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 .