sprague dawley adult male rats ( n = 17, 275g – 310g, charles river laboratories, wilmington, massachusetts, united states ) were subjected to one and a half hour suture occlusion of the middle cerebral artery ( mcao ), as previously described [ 49 ] and immunosuppressed 2 days before cell transplantation and daily thereafter for one week with i. p. injections of cyclosporine a ( 20 mg / ml, sandimmune, novartis pharmaceuticals ). thereafter oral cyclosporine was used at 210 µg / ml in drinking water until euthanasia. undifferentiated sd56 hnscs from passages between p9 and p13 were single cell dissociated using trypsin - edta in preparation for cell transplantation. one week after the stroke lesion, 2 µl of the hnscs, at a concentration of 50, 000 cell / µl, were stereotaxically transplanted into 4 sites within the lesioned striatum ( n = 10 ) 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. as a control group, we used rats subjected to ischemia and injected with the vehicle ( n = 7 ). all animals underwent baseline motor behavioral assessment before and after the ischemic lesion, and 4 & 8 weeks after cell transplantation. the animals were killed after 2 - month survival time by transcardial perfusion with phosphate buffered saline ( pbs ) followed by 4 % paraformaldehyde. the brains were cryoprotected in an increasing gra dien t of 10, 20 and 30 % sucrose solution and cryostat sectioned at 40 µm and processed for immunocytochemistry. immunocytochemistrycultures were fixed with 4 % paraformaldehyde for 15 min. both cells and brain sections were rinsed in pbs for 3×5 min then incubated for 2 hrs ( cultures ) or overnight ( brain sections ) with the appropriate primary antibod ies for multiple labeling. secondary antibod ies raised in the appropriate hosts and conjugated to fitc, ritc, amca, cy3 or cy5 chromogenes ( jackson immunorese arch ) were used. cells and sections were counterstained with the nuclear marker 4 ′, 6 - diamidine - 2 ′ - phenylindol e dihydrochloride ( dapi ). positive and negative controls were included in each run. immunostained sections were coverslipped using fluorsave ( calbiochem ) as the mounting medium. the following antibod ies were used: anti-human nuclei ( hnuc, monoclonal 1∶100, chemicon ), anti-tuj1 ( monoclonal 1∶100, covance; polyclonal 1∶200, aves labs ); anti-gad65 / 67 ( polyclonal 1∶1000, chemicon ); anti-glial fibrillary acidic protein ( gfap, monoclonal 1∶1000, chemicon; polyclonal 1∶200, aves labs ); anti-galactocerebrocide ( gc, monoclonal 1∶250, chemicon ); anti-cnpase ( polyclonal 1∶200, aves labs ); anti-glucose transporter type 1 ( glut - 1 polyclonal, 1∶500, chemicon ); anti-nestin ( polyclonal 1∶1000, chemicon ); anti-vimentin ( monoclonal 1∶500, calbiochem ); anti-3cb2 ( monoclonal 1∶500, developmental studies hybridoma bank ); anti-doublecortin ( dcx, polyclonal 1∶250, santacruz biotechnology ); anti-ki67 ( polyclonal 1∶250, santacruz biotechnology ). fluorescence was detected, analyzed and photographed with a zeiss lsm550 laser scanning confocal photomicroscope. for each animal, quantitative estimates of the total number of grafted cells were ster eol ogically determined using the optical fractionator procedure [ 50 ]. a computer - assisted image analysis system was performed using stereo investigator software ( microbrightfield, inc. ). the rostral and caudal limits of the reference volume were determined by first and last frontal sections containing grafted cells. the striatum and cortex were ac curate ly outlined at low magnification ( 2. 5× objective ). the optical fractionator probe was selected to perform systematic sampling of the immunoreactive cell population distributed within the serial sections to estimate the population number in the volume of tissue. the counting frame of the optical fractionator was defined at 50×50 µm squares and the systematic sampling was performed by translating a grid with 200×200 µm squares onto the sections of interest using the stereo investigator software. the sample sites were systematically and automatically generated by the computer and examined using a 60× objective of a nikon eclipse te 300 microscope. the counting frame displayed inclusion and exclusion lines and only immunoreactive cell bodies falling within the counting frame with no contact with the exclusion lines were counted. the cell dispersion was measured by counting the number of cells within 200 µm distance from the graft site. the number and distance in µm of cells dispersed beyond 200 µm was also measured. an average of 2, 000 cells was counted per animal. double labeling was determined using the confocal laser scanning microscope by random sampling of 100 or more cells per marker for each animal, scoring first for hnuc +, followed by dapi + nuclei and then the marker of choice. the double labeling was always confirmed in x - z and y - z cross - sections produced by the orthogonal projections of z - series. reverse transcription - polymerase chain reaction ( rt - pcr ) analysistotal rna was extracted from cultured cells using rnaeasy kit ( quiagen ). aliquots ( 1 µg ) of total rna from the cells were reverse transcribed in the presence of 50 mm tris-hcl, ph 8. 3, 75 mm kcl, 3 mm mgcl2, 10 mm dtt, 0. 5 µm dntps, and 0. 5 µg oligo - dt ( 12 – 18 ) with 200 u superscript rnase h - reverse transcriptase ( invitrogen ). pcr amplification was performed using standard procedure with taq polymerase. aliquots of cdna equivalent to 50 ng of total rna were amplified in 25 µl reactions containing 10 mm tris-hcl, ph 8. 3, 50 mm kcl, 1. 5 mm mgcl2, 50 pmol of each primer, 400 µm dntps, and 0. 5 u amplitaq dna polymerase ( perkin - elmer ). pcr was performed using the following thermal profile: 4 min at 94°c; 1 min at 94°c, 1 min at 60°c, 1. 5 min at 72°c, for 30 – 40 cycles; 7 min at 72°c, and finally a soak at 4°c overnight. the following day, 10 µl aliquots of the amplified products were run on a 2 % agarose tris – acetate gel containing 0. 5 mg / ml ethidium bromide. the products were visualized through a uv transilluminator, captured in a digital format using quantify one gel analysis software ( bio - rad laboratories ) on a macintosh g4 computer. the pcr primers specific to each transcript were as follows: gfap, forward ( f ), 5 ′ - tcatcgctcaggaggtcctt – 3 ′ reverse ( r ), 5 ′ - ctg ttgccagagatggaggtt – 3 ′; map2 ( f ) 5 ′ - gaagactcgcatccgaatgg – 3 ′, ( r ) 5 ′ - cgcaggataggaggaagagact – 3 ′; mbp ( f ) 5 ′ - ttagctgaattc gcgtgtgg – 3 ′, ( r ) 5 ′ - gaggaagtgaatgagccggtta - 3 ′ were deigned using the primer designer software, version 2. 0 ( scientific and educational software ) [ 48 ]. 18s, β - tubulin class iii, n - cam, nestin, nf - m, notch - 1 primers [ 51 ]. foxa2 ( hnf3b ), brachyury primers [ 52 ]. behavioral teststhe cylinder test was used to assess the spontaneous forelimb use during lateral exploration movement [ 22 ]. rats were placed in a transparent acrylic cylinder ( 20 cm diameter ) for 5 minutes. the cylinder encourages use of the forelimbs for vertical exploration. a mirror was placed behind the cylinder so that the forelimbs could be viewed at all times. testing sessions were videotaped and forelimb use was scored by a blinded operator. movements scored were the independent use of the left or right forelimb or simultaneous use of both the left and right forelimb to contact the wall of the cylinder during a full rear, to initiate a weight - shifting movement, or to regain center of gravity while moving laterally in a vertical posture along the wall. animals were tested for their baselines after stroke and 4 and 8 weeks after cell transplantation.