furthermore, the sd56 hnscs described here exhibited the multipotential characteristic to differentiate into neurons, astrocytes and oligodendrocytes both in vitro and upon transplantation. together these findings suggest that the hnsc line we isolated are appropriately programmed and share similar characteristics with the definitive nscs of the developing brain. the sd56 hnscs demonstrated a remarkable ability to migrate toward the stroke - damaged parenchyma of the adult rat brain. this directed migration by the majority of the grafted cells could be due to their uniform cellular composition, which results in an equal response to the host microenvironment. in previous studies, subpopulations of transplanted hescs that were enriched in neural cells migrated in host microenvironments conducive to cell migration, such as the developing brain or in structures such as the rostral migratory stream [ 13 ], [ 20 ]. in the adult lesioned brain, the grafted hesc - derived neural cells proliferated and formed rosettes [ 14 ], teratomas [ 12 ], [ 15 ] or a cellular mass that induced a gliotic host response whereby local astrocytes demarcated the grafts [ 16 ]. enriched neural cultures derived from mouse [ 42 ] and monkey escs [ 43 ] have produced behavioral improvements when transplanted into animal models of stroke and brain injury. however, in these cases, the transplanted non - human escs also formed a mass with signs of overgrowth in the core, as well as deformations [ 44 ], [ 45 ], [ 46 ]. escs plated at low density acquire neural identity within few hours after plating [ 47 ]. interestingly, nearly all viable cells expressed nestin, the early neural fate marker sox1, and the pluripotency marker oct4. together, these studies are seminal and suggest that complete neuralization may not be achieved through certain enrichment processes, consequently the neural cells could revert to a pluripotent stage [ 17 ]. the dispersion of the grafted hnscs within host parenchyma may allow for more graft - host interactions that could stabilize differentiation, inhibit growth and prevent gliotic host response. in the present study, sd56 hnsc - transplanted animals demonstrated a stable improvement in the sensorimotor function when evaluated for spontaneous exploratory activity in the cylinder test that detects long - lasting deficits in forelimb use in the experimental models of stroke [ 22 ]. the transplantation of hnscs significantly enhanced the independent use of the impaired contralateral forelimb 8 weeks post transplantation. this is the first report demonstrating that the transplantation of hnscs derived from hescs can improve neurologic behavior after experimental stroke. together, these findings are encouraging and suggest that these cells are promising for future development. in addition to their therapeutic application, the hnscs isolated under the reported conditions offer a means to interrogate host environments and unravel mechanistic features of self - renewal, non-tumorigenic ity and functional engraftability in animal models of neurological disor ders. hesc and nsc culturesthe hesc line h9 ( wicell research institute ) was propagated every 5 to 7 days on irradiated mouse embryonic fibroblasts. the cell culture media consisted of a 1∶1 mixture of dulbecco ' s modified eagle ' s medium ( dmem ) and f12 nutrient, 20 % serum replacement ( invitrogen ), 0. 1 mm β - mercaptoethanol, 2 µg / ml heparin and 4 ng / ml bfgf ( r & d systems ). to generate the nscs, dissociated hescs were cultured in a chemically defined medium compose d of dmem / f12 ( 1∶1 ) including glucose ( 0. 6 % ), glutamine ( 2 mm ), sodium bicarbonate ( 3 mm ), and hepes buffer ( 5 mm ) [ all from sigma except glutamine ( invitrogen ) ]. a defined hormone mix and salt mixture ( sigma ), including insulin ( 25 mg / ml ), transferrin ( 100 mg / ml ), progesterone ( 20 nm ), putrescine ( 60 mm ), and selenium chloride ( 30 nm ) was used in place of serum. the medium was supplemented with egf ( 20 ng / ml ), bfgf ( 10 ng / ml ) and lif ( 10 ng / ml ). dissociated hnscs were plated at a density of 100, 000 cell / ml in corning t75 ( invitrogen ) culture flasks in the defined media together with the growth factors. after 5 – 7 div, the adherent culture was incubated in 0. 025 % trypsin / 0. 01 % edta ( w / v ) for 1 min followed by the addition of trypsin inhibitor ( invitrogen ) then gently triturated to achieve single cell suspension. the cells were then washed twice with fresh medium and reseeded in fresh growth factor - containing media at 100, 000 cells / ml. this procedure was performed for 21 passages and the fold of increase and population doubling were calculated at each passage. for clonal analysis, single spheres or confluent hnsc cultures were single cell dissociated and serially diluted to yield 1 – 2 cell / 10 µl. a 10 - µl - cell suspension was then added to each of 96 or 24 well plates containing 200 – 300 µl of growth media. wells containing one viable cell were marked the next day and re - scored 5 to 7 days later for cell proliferation. the differentiation of the hnscs was performed as previously described [ 48 ]. dissociated hnscs were plated at a density of 106 cells / ml in control ( media / hormone mix ) medium devoid of any growth factors and supplemented with 1 % fetal bovine serum ( fbs ) on poly-l-ornithine-coated ( 15 mg / ml; sigma ) glass coverslips in 24 - well nunclon culture dishes with 0. 5 ml / well. after 2, 7 – 15 div cultures were fixed and processed for immunocytochemistry or used for rt - pcr analysis. karyotype analysislong - term cultures of hnscs were incubated at 37°c and harvested for metaphase chromosomes when the cultures were 75 % confluent. metaphase chromosomes were obtained by standard chromosome harvest methods by exposure to colcemid at 0. 1 µg / ml for 1 hour at 37°c, a 2 - minute exposure to trypsin / edta, hypotonized with 0. 057 m kcl and fixed with 3∶1 methanol: acetic acid. slide preparations were made by dropping the fixed cell pellet onto cold, wet slides and air - dried. after incubating the slides at 90°c for 30 minutes, chromosomes were trypsin banded and then wright / giemsa stained for g - banding analysis. twenty metaphase cells were completely analyzed and a normal female chromosome complement was found ( 46, xx ). tumorigenicity in nude ratsall animal experiments were conducted according to the national institute of health ( nih ) guidelines and approved by the iacuc. normal adult nih - nude rats ( n = 5, 8 week - old, taconic, germantown, new york, united states ) were used to test the tumorigenic potential of the sd56 hnscs. undifferentiated hnscs from passage 9 were single cell dissociated using trypsin - edta and suspended at the concentration of 125, 000 cell / µl in preparation for cell transplantation. two µl of the cell suspension were stereotaxically transplanted into 4 sites within the striatum at the following coordinates: ap: + 1. 0 mm, ml: + 3. 2 mm, dv: −5. 0; ap: + 0. 5 mm, ml: + 3. 0 mm, dv: −5. 0; ap: −0. 5 mm, ml: + 3. 0 mm, dv: −5. 0; ap: −1. 0 mm, ml: + 3. 5 mm, dv: −5. 0 mm with the incisor bar set at 3. 4 mm. the injection rate was 1 µl / min, and the cannula was left in place for an additional 5 min before retraction. for the flank tumor assay, 2×106 cells ( 125, 000 cell / µl ) were injected subcutaneously to the side of the adult nude rats. to label mitotically active cells in vivo during s-phase, the rats were injected ip with the brdu ( 50 mg / kg, sigma ) every 8 hours during the last 24 hours before euthanasia. after 2 - month survival time, rats were euthanized and a postmortem examination for tumor formation was performed. induction of focal ischemia and cell transplantationall animal experimentations were conducted according to the national institute of health ( nih ) guidelines and approved by the iacuc.