increase in the prevalence of neurodegenerative diseases and neural damage has caused the scientists to search for various approaches such as cell therapy to improve the neurogenesis techniques .
a lot of investigations confirmed the presence of adult stem cells in various tissues including breast milk .
breast milk contains a heterogeneous cell population ; besides , a subpopulation with stem cell properties including the ability to be differentiated into different cell lineages has been isolated from fresh human milk [ 2 , 3 ] .
a bipotential stem cell with the differentiation capacity into mammary epithelial cell and myoepithelial cells has been detected in mammary gland tissue .
maternal mammary stem cells have been considered as one of the cell sources in breast milk .
the presence of exfoliated epithelial cells from alveoli , macrophages , and lymphocytes was also reported [ 6 , 7 ] .
it has been also reported that 1015% of the cells isolated from fresh breast milk expressed mesenchymal stem cell ( msc ) markers and culturing the isolated cells led to an increase in the msc population due to their higher capacity of cell proliferation .
the presence of a nestin - positive subpopulation was also reported in the breast milk - derived cells ; however , the frequency of these cells was low [ 8 , 9 ] .
previous studies identified the presence of activated mammary stem cell ( masc ) in breast milk through staining cells for masc markers .
hassiotou et al . named these cells as human breast - milk stem cells ( hbscs ) .
their results demonstrated that these stem cells have a capability to be differentiated into mammary cells ( luminal and myoepithelial ) .
furthermore , they determined the expression of a group of embryonic stem cell- ( esc- ) associated genes such as oct4 , klf4 , nanog , and sox2 in hbscs .
also , it has been reported that the phenotype , colony morphology , and differentiating capability of hbscs are similar to those in esc [ 3 , 10 ] .
consequently , as breast milk contains these special pluripotent stem cells , it can be utilized as a valuable and a new easily available source for regenerative medicine [ 3 , 10 ] .
the presence of nestin - positive cell population and also a subpopulation of expressed esc markers in breast milk caused this noninvasive source of stem cells to be considered as a good candidate for differentiation into neural cell lineage .
it has been demonstrated that the common regulators play role in the development of both mammary gland and neuroepithelium , and these regulators are also involved in escs differentiation and self - renewal .
these common embryonic origins and also common regulators may suggest breast milk - derived cells as a good source for neural cell lineages differentiation .
previous studies reported that neural stem cells showed the capability to be differentiated toward neuronal cells [ 12 , 13 ] .
they may be considered as an appropriate source for cell replacement therapies ( crts ) of the brain diseases . however , there have been various reports which show the risks of neural stem cell application for the patients including tumor formation , insufficient migration , immune rejection , surgical threats , and transmission of infections possibility during transplantation . besides , neural stem cell isolation is invasive .
therefore , finding an alternative stem cell source is essential to overcome such issues and barriers .
the pluripotency of the breast milk - derived cells was evaluated by exposing the cells to various culture conditions .
the cells treated with neurogenic medium expressed nestin and tubulin which indicated the cell differentiation into neural progenitor cell and neuron - like cells , respectively .
this study tried to find the breast milk - derived cell ability to be differentiated into three neural cell lineages , neurons , astrocytes , and oligodendrocytes .
also , the current study showed that the breast milk - derived cell behaved in a similar way to neural stem cells in vitro .
this study was approved by the ethics committee of shiraz university of medical sciences , and all participants provided informed written consent .
women , who were healthy breastfeeding participants , submitted informed written consent prior to sample collection . in aseptic procedures , mature breast milk ( 5200  ml )
disinfected phosphate buffered saline ( pbs ) ( ph 7.4 , gibco ) equal volumes were used to dilute the breast milk and they were centrifuged for 20 minutes at 20c at 805  g. then , skim milk liquid component and fat layer were taken out . in pbs ,
after that , the cell pellet was resuspended in 10% fetal bovine serum ( fbs , certified , invitrogen ) within pbs ( blocking buffer ) . to determine each sample 's cell viability and cell concentration ,
breast milk cells ( 40000  cells / ml ) were cultured in plates coated with gelatin .
they were incubated at 5% co2 and 37c ; also , the media were changed daily .
after 5 days of culturing , each colony was separately selected and relocated in new dishes and cultured in medium in order to make feeder culture of the second and third ones .
furthermore , ultralow binding plates were used to seed cells on them to have spheroid culture and the es media ( dmem / f12 , kosr ( knockout serum replacement ) 10% , nonessential amino acid 1% , bfgf 10  g / ml , and pen / strep 1% ) were added to the plates . whenever the cells needed to be passaged , they were trypsinized ( gibco ) at 37c in 5 minutes and were divided to 1  :  2 . some embryonic and mesenchymal stem cell markers antibodies such as nanog , oct4 ( abcam ab19857 1  :  500 ) , sox2 , cd44 ( abcam ab119863 1  :  500 ) , cd105 ( abcam ab44967 1  :  250 ) , cd106 ( abcam ab19569 1  :  500 ) , cd90 ( abcam ab225 1  :  250 ) , and cd133 ( millipore mab4399 ) were used to characterize the isolated cells from human breast milk by immunocytochemistry method . briefly
, the cultured cells were fixed by paraformaldehyde 4% in 4c for 20 minutes ; after washing with pbs , primary antibodies were diluted in pbs containing 0.3% triton and 5% goat serum and the samples were kept in room temperature for 60 minutes .
after that , washing with pbs was performed for 3 times . then secondary antibodies were added and , after 45 minutes of incubation in room temperature , the last washing with pbs was done .
the isolated cells were cultured in gelatin coated plates with concentration of 10000  cells / cm with dmem / f12 , 1% b27 , and 2% n2 for 710 days .
these spheres were separated and passaged in a different plate using dmem / f12 containing b27 , n2 , bfgf 10  g / ml , and egf 20  g / ml ( ns - a media ) .
then , the spheres were formed and each cell of these spheres was capable of providing another sphere by passaging . for neural stem cells identification
, immunocytochemistry was performed for nestin antibody ( millipore , ab5922 ) on neurospheres and cd133 ( millipore mab4399 )   for neural stem cells with protocol mentioned above . neural stem cells ( nscs ) ( 5000  cells / ml )
were seeded in poly - l - ornithine - coated culture dishes to be differentiated into neuron , oligodendrocyte , and astrocyte . for differentiation , bfgf and egf
were removed from the media and fcs 5% was added to them and after 5 days the differentiated neural stem cells provided neurons , oligodendrocytes , and astrocytes . for neuron , oligodendrocyte , and astrocyte detection , antibodies , -tubulin iii ( promega g7121 , 1  :  2000 ) , o4   ( millipore , mab345 1  :  50 ) , and gfap   ( dakocytomation , code number z0334 1  :  500 ) , respectively , were assessed by an immunocytochemistry method similar to the one previously described .
to detect the cells ' nucleus , they were fixed with paraformaldehyde 4% and then 4,6-diamino-2-phenylindole dihydrochloride ( millipore s7113 1  :  1000 ) was added to the fixed cells and they were kept in room temperature for 30 minutes .
the isolated breast - milk - derived cells were adherent to the plates and their morphology was similar to the cells of myoepithelium of the breast ( figure 1 ) .
they were cultured to the 10th passage and after each passage the provided cells were capable of proliferating and kept their expansion potential .
the immunohistochemistry staining showed a subpopulation of breast milk - derived cells that expressed embryonic stem cells markers such as nanog ( 66.2%  6.52 ) , oct4 ( 42.9%  6.99 ) , and sox2 ( 57.3%  7.74 ) ( figure 2 ) .
a subpopulation of the isolated cells also expressed markers that were also detected on the surface of mesenchymal stem cells including cd44 ( 67%  10.7 ) , cd105 ( 68.3%  3.91 ) , cd106 ( 8.4%  2.36 ) , and cd133 ( 2.76%  1.93 ) .  
table 1 summarizes the frequency of the positive cell for each marker . after exposing the breast - milk stem cells to ns - a media ( dmem / f12 , n2 , b27 , bfgf , and egf ) for 5 days
, they formed shiny , floating , sphere - like cell aggregations , neurospheres , measuring about 100  m in diameter ( figure 3 ) .
the enzymatically dissociated neurospheres showed the capability of forming a new sphere within 57 days .
in addition , the cells kept their sphere - forming ability till the 8th passage without any significant change in their sphere - forming frequency ( figure 8) .
a small subpopulation of the breast milk - derived cells expressed nestin ( 7.4%  3.30 ) and cd133 ( 2.76%  1.93 ) as markers for neural stem cells .
the frequency of the nestin - positive ( 58.20%  6.71 ) ( figures 6 and 9 ) and cd133-positive ( 58.74%  3.36 ) ( figures 7 and 10 ) cells was increased significantly after exposing the cells to neurogenic media and neurosphere formation .
the behavior of the nestin - positive breast milk - derived cells treated with neurogenic media , including the formation of spheroid aggregates , was similar to the neural stem cells .
the most important characteristic for specification of neural stem cells is their ability to produce three neural lineages consisting of neurons , oligodendrocytes , and astrocytes .
differentiated astrocytes showed a flat or polygonal cell body with several processes . after adding 5% fetal bovine serum , removing growth factors ( bfgf and egf ) , to the cells for 5 days , the neural stem cell - like cells proliferated and attached to the plate totally and their appearances
were completely changed into three neural cell lineages ( figure 4 ) . to detect neurons , the expression of neuronal marker , -tubulin iii , was evaluated .
the percentage of the tubulin iii was 19%  2.44 of the cells treated with neurogenic media . for detection of oligodendrocyte , anti - o4 antibody  
was used and the frequency of the o4-positive cells was 14.31%  2.31 ; and for detection of astrocytes , anti - gfap antibody   was administrated and it was shown that 66.69%  7.50 of the cells were astrocytes ( figure 5 , table 2 ) .
therefore , breast milk cells have been suggested to be used for treating neonatal disorders .
breast - milk stem cells have been previously demonstrated to express nestin , a neuroectoderm marker ; however , a few samples contained the cells expressing neurofilament , a late neural differentiation marker .
the presence of nestin - positive cells indicated that the breast - milk stem cells can be an appropriate candidate for differentiation toward neurons or neuroglia .
the results of the current study revealed that the exposure of the whole cell population of breast milk to neurogenic medium led to an increase in the presence of nestin - positive cells and that the cell behavior in neurosphere formation is the same as what happened in the culture of neural stem cells .
further differentiation of nestin - positive cells into the neuron as well as neuroglia was shown in this study as indicated by the presence of cells that expressed -tubulin as neuron marker , o4 as oligodendrocyte marker , and gfap as astrocyte marker .
several studies also showed that nestin - positive cells were derived from the neurosphere of the neural stem cells isolated from the avian species , mouse embryo , adult mouse , and human   and differentiated into three neural lineages .
it seems that the cells derived from breast milk were differentiated in the same way as in the case of neural stem cells .
mesenchymal stem cells derived from human adipose tissue [ 25 , 26 ] , bone marrow , muscle , and umbilical cord   have been shown to be able to be differentiated toward neuron - like cells .
neuron - like cells differentiated from wharton jelly of the umbilical cord were also transplanted to the app / ps1 mouse .
the transplantation improved the cognitive reactions of the mouse and decreased the level of amyloid -peptides deposition .
human umbilical mesenchymal stem cells - derived neurospheres with bdnf were transplanted to the injured spinal cord and the morphologic and functional recoveries have been demonstrated .
these data indicated that nonneural stem cells may be functional when they were transplanted to the animal models .
therefore , the neural - like cells or neuroglia derived from breast milk also may be as effective as those derived from the other sources ; however , it needs further investigations .
our results indicated that breast milk - derived stem cells express embryonic cell markers such as nanog , oct4 , sox2 , seea4 , and tra 160/81 .
embryonic stem cells have a great potential to be differentiated toward neural cell lineages [ 31 , 32 ] .
the stability in electrical phenotype of stem cell - derived neuron has been shown previously , revealing that the differentiated cells may be functional . the transplanted mesenchymal stem cells in cns has been shown they are capable to survive and differentiate to neural lineages . the breast milk - derived stem cells showed the capability to be differentiated into neural cell lineages and their similarity to both embryonic and mesenchymal stem cells makes them a good candidate for cell therapy in neurodegenerative diseases .
as the isolation of neural stem cells is an invasive procedure , using breast milk - derived cells as a source of cell therapy may be preferable especially for the patients in child - bearing ages .
however , the physiological test and also in vivo studies should be done to ensure that the cells do not dedifferentiate and are functional when they are transplanted .
 
 objectives . human breast milk contains a heterogeneous population of cells that have the potential to provide a noninvasive source of cells for cell therapy in many neurodegenerative diseases without any ethical concern . 
 the objectives of this study were to differentiate the breast milk - derived stem cells ( bmdsc ) toward neural stem cells and then into the neurons and neuroglia . 
 materials and methods . to do this , 
 the bmdsc were isolated from human breast milk and cultured in dulbecco 's modified eagle medium / f12 ( dmem / f12 ) containing fibroblast growth factor ( bfgf ) . 
 the cells were then characterized by evaluation of the embryonic and stem cell markers . 
 then , the cells were exposed to culture medium containing 1% b27 and 2% n2 for 710 days followed by medium supplemented with b27 , n2 , bfgf 10  g / ml , and endothelial growth factor ( egf ) 20  g / ml . then , the sphere - forming assay was performed . 
 the spheres were then differentiated into three neural lineages by withdrawing growth factor in the presence of 5% fbs ( fetal bovine serum ) . 
 the immunofluorescence was done for -tubulin iii , o4 , and gfap ( glial fibrillary acidic protein ) . 
 results . 
 the results indicated that the cells expressed both embryonic and mesenchymal stem cell ( msc ) markers . 
 they also showed neurospheres formation that was nestin - positive . 
 the cells were also differentiated into all three neural lineages . conclusion . 
 the bmdsc can behave in the same way with neural stem cells . 
 they were differentiated into oligodendrocytes , and astrocytes as well as neurons . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion