erectile dysfunction ( ed ) remains a common cause of significant postoperative morbidity in patients undergoing radical therapies for prostate cancers or other pelvic malignancies , because the cavernous nerve can be inadvertently axotomized , lacerated , or stretched at the time of surgery .
although techniques such as nerve - sparing radical prostatectomy , nerve grafting , and robotic - assisted radical prostatectomy have been developed in which the cavernous nerve is largely preserved , postoperatively , patients still face a high incidence of ed . in recent years
, stem cell therapy has evolved as a potential therapy in the prevention of ed following cavernous nerve injury .
the multilineage differentiation potential of stem cells can contribute to regenerate the damaged cavernous nerve , thereby providing an autologous and curative therapeutic option .
although bone marrow cells have the ability to differentiate into neurons , limitations hinder the widespread application of these cells . among the various stem cells , human muscle - derived stem cells ( hmdscs ) and
human - adipose tissue derived stem cells ( hadscs ) have great potential in stem cell therapy because of their abundance , ease of isolation , and low immunogenicity .
in addition , hmdscs and hadscs have a considerable capacity for self - renewal and can differentiate into neural lineages [ 5 - 9 ] .
although many studies on the neurogenic trans - differentiation of hmdscs and hadscs have been conducted , no in vitro studies are available for comparison of the neurogenic capacity of these cells . in this study , therefore , we compared the neuronal differentiation capacity of hmdscs with that of hadscs in vitro .
human adipose tissues were obtained by simple liposuction from the abdominal subcutaneous fat of donors .
subcutaneous adipose tissues were washed with phosphate- buffered saline ( pbs ) and digested with 1 mg / ml collagenase under gentle agitation for 60 minutes at 37. next , the digested tissues were filtered through a 100 m nylon mesh in order to remove cellular debris and centrifuged at 1,500 rpm for 5 min to obtain a pellet .
the pellet was re - suspended in rcme ( rnl bio , seoul , korea ) or dmem containing 10% fetal bovine serum ( fbs ) .
the cell fraction was cultured overnight at 37 in 5% co2 in rcme or dmem .
adhesion of cells was checked under a microscope the next day . the procedure for preparation of hadscs
using scalpels , hmdscs isolated from the rectus muscles of humans were removed and minced into a coarse slurry .
louis , mo , usa ) , dispase ( invitrogen , carlsbad , ca , usa ) , and trypsin - edta ( invitrogen ) . after physical and enzymatic dissociations
, muscle - derived cells were centrifuged and re - suspended in proliferation medium ( containing dmem [ gibco , grand island , ny , usa ] , 10% horse serum , 10% fbs , 1% chick embryo extract , and 1% penicillin - streptomycin ) . for isolation of smnps , cells were plated in collagen - coated flasks ( pp1 ) .
after 2 hours , cells non - adherent in pp1 were transferred into the next flasks ( pp2 ) and incubated for 16 hours .
no adherent cells were observed in pp3 ; therefore , the serial transfer of non - adherent cells was terminated at this point . during cultivation , two morphologically
pp2 and pp3 flasks contained approximately 30% , 10% , and < 1% of fibroblast - like cells , respectively .
further purification of weakly adherent cells ( primarily round - shaped cells ) was performed on cells adherent in pp2 and pp3 flasks .
cells were trypsinized for 3 min , after which suspended cells from pp2 and pp3 flasks were combined and re - plated in a fresh flask . as these cells proliferated in proliferation medium for 3 to 4 days ,
were grown as adherent cultures in dmem supplemented with 10% fbs and 1% penicillin - streptomycin at 37 in 5% co2 .
after 24 hours , cells were washed with pbs and plated in neurobasal media , 1% penicillin , and 50 m / ml of forskolin and 1 g / ml laminin , 20 ng / ml basic fibroblast growth factor ( bfgf ) , and 20 ng / ml epidermal growth factor ( egf ) with 2% fbs .
both hmdscs and hadscs were placed and grown on poly - l - lysine - coated cover slips for 5 days .
after 5 days , these cells were washed with pbs , fixed with 4% paraformaldehyde for 10 minutes at room temperature , washed with pbs again , and then permeabilized with 0.1% triton - x 100 .
after washing twice with pbs , cells were blocked by incubation with 10% normal goat serum for 1 hour at room temperature .
nestin ( 1:100 ; abcam , combride , uk ) , -tubulin iii ( tuj1 , 1:500 ; abcam , combride , uk ) , and gfap ( 1:100 ; abcam , combride , uk ) were then added .
primary antibodies were incubated overnight at 4. after washing , the cells were treated with alexa 488-conjugated goat anti - mouse antibody as a secondary antibody , followed by incubation at room temperature for 1 hour .
nuclei were stained with dapi for cell counting , and the cells were observed under a fluorescence microscope .
rna was extracted from hmdscs , differentiated hmdscs ( d - hmdscs ) , hadscs , and differentiated hadscs ( dhadscs ) .
first - strand cdna was synthesized by using oligo dt primer and superscript ii reverse transcriptase according to the manufacturer 's instructions .
real - time polymerase chain reaction analysis ( pcr ) was performed by using rt sybr green / rox qpcr master mix using the real - time thermal cycler ( 50/2 min , 95/10 min , [ 95/15 s , 60/30 s , 72/30 s - 40 cycles ] , 95/15 s , 60/1 min , 95/15 s ) .
protein was homogenized in ice - cold lysis buffer containing 20 mm tris - cl , ph 8.0 , 150 mm nacl , 1 mm edta , 1% triton x-100 , 10 m leupeptin , 20 g / ml chymostatin , and 2 mm pmsf .
following centrifugation at 12,000 xg for 30 minutes at 4 , the supernatant was extracted and quantified by use of the bicinchoninic acid ( bca ) protein assay kit ( pierce , rockford , il , usa ) and then electrophoresed on an sds - page gel .
after transfer to a nitrocellulose membrane , the membrane was blocked with 5% skim milk and then incubated with antibody against nestin ( 1:1,000 ; abcam ) , -tubulin iii
( 1:1,000 ; abcam ) , gfap ( 1:1,000;abcam ) , or -actin , followed by incubation with the corresponding secondary antibody conjugated to horseradish peroxidase .
the ecl method ( amersham , arlington heights , il , usa ) was used for development of protein bands .
statistical analyses were performed by using the two - tailed student 's t - test .
human adipose tissues were obtained by simple liposuction from the abdominal subcutaneous fat of donors .
subcutaneous adipose tissues were washed with phosphate- buffered saline ( pbs ) and digested with 1 mg / ml collagenase under gentle agitation for 60 minutes at 37. next , the digested tissues were filtered through a 100 m nylon mesh in order to remove cellular debris and centrifuged at 1,500 rpm for 5 min to obtain a pellet .
the pellet was re - suspended in rcme ( rnl bio , seoul , korea ) or dmem containing 10% fetal bovine serum ( fbs ) .
the cell fraction was cultured overnight at 37 in 5% co2 in rcme or dmem .
adhesion of cells was checked under a microscope the next day . the procedure for preparation of hadscs
using scalpels , hmdscs isolated from the rectus muscles of humans were removed and minced into a coarse slurry .
louis , mo , usa ) , dispase ( invitrogen , carlsbad , ca , usa ) , and trypsin - edta ( invitrogen ) . after physical and enzymatic dissociations
, muscle - derived cells were centrifuged and re - suspended in proliferation medium ( containing dmem [ gibco , grand island , ny , usa ] , 10% horse serum , 10% fbs , 1% chick embryo extract , and 1% penicillin - streptomycin ) . for isolation of smnps , cells were plated in collagen - coated flasks ( pp1 ) .
after 2 hours , cells non - adherent in pp1 were transferred into the next flasks ( pp2 ) and incubated for 16 hours .
no adherent cells were observed in pp3 ; therefore , the serial transfer of non - adherent cells was terminated at this point . during cultivation , two morphologically
pp2 and pp3 flasks contained approximately 30% , 10% , and < 1% of fibroblast - like cells , respectively .
further purification of weakly adherent cells ( primarily round - shaped cells ) was performed on cells adherent in pp2 and pp3 flasks .
cells were trypsinized for 3 min , after which suspended cells from pp2 and pp3 flasks were combined and re - plated in a fresh flask . as these cells proliferated in proliferation medium for 3 to 4 days , the cells formed additional colonies .
human adipose tissues were obtained by simple liposuction from the abdominal subcutaneous fat of donors .
subcutaneous adipose tissues were washed with phosphate- buffered saline ( pbs ) and digested with 1 mg / ml collagenase under gentle agitation for 60 minutes at 37. next , the digested tissues were filtered through a 100 m nylon mesh in order to remove cellular debris and centrifuged at 1,500 rpm for 5 min to obtain a pellet .
the pellet was re - suspended in rcme ( rnl bio , seoul , korea ) or dmem containing 10% fetal bovine serum ( fbs ) .
the cell fraction was cultured overnight at 37 in 5% co2 in rcme or dmem .
adhesion of cells was checked under a microscope the next day . the procedure for preparation of hadscs
using scalpels , hmdscs isolated from the rectus muscles of humans were removed and minced into a coarse slurry .
louis , mo , usa ) , dispase ( invitrogen , carlsbad , ca , usa ) , and trypsin - edta ( invitrogen ) .
after physical and enzymatic dissociations , muscle - derived cells were centrifuged and re - suspended in proliferation medium ( containing dmem [ gibco , grand island , ny , usa ] , 10% horse serum , 10% fbs , 1% chick embryo extract , and 1% penicillin - streptomycin ) . for isolation of smnps
after 2 hours , cells non - adherent in pp1 were transferred into the next flasks ( pp2 ) and incubated for 16 hours .
no adherent cells were observed in pp3 ; therefore , the serial transfer of non - adherent cells was terminated at this point . during cultivation , two morphologically
pp2 and pp3 flasks contained approximately 30% , 10% , and < 1% of fibroblast - like cells , respectively .
further purification of weakly adherent cells ( primarily round - shaped cells ) was performed on cells adherent in pp2 and pp3 flasks .
cells were trypsinized for 3 min , after which suspended cells from pp2 and pp3 flasks were combined and re - plated in a fresh flask . as these cells proliferated in proliferation medium for 3 to 4 days , the cells formed additional colonies .
both hmdscs and hadscs ( 1x10cells ) were grown as adherent cultures in dmem supplemented with 10% fbs and 1% penicillin - streptomycin at 37 in 5% co2 . after 24 hours , cells were washed with pbs and plated in neurobasal media , 1% penicillin , and 50 m / ml of forskolin and 1 g / ml laminin , 20 ng / ml basic fibroblast growth factor ( bfgf ) , and 20 ng / ml epidermal growth factor ( egf ) with 2% fbs .
both hmdscs and hadscs were placed and grown on poly - l - lysine - coated cover slips for 5 days .
after 5 days , these cells were washed with pbs , fixed with 4% paraformaldehyde for 10 minutes at room temperature , washed with pbs again , and then permeabilized with 0.1% triton - x 100 .
after washing twice with pbs , cells were blocked by incubation with 10% normal goat serum for 1 hour at room temperature .
nestin ( 1:100 ; abcam , combride , uk ) , -tubulin iii ( tuj1 , 1:500 ; abcam , combride , uk ) , and gfap ( 1:100 ; abcam , combride , uk ) were then added .
primary antibodies were incubated overnight at 4. after washing , the cells were treated with alexa 488-conjugated goat anti - mouse antibody as a secondary antibody , followed by incubation at room temperature for 1 hour .
nuclei were stained with dapi for cell counting , and the cells were observed under a fluorescence microscope .
rna was extracted from hmdscs , differentiated hmdscs ( d - hmdscs ) , hadscs , and differentiated hadscs ( dhadscs ) .
first - strand cdna was synthesized by using oligo dt primer and superscript ii reverse transcriptase according to the manufacturer 's instructions . real - time polymerase chain reaction analysis ( pcr ) was performed by using rt sybr green / rox qpcr master mix using the real - time thermal cycler ( 50/2 min , 95/10 min , [ 95/15 s , 60/30 s , 72/30 s - 40 cycles ] , 95/15 s , 60/1 min , 95/15 s ) .
protein was homogenized in ice - cold lysis buffer containing 20 mm tris - cl , ph 8.0 , 150 mm nacl , 1 mm edta , 1% triton x-100 , 10 m leupeptin , 20 g / ml chymostatin , and 2 mm pmsf .
following centrifugation at 12,000 xg for 30 minutes at 4 , the supernatant was extracted and quantified by use of the bicinchoninic acid ( bca ) protein assay kit ( pierce , rockford , il , usa ) and then electrophoresed on an sds - page gel .
after transfer to a nitrocellulose membrane , the membrane was blocked with 5% skim milk and then incubated with antibody against nestin ( 1:1,000 ; abcam ) , -tubulin iii ( 1:1,000 ; abcam ) , gfap ( 1:1,000;abcam ) , or -actin , followed by incubation with the corresponding secondary antibody conjugated to horseradish peroxidase . the ecl method ( amersham , arlington heights , il , usa )
statistical analyses were performed by using the two - tailed student 's t - test .
1a and i ) . in immunohistochemical staining , hmdscs and hmdscs were strongly positive for neural stem cell marker ( nestin ) , but weakly positive for neuronal marker ( -tubulin iii ) and glial marker ( gfap ) ( fig .
following 5 days of neural induction , hmdscs and hadscs displayed a differentiated phenotype in morphology ( fig .
immunohistochemistry showed that nestin expression was decreased , and -tubulin iii and gfap expression were increased ( fig .
there was no significant difference in nestin expression between d - hmdscs and d - hadscs ; however , -tubulin iii and gfap expression in d - hadscs was increased compared with that in d - hmdscs .
according to cell morphology and staining , hadscs had a higher phenotype and neurogenic potential than did hmdscs . to dissect the neurogenic potential of hmdscs and hadscs at the molecular level , the mrna levels of nestin , -tubulin iii , and gfap were detected by real - time pcr , normalized to the housekeeping gapdh .
nestin mrna levels in d - hmdscs and d - hadscs were approximately 50% lower than the expression in hmdscs and hadscs , respectively .
there was no significant difference in nestin mrna expression between d - hmdscs and d - hadscs ( fig .
however , -tubulin iii and gfap mrna expression in d - hadscs was significantly higher than that in d - hmdscs ( fig . 2b and 2c )
. we also investigated the protein expression of nestin , -tubulin iii , and gfap in differentiated hmdscs and hadscs by western blot analysis ( fig .
the protein expression of nestin , -tubulin iii , and gfap before and after differentiation showed almost the same tendency with mrna expression .
protein expression of nestin in the hmdscs and hadscs was significantly decreased after neuronal induction ( fig .
1a and i ) . in immunohistochemical staining , hmdscs and hmdscs were strongly positive for neural stem cell marker ( nestin ) , but weakly positive for neuronal marker ( -tubulin iii ) and glial marker ( gfap ) ( fig .
following 5 days of neural induction , hmdscs and hadscs displayed a differentiated phenotype in morphology ( fig .
immunohistochemistry showed that nestin expression was decreased , and -tubulin iii and gfap expression were increased ( fig .
there was no significant difference in nestin expression between d - hmdscs and d - hadscs ; however , -tubulin iii and gfap expression in d - hadscs was increased compared with that in d - hmdscs .
according to cell morphology and staining , hadscs had a higher phenotype and neurogenic potential than did hmdscs .
to dissect the neurogenic potential of hmdscs and hadscs at the molecular level , the mrna levels of nestin , -tubulin iii , and gfap were detected by real - time pcr , normalized to the housekeeping gapdh .
nestin mrna levels in d - hmdscs and d - hadscs were approximately 50% lower than the expression in hmdscs and hadscs , respectively .
there was no significant difference in nestin mrna expression between d - hmdscs and d - hadscs ( fig .
however , -tubulin iii and gfap mrna expression in d - hadscs was significantly higher than that in d - hmdscs ( fig . 2b and 2c ) .
we also investigated the protein expression of nestin , -tubulin iii , and gfap in differentiated hmdscs and hadscs by western blot analysis ( fig .
the protein expression of nestin , -tubulin iii , and gfap before and after differentiation showed almost the same tendency with mrna expression .
protein expression of nestin in the hmdscs and hadscs was significantly decreased after neuronal induction ( fig .
-tubulin iii and gfap protein expression in d - hadscs was significantly higher than that in d - hmdscs ( fig .
this study compared the neurogenic potential of hmdscs and hadscs by detecting several neuron cell markers .
our data suggest that differentiated hadscs have higher neuronal / glial marker expression than do hmdscs and thereby are more suitable for nerve regeneration .
recently , stem cell treatment has been regarded as a potential treatment for ed . in recent years
, tissue engineering strategies have combined living cells and scaffold materials for the development of biological substitutes that can restore tissue functions .
both natural and synthetic materials have been fabricated for transplantation of stem cells and their specific differentiation into muscles , bones , cartilage , and neuron - like cells .
studies have shown that hmdscs and hadscs can be induced into adipocytes , osteoblasts , chondrocytes , myocytes , hepatocytes , endotheliocytes [ 5 - 9 ] , and neuron - like cells [ 2,10 - 13 ] .
the surface phenotype of hmdscs and hadscs is similar to that of bone marrow - derived stromal cells .
previous reports have indicated that neural differentiation of hmdscs and hadscs is achieved with different experimental protocols , including the use of chemical agents , such as forskolin [ 11,14 - 17 ] , laminin , egf , and bfgf .
bfgf is known to generate neural precursor cells with a greater capacity for neuronal differentiation .
forskolin is a commonly used agent to increase the intracellular levels of cyclic adenosine monophosphate ( camp ) by activating the enzyme adenylyl cyclase .
laminin - stimulated levels of tyrosine hydroxylase of chromaffin cells were associated with their conversion to adrenergic sympathetic cells .
laminin has differential action in the proliferation , survival , and differentiation of cultured neuronal cells .
although forskolin , laminin , egf , and bfgf enhanced neuron - like cell production , the results were greatly improved when the combination was added to the medium . we succeeded in inducing the neural differentiation of hmdscs and hadscs by treatment with forskolin , laminin , b - fgf , and egf . in the present study ,
the neuronal phenotype at 5 days following neural induction was observed by staining for nestin - negative and -tubulin iii- and gfap - positive .
we found that d - hmdscs and d - hadscs displayed similar immunocytochemical changes following neural induction .
hmdscs and hadscs grown under control conditions expressed low -tubulin iii and gfap but detectable levels of nestin .
after 5 days of neural induction , we detected strong staining for -tubulin iii as well as gfap in d - hadscs .
real - time pcr and western blotting confirmed that the number of undifferentiated hmdscs and hadscs expressing cell type - specific markers was very low .
neurons were observed in cultures when compared with that of nestin - negative and -tubulin iii- , gfap - positive .
our results showed that differentiated muscle- or adipose - derived stem cells have morphological and phenotypic characteristics that are similar to those of neural cells . in this study
, we confirmed that the ability of hadscs to differentiate into neural cells was better than that of hmdscs .
in summary , we have shown that both hmdscs and hadscs have morphological and phenotypic characteristics similar to those of neural cells .
therefore , hadscs seem to be an ideal alternative source of stem cells for cavernous nerve regeneration .
 purposeerectile dysfunction ( ed ) remains a major complication from cavernous nerve injury during radical prostatectomy . 
 recently , stem cell treatment for ed has been widely reported . 
 this study was conducted to investigate the availability , differentiation into functional cells , and potential of human muscle - derived stem cells ( hmdscs ) and human adipose - derived stem cells ( hadscs ) for ed treatment.materials and methodswe compared the neural differentiation of hmdscs and hadscs . 
 human muscle and adipose tissues were digested with collagenase , followed by filtering and centrifugation . for neural induction , isolated hmdscs and hadscs 
 were incubated in neurobasal media containing forskolin , laminin , basic - fibroblast growth factor , and epidermal growth factor for 5 days . following neural induction , hmdscs and hadscs 
 were differentiated into neural cells , including neurons and glia , in vitro.resultsin neural differentiated hmdscs ( d - hmdscs ) and differentiated hadscs ( d - hadscs ) , neural stem cell marker ( nestin ) showed a significant decrease by immunocytochemistry , and neuronal marker ( -tubulin iii ) and glial marker ( gfap ) showed a significant increase , compared with primary hmdscs and hadscs 
 . real - time chain reaction analysis and western blotting demonstrated significantly elevated levels of mrna and protein of -tubulin iii and gfap in d - hadscs compared with d-hmdscs.conclusionswe demonstrated that hmdscs and hadscs can be induced to undergo phenotypic and molecular changes consistent with neurons . 
 the neural differentiation capacity of hadscs was better than that of hmdscs . 
INTRODUCTION
MATERIALS AND METHODS
1. Cell isolation
Human adipose-derived stem cells
Human muscle-derived stem cells
2. Neural induction
3. Immunocytochemistry
4. Real-time polymerase chain reaction analysis
5. Western blot analysis
6. Statistical analysis
RESULTS
1. Morphologic changes after neural induction
2. Real-time polymerase chain reaction analysis
3. Western blot analysis
DISCUSSION
CONCLUSIONS