the grafted cells rarely expressed the proliferation marker ki67 ( figure 3d ), 29. 8±4. 4 % expressed nestin ( figure 3e ), 6. 5±0. 9 % expressed doublecortin ( dcx ) and 60. 8±8. 1 % were tuj1 + ( figure 3f, g ). grafted cells rarely co - expressed the astroglial marker gfap ( figure 3h ) or differentiated into cnpase - expressing oligodendrocytes ( figure 3i ). immunostaining for gad demonstrated that 25. 1±2. 3 % of grafted hnscs differentiated into gabaergic neurons while less than 2 % were positive for glutamate ( figure 3j, k ). 10. 1371 / journal. pone. 0001644. g003figure 3dispersion, engraftment and differentiation of the hnscs in stroke - lesioned animals. ( a ) schematic drawing of a frontal section through the striatum illustrating the dispersion of grafted hnscs in the focal ischemia - lesioned parenchyma ( shaded area ). ( b, c ) photos show frontal sections through the graft in the striatum immunostained with the human specific antibod ies: anti-hnuc ( green in b & c ) and anti-glut1 ( red, b & c ) showing blood vessels and dispersed hnscs in the graft zone. c: higher magnification of the inset in b. ( d – i ) photos taken from frontal sections through the graft in the striatum double immunoprocessed for cell proliferation and neural lineage markers. ( d ) note the endogenous ki67 + cells ( red cells, arrow ) in the stroke damaged area and the hnuc + ( green ) / ki67 - grafted hnscs ( arrowheads ). ( e ) examples of grafted sd56 hnscs showing co - expression of hnuc ( green ) and nestin ( red ). ( f ) confocal 3d reconstructed orthogonal images of the hnuc + ( green ) / dcx + ( red ) nscs ( arrowheads ) viewed in the x - z plan on the top and y - z plan on the right. ( g ) examples show the majority of grafted nsc progeny co - expressing hnuc ( red ) and the neuronal marker tuj1 ( green ). grafted nscs rarely differentiate into gfap + astrocytes ( h ). in i, rare example of grafted nsc progeny becoming an oligodendrocyte identified by the expression of cnpase ( green ). grafted nscs expressed the gabaergic marker gad65 / 67 ( j ) and rarely expressed glutamate ( k ). ( abbreviations: cx: cortex, str: striatum ). bars: ( b, c ) 100 µm; ( d, f ) 20 µm; ( e, g – k ) 10 µm. transplanted cells improve sensorimotor function of the stroke - disabled forelimbwe asked whether transplanted sd56 hnscs could enhance the recovery of sensorimotor function that is compromised in the stroke - injured rats. we used the cylinder test to measure the sensorimotor asymmetry in forelimb use during spontaneous exploration [ 22 ]. to establish the baseline of the stroke - induced sensorimotor deficit, spontaneous behavior of rats in a transparent cylinder was videotaped one week after stroke ( pre - transplant, figure 4 ). tests were then conducted 4 and 8 weeks after vehicle and sd56 hnscs transplantation. stable asymmetry in forelimb use was observed 7 days post - stroke ( pre - transplant, figure 4 ). ischemic rats used their impaired forelimbs ( contralateral to lesion ) during lateral exploration less than they did before stroke. transplantation of sd56 hnscs significantly enhanced the independent use of the impaired contralateral forelimb 4 weeks post transplantation ( p < 0. 05 vs pre - transplant ). eight weeks after transplantation the improvement in the use of the impaired forelimb was stable and significant when compared to the pre - transplant group and significantly improved in comparison to vehicle treated group at 8 weeks ( figure 4 ). in the vehicle treated group, the independent use of the contralateral forelimb remained impaired 4 and 8 weeks post - injection. in an independent study and using the same mcao rat animal model, we found that transplantation of dermal fibroblasts did not improve the stroke - induced motor deficits ( unpublished data ). 10. 1371 / journal. pone. 0001644. g004figure 4transplantation of nscs improves sensorimotor function of the stroke - disabled forelimb. forelimb use during spontaneous lateral exploration was measured in the cylinder test ( see method and results sections for details ). groups of vehicle injected ( n = 7 ) and hnscs ( n = 10 ) transplanted are represented. the animals were tested as described in method section. note the significant increase in the independent use of the impaired contralateral forelimb at 4 and 8 weeks post transplantation ( p < 0. 05 vs pre - transplant group ). the contralateral forelimb remained impaired in the vehicle treated group at 4 and 8 weeks post - injection. of steps taken by the ipsilateral, contralateral and both forelimbs simultaneously. * p < 0. 05 vs pre - transplant group; # p < 0. 05 vs vehicle groups. our results indicate that a self - renewable and homogenous population of hnscs, sd56, was derived from hescs using defined media supplemented with a specific combination of growth factors. the sd56 hnscs grew as an adherent monolayer culture, uniformly expressed molecular features of hnscs including nestin, vimentin and the radial glial marker 3cb2, and did not express the pluripotency markers oct4 or nanog. the self - renewal property of the hnscs was characterized by a predominant symmetrical mode of cell division. they exhibited no chromosomal abnormalities and demonstrated non-tumorigenic properties after implantation into ischemic brains and into naive nude rat brains and flanks. furthermore, the transplanted sd56 hnscs migrated toward the stroke - damaged adult brain parenchyma, engrafted and improved the independent use of the stroke - impaired forelimb. maintenance of stem cells requires symmetrical and asymmetrical cell divisions to both expand and to give rise to specialized progeny of a specific tissue ( reviewed in [ 23 ] ). in vivo, a complex cellular micro - environment or niche ensures the self - maintenance property of nscs [ 24 ], [ 25 ], [ 26 ], [ 27 ]. in vitro, defined growth factors and extracellular matrices support stem cell self - renewal [ 28 ], [ 29 ]. the embryonic stem cells can propagate in a predominantly proliferative symmetrical mode, leading to homogeneous cell cultures growing relatively quickly with minimal cell differentiation [ 30 ], [ 31 ], [ 32 ], [ 33 ], [ 34 ]. tissue specific stem cells, however, self - renew in a predominant asymmetric mode to maintain them selves and compensate for the loss of differentiated cells due to disease or injury. thus, nscs isolated from developing or adult brain grow as a mixture of undifferentiated and differentiated cells due the predominant asymmetrical mode of cell division [ 35 ], [ 36 ], [ 37 ], [ 38 ], [ 39 ], [ 40 ], [ 41 ]. a recent study has reported that a murine esc - derived nsc line ( lc1 ) is propagated as an adherent homogenous culture with a dominant mode of symmetrical self - renewal [ 21 ]. a combination of egf and fgf2 was sufficient to propagate these nscs as an adherent monolayer. however, the sd56 hnsc line described here required the combination of egf, bfgf and lif for self - maintenance. although there are morphological and molecular similarities between our hnscs and the nscs previously described [ 21 ], the methods of isolation and growth are different. in addition to the different combination of growth factors used, the hnsc line we have isolated did not go through the rosette - structure stage. the in vitro analysis of brdu incorporation and nestin expression indicated that our hnscs divide predominantly symmetrically. this type of growth pattern is characteristic of primitive normal stem cells undergoing mostly symmetric cell division to increase the stem cell pool at the early stage of development or during tissue regeneration after injury [ 23 ]. rt - pcr and immunocytochemistry analysis demonstrated that these undifferentiated sd56 cells did not express any pluripotency, endodermal or mesodermal markers.