiation of primary hippocampal neurons by up-regulating bdnf mrna expression and protein synthesis. ngf is critical for the survival and maintenance of sympathetic and sensory neurons. without it, these neurons undergo apoptosis. nerve growth factor induces axonal growth including axonal branching and a bit of elongation. bdnf is the second neurotrophic factor to be characterized after ngf. they help support the survival of existing neurons and encourage the growth and differentiation of new neurons and synapses. emerging evidence established that growth factors such as bdnf and ngf are physiological inductors for neural differentiation of mscs not only in the mscs, recipients but also in the mscs, co-cultured medium. this study tested the hypothesis that cart could promote the differentiation of mscs into neural cells through increasing neurofactors such as bndf and ngf.  identification of mouse mscs firstly, cell morphology was observed daily by phase contrast invert microscopy. in the early days, individual adherent cells appeared. among the adherent cells, some were fibroblastic in shape and the others were round with dark centers and transparent peripheries. in the subsequent days, some fibroblastic cells proliferated. they gave rise to clones of pure fibroblastic cells, each of which was composed of several cells. finally, these cells were almost completely obscured by the fibroblastic cells. fluorescent activated cell sorting analysis demonstrated that the expanded plastic adherent cells were positive for the mesenchymal stem cell-associated surface markers cd cd and cd but negative for the hematopoietic progenitor's specific surface maker cd. thus, the cells used in this study fulfilled all criterion to be defined as mscs. neuronal induction of mouse mscs treated by cart cell culture 48- h after induction, some cells became shorter. in addition, the cytoplasm gathered towards the nucleus and formed the axons and dendrites. another days later, the majority of cells turned into neuron-like cells. they had large and round cell bodies with longer axons similar to the shape of cells induced by endothelial growth factor and basic fibroblast grow factor. the uninduced group remained in fiber-like cells. immunofluorescence assay of neural marker proteins mscs treated or not treated by cart were prepared for immunofluorescence analysis by antibodies against the neural precursor nestin, the markers of neurons map- and neun, and the marker of astrocytes gfap. quantification of immunofluorescence staining from three independent experiments revealed that nestin, map- gfap and neun were ± %, ± %, ± %, ± % of all cells in days and ± %, ± %, ± %, ± % in days, respectively. a similar rate of conversion was induced by egf/bfgf, with ± %, ± %, ± %, ± % in days and ± %, ± %, ± %, ± % in days. only a trace amount of mscs in the untreated group expressed low levels of nestin but no map- gfap or neun. * p < versus control group function of these differentiated cells to investigate the function of differentiated neurons, chat and th were detected by immunofluorescence. results showed that a few neun positive cells co-expressed chat or th. the mscs in the control and the growth factor-treated groups hardly expressed either of them. also, we carried out the nissl stain to look for nissl bodies among the mscs with cart for days. we found that nissl bodies existed as dark blue particles in the cytoplasm of several differentiated mscs. no similar structure was detected in control group. what is more, excitable properties of msc-derived neural like cells were detected by whole cell recording. voltage-gated potassium current and voltage-gated calcium current were elicited in cart treated mscs. cart up-regulated neurofactors bdnf and ngf in addition to immunofluorescence assay, the neural specific rna transcripts bdnf and ngf were amplified by rt-pcr. for each marker, the rna extracts of mscs were used as controls. this study showed that bdnf and ngf were highly induced on days after cart treatment. compared to the control group, the amount of bdnf was folds and folds higher with the incubation of cart days and days. ngf was also higher with the incubation of cart, with fold of control group in day and folds of control group in day. there were no significant differences between cart treated-group and growth factor treated-group. discussion in the present study, we thoroughly assessed for the first time the capacity of cart to enhance the neural differentiation of mscs. we demonstrated that, when exposed to cart, map- gfap or neun positive cells were traced in the cultured mscs. furthermore, the proposed differentiated neurons not only have the potency to express chat and th, but also display the nissl bodies in the cytoplasm. in addition, neurotrophic factors including ngf and bdnf were up-regulated in cart-treated group. our results suggest that cart may promote the differentiation of mscs in vitro by enhancing the endogenous expression of ngf and bdnf. mscs extracted from mouse bone marrow have great multiplication potency. cell-doubling time is 48- h and cells can be expanded in culture for more than doublings. herein, we isolated bm-mscs with the characteristics involving adherence to cell culture plastic, specific surface antigen expression, and multipotent differentiation which met the criterion defined by the mesenchymal and tissue stem cell committee of the international society for cellular therapy. also, bm-mscs possess pluripotency defined by their ability to differentiate into bone, fat, cartilage and muscle. compelling evidences unveiled that mscs are competent to break germ layer commitment and differentiate into cells expressing neuroectodermal properties. such a capacity opens extensive possibilities for autologous therapeutic treatments in a variety of neurological disorders. also, as there are ethical issues related to isolating stem cells from fetal tissue and from adults, alternative sources of neuron-like cells for cell therapy are necessary. indeed, a portion of studies indicate that, in contrast to the primary stem cells, the transplantation of pre-differentiated stem cells can diminish the risk of tumogenesis in the host patients. however, current inducing methods obtain a lower percentage of neuronal differentiation by a long inducing time. in sanchez-ramos et al used ra in combination with bdnf to introduce differentiation of bmsc to neural phenotypes. neun and gfap positive cells only rank and 1% respectively. likewise, woodbury et al found β-mercaptoethanol can rapidly induce the transition from mscs into neurons but not glial cells. there is also a debate about whether or not the converted neural phenotyped cells are able to work. this premise provided the impetus to investigate better and new reagent which can yield functional neuronal cells at a high rate. here we extended our study on the neuroectodermal conversion of bm-mscs in vitro by exposure to the prospective peptide cart. after treatment with cart, we found that cart could successfully differentiate mscs into neural cells in vitro. these cells were morphologically similar to neural stem cells. more importantly, they could express neural progenitor stem cell marker nestin and mature glial marker gfap as well as mature neuron markers like map- and neun. it indicated that the protocol of using cart for three or six days induced significant changes in morphology and expression of markers of early and mature neural cell types. similar with induction rate of bfgf plus egf, cart exposure obtained that map- gfap and neun expressions were found in ± %, ± %, ± % of all cells in days, and ± %, ± %, ± % in days. however, the expression of one or even two neuronal proteins does not prove that the cell bearing these "neuronal markers" is capable of all the complex functions of a neuron. it will be important to determine whether these cells possess functional characteristics of neurons. subsequently, double-labeled immunofluorescence was performed to calculate the function of the converted cells. it was intriguing that the differentiated neun positive cells co-expressed chat or th. our study on excitable property of msc-derived neural like cells demonstrated that the differentiated cells pocess founctional patassium and cacium channels. recent research discovered that the neuron-like cells differentiated from bm-mscs resemble the endogenous neural progenitors in morphological, immunocytochemical, and functional characteristics. another independent investigator also reported that the transformed bm-mscs exhibit both neuron-like biochemical function and some corresponding electrophysiological properties. cart is an endogenous neuropeptide which is widely distributed in the brain and peripheral nervous system. it has been well-established that cart can promote the survival and differentiation of neurons. some investigators disclosed that cart has the capability to modulate the expression of growth factors and neurotrophic factors, which probably participated in the underlying effect of cart. based on this theory, we made an assumption that neurotrophic factors could be the bridge between cart and stem cell differentiation. in order to verify this hypothesis, the amount of ngf and bdnf was determined by rt-pcr. the result that neurotrophic factors are significant higher in cart treated group implied cart could promote the endogenous production of neurotrophic factors. neurotrophic factors in cell and tissue culture have been shown to promote neuronal survival and differentiation and also regulate the growth of axons and dendrites. the presence of growth factors such as bdnf or ngf in the transplantation or the co-culture experiment are considered as physiological inductors for neural differentiation of mscs. mscs can induce a variety of neuro-regulatory proteins in addition to bdnf and β-ngf. further study demonstrated that the ngf and bdnf receptor trka and trkb do exist on the surface of mscs. moreover, the trkb receptor modified by bdnf may trigger the mapk signal pathway. this process contributes to the maturation and neural differentiation of mscs. early studies confirmed that bdnf+ra adult bone marrow stromal cells can be induced to differentiate into neural cells in vitro. however, cart stimulated the endogenous bdnf and ngf expression of mscs. this stimulation may imitate the natural physiological pattern. as a result, we obtained a higher yield rate than in previous studies. in the hippocampus, bdnf and other neurotrophins are expressed from early stage of neuron development. this process is a prerequisite for the differentiation of neurons derived from hippocampal precursor cells in vitro. overall, findings suggest that the bdnf and ngf enhanced by cart may be the foundation of stem cell differentiation and maturation.  in summary, we identified cart as a potential neurotrophic factor. cart can promote the neural differentiation of bm-mscs, probably through enhancing the endogeneous expression of neurotrophic factors. combined our previous studies, this study further confirmed the benefitial role of cart in the central nervous system. besides the protective effect on neurons through complicated interaction with immunology and inflammation, cart also contributes to neuronal cells differentiation and generation. in the future, cart pre-treatment may become an attractive and implemental method in stem cell therapy for neurological disease. 
in this work, we isolated and identified mscs from mouse bone marrow. in addition, neun positive cells could co-localize with th or chat by double-labled immunofluorescence and nissl bodies were found in several differentiated cells by nissl stain. this study demonstrated that cart could promote the differentiation of bm-mscs into neural cells through increasing neurofactors, including bndf and ngf. co-cultured with cart for six days, bm-mscs were differentiated into neuron-like cells by the observation of optical microscopy. however, the neural differentiation of stem cells influenced by massive factors and interactions is not well studied at present. cell therapy using bm-mscs seems like a promising approach for the treatment of neurological diseases. combined application of cart and bm-mscs may be a promising cell-based therapy for neurological diseases.
 neural tissue has limited potential to self-renew after neurological damage. cell therapy using bm-mscs seems like a promising approach for the treatment of neurological diseases. however, the neural differentiation of stem cells influenced by massive factors and interactions is not well studied at present.  in this work, we isolated and identified mscs from mouse bone marrow. co-cultured with cart for six days, bm-mscs were differentiated into neuron-like cells by the observation of optical microscopy. immunofluorescence demonstrated that the differentiated bm-mscs expressed neural specific markers including map- nestin, neun and gfap. in addition, neun positive cells could co-localize with th or chat by double-labled immunofluorescence and nissl bodies were found in several differentiated cells by nissl stain. furthermore, bdnf and ngf were increased by cart using rt-pcr.  this study demonstrated that cart could promote the differentiation of bm-mscs into neural cells through increasing neurofactors, including bndf and ngf. combined application of cart and bm-mscs may be a promising cell-based therapy for neurological diseases. 